Antibiotics doi: 10.3390/antibiotics15090886

Authors: Tim Klomp Andreas Braun Georg Conrads

Background: Porphyromonas gingivalis is a Gram-negative, anaerobic keystone pathogen strongly implicated in the pathogenesis of periodontitis. Its pathogenicity is mediated through virulence factors, such as lipopolysaccharides (PG-LPS), which have been reported to display conflicting results regarding structural composition and immunogenicity. Objectives: This scoping review investigates how PG-LPS modulates host immunity and contributes to advanced periodontitis, with emphasis on its structural composition, divergent immunogenic properties, and the possible regulatory factors underlying these observations. Furthermore, PG-LPS interactions with periodontal tissues and associations with outer membrane vesicles (OMV) are examined. Materials and Methods: A structured literature search was conducted in PubMed following the PRISMA-ScR guidelines and PCC frameworks. Results: Structural heterogeneity, especially within the lipid A moiety of PG-LPS, accounts for previously inconsistent findings regarding its immunogenicity. Minor alterations in lipid A composition through enzymatic dephosphorylation or deacylation profoundly affect Toll-like receptor (TLR) engagement, antimicrobial resistance, and cytokine induction, and contribute to the biogenesis of OMVs. Environmental factors such as hemin availability and temperature influence enzymatic activity, resulting in lipid A isoforms that act as TLR4 agonists, antagonists, or remain immunologically inert. This structural plasticity mediates dynamic immune modulation that fosters polymicrobial dysbiosis and perpetuates non-resolving inflammation, leading to progressive tissue destruction, while concurrently contributing to the systemic inflammatory burden. Conclusions: Lipid A heterogeneity represents a central adaptive strategy potentially linking environmental cues and the bioenergetic status of P. gingivalis to immunomodulative properties. Verification of in vivo lipid A structural shifts in clinical isolates could inform novel therapeutic approaches targeting P. gingivalis-mediated inflammation.

Antibiotics doi: 10.3390/antibiotics15090885

Authors: Hülya Arık

The rise in carbapenem resistance is well documented, but a less conspicuous trend has received far less attention: the gradual upward shift in minimum inhibitory concentration (MIC) values among isolates that remain phenotypically “susceptible.” We propose the term carbapenem floor for this phenomenon: an intermediate zone in which standard dosing regimens become progressively less reliable at achieving target exposure, yet which remains invisible to breakpoint-based surveillance; we present this as a hypothesis-generating framework consistent with existing pharmacokinetic and epidemiological evidence rather than as an empirically established trend, since no systematic longitudinal quantification of carbapenem MIC drift has yet been undertaken. Because carbapenems exhibit time-dependent killing and their pharmacokinetic/pharmacodynamic (PK/PD) target has MIC as its denominator, even a shift within the susceptible range can substantially reduce target attainment; augmented renal clearance in critically ill patients deepens this erosion. This review integrates resistance surveillance evidence with PK/PD principles to evaluate prolonged infusion, loading doses, therapeutic drug monitoring (TDM), and empirical protocols derived from population pharmacokinetic models and local MIC distributions, examining for each the evidence and the practical constraints. We also consider the reverse relationship: failure to attain PK/PD targets appears to select for elevated MICs, suggesting that dosing practice may contribute to resistance selection and potentially influence resistance epidemiology, not only respond to it. Extending antibiogram reporting to include MIC distributions, and translating those data into dosing protocols through antimicrobial stewardship, is a feasible priority where TDM infrastructure is limited.

Antibiotics doi: 10.3390/antibiotics15090884

Authors: Magdalena Szemraj Monika Sienkiewicz

Background/Objectives: Skin and soft tissue infections (SSTIs) remain a major clinical challenge due to the increasing prevalence of antimicrobial resistance and biofilm-associated pathogens. Conventional antibiotics are often limited by reduced efficacy, recurrent infections, and disruption of the resident skin microbiota. Consequently, bacteriocins have emerged as promising alternative or adjunctive antimicrobial agents for the treatment of skin and wound infections. Methods: A literature review was conducted using the PubMed, Scopus, and Web of Science databases. Experimental in vitro, ex vivo, and in vivo studies investigating bacteriocins in skin and wound infection models were analyzed, with a focus on antimicrobial activity, antibiofilm efficacy, activity against antimicrobial-resistant pathogens, and formulation strategies designed to improve therapeutic performance. Results: Available evidence demonstrates that numerous bacteriocins exhibit potent antimicrobial activity against clinically relevant skin-associated pathogens, particularly Staphylococcus aureus, including methicillin-resistant strains (MRSA). Several bacteriocins also showed significant antibiofilm properties and synergistic interactions with conventional antibiotics, resulting in enhanced bacterial eradication and reduced risk of resistance development. Experimental infection models further support their therapeutic potential in wound-associated infections. Additionally, advanced delivery platforms, including hydrogels, wound dressings, nanofibers, and lipid-based nanoparticles, improved peptide stability, sustained release, and local antimicrobial efficacy. Conclusions: Bacteriocins are promising candidates for the prevention and treatment of skin and wound infections due to their antimicrobial and antibiofilm activity, low propensity for resistance development, and suitability for topical administration. However, further studies addressing formulation optimization, safety, pharmacokinetics, and clinical validation are required before bacteriocin-based therapies can enter routine clinical practice.

Antibiotics doi: 10.3390/antibiotics15090883

Authors: Alessandra Romandini Chiara Resnati Stefania Crucitta Stefano Agliardi Federico D’Amico Giulia Angela Carla Pattarino Elena Altieri Romano Danesi Costantino De Giacomo

The rise of multidrug-resistant (MDR) pathogens in the pediatric population represents a significant global health challenge, compounded by a historically stagnant antibiotic pipeline for children. While several novel antibiotics have been approved for adults in recent decades, pediatric labeling is often deferred due to the complexities of developmental pharmacology and due to a certain precautionary prudence in introducing new drugs onto the market for this population. This review explores the landscape of the most recent antibiotics, including advanced cephalosporins (ceftaroline, cefiderocol, and ceftobiprole), novel beta-lactam/beta-lactamase inhibitor combinations (e.g., ceftazidime/avibactam, meropenem/vaborbactam), and long-acting lipoglycopeptides. We analyze their approval trials, pediatric-specific PK/PD profiles, and the balance between on-label use and evidence-based off-label prescriptions. Furthermore, we emphasize the role of antimicrobial stewardship through the “3 D’s” rule and the evolution of Therapeutic Drug Monitoring (TDM) from reactive safety controls to proactive, model-informed precision dosing (MIPD). Finally, we advocate for a multidisciplinary synergy between pediatricians and pharmacologists as the cornerstone for optimizing outcomes and preserving the efficacy of the future antibiotic pipeline.

Antibiotics doi: 10.3390/antibiotics15090882

Authors: Noran Magdy Zahran Usman Abubakar Asmaa Ezzeldin Mohd. Mohamed Hani Jaouni Sayed Tarique Kazi Muna Al-Maslamani

Background/Objectives: The prescription of aerosolized antimicrobial agents for lower respiratory tract infections varies significantly among clinicians. This study investigated the practices and perceptions of clinicians regarding aerosolized antimicrobial therapy. Methods: A cross-sectional study was conducted among infectious diseases (ID) and critical care clinicians (physicians and clinical pharmacists) in eight acute care hospitals in Qatar using a validated and pretested self-administered online questionnaire. Data was collected between September and October 2025, and analyzed using the Statistical Package for the Social Sciences (SPSS). Results: The majority of the respondents were physicians (70.5%), and about one-third (31.8%) had more than 15 years of work experience. A vast majority (97.7%) had previously prescribed or recommended aerosolized antimicrobial agents. Some respondents (34.1%) reported using aerosolized antimicrobial agents at least once every three months. The indications for aerosolized antimicrobial prescribing were for the treatment of bacterial infections (86.4%) and prophylaxis against bacterial infections (38.6%). Aerosolized antimicrobials were used for the treatment of ventilator-associated pneumonia (68.2%), cystic fibrosis (61.4%) and ventilator-associated tracheobronchitis (34.1%). Gentamicin (88.6%) and colistin (72.7%) were the most frequently used aerosolized antimicrobial agents. The majority (88.6%) of the respondents combined aerosolized antimicrobial agents with systemic antimicrobial agents, 65.9% of whom combined different antimicrobials for aerosolized and systemic routes. Pseudomonas aeruginosa (97.7%) and Acinetobacter baumannii (56.8%) were the most common microorganisms treated with aerosolized antimicrobial agents. Most respondents (61.4%) did not provide pre-treatment prior to aerosolized antimicrobial therapy. However, 38.6% used bronchodilators for pre-treatment. Bronchospasm (79.5%) and hypoxemia (20.5%) were the adverse effects reported by the respondents. Some respondents (40.9%) strongly agreed/agreed that aerosolized antimicrobial therapy improves clinical and microbial cure rates. While 81.8% strongly agreed/agreed that aerosolized antimicrobial therapy reduces systemic toxicity. Practices and perceptions varied significantly between ID and critical care specialties, and between physicians and pharmacists. Conclusions: Clinicians need training on aerosolized antimicrobial therapy for lower respiratory tract infections. Immunosuppression, renal function and infections due to multidrug-resistant organisms were reported by clinicians as factors that influenced their decision to use aerosolized antimicrobial therapy. A protocol for the administration of aerosolized antimicrobial therapy is recommended.

Antibiotics doi: 10.3390/antibiotics15090881

Authors: Jarett Worden Tyler Baumeister Ellen Eaton

The treatment of infective endocarditis (IE) in patients who inject drugs (PWID) is complex and challenging. These patients face stigma in the healthcare system, as well as higher mortality, longer length of hospital stay, higher rates of recurrent infections, and lower likelihood of antibiotic completion. Standard treatment for IE involves several weeks of antibiotics. For PWID, these prolonged courses often mean intravenous antibiotics administered in the inpatient setting for at least 4 to 6 weeks, which increases both healthcare costs and the risk of patient-directed discharge against medical advice. Effective treatment of IE in PWID involves a combination of addiction management and appropriate antibiotic therapy. To promote rational use of healthcare resources and optimize antibiotic stewardship, novel approaches to antibiotic therapy must be utilized. This review discusses evidence supporting treatment strategies outside of the inpatient hospital setting, including outpatient parenteral antibiotic therapy (OPAT), long-acting lipoglycopeptides, and oral antibiotic therapy for the treatment of endocarditis. Optimal treatment of IE in PWID involves support from a multidisciplinary healthcare team, management of substance use disorder, and effective antibiotic therapy.

Antibiotics doi: 10.3390/antibiotics15090880

Authors: Aghilas Akkache Mattéo Védère Skander Hathroubi

Antimicrobial peptides (AMPs) are increasingly regarded as next-generation antimicrobial agents because of their broad-spectrum activity, rapid killing, antibiofilm potential, immunomodulatory properties, and mechanisms of action that differ from those of many conventional antibiotics. Despite these advantages, their clinical translation remains limited by proteolytic instability, hemolysis or cytotoxicity, poor pharmacokinetics, salt and serum sensitivity, production costs, and delivery challenges. The AMP field is therefore shifting from natural peptide discovery toward integrated engineering pipelines that combine rational peptide modification, biomaterial-based delivery, high-throughput screening, and artificial intelligence (AI), particularly machine learning (ML) and deep learning approaches. Chemical and structural modifications, including D-amino acid substitution, N-glycine substitution, cyclization, lipidation, PEGylation, terminal amidation, hydrocarbon stapling, hybridization, sequence truncation, metal coordination, and biomaterial immobilization, are being used to improve stability, potency, selectivity, antibiofilm activity, and tissue localization. In parallel, AI-guided approaches, including ML, deep learning, and generative modeling, enable large-scale exploration of diverse peptide sources, including microbiomes and extinct proteomes, to entirely new sequences, while supporting optimization of potency, selectivity, stability, toxicity, and synthesizability. This focused review summarizes recent advances in AMP engineering, biomaterial-assisted delivery, and AI-guided discovery for biofilm-associated infections in the context of antimicrobial resistance.

Antibiotics doi: 10.3390/antibiotics15090879

Authors: Aryaan P. Duggal Adit B. Alreja Isha Vashee Hayley Nordstrom Erin Harrelson Nakia Fallen Kari-Ann Takano Ryan A. Blaustein Derrick E. Fouts Norberto Gonzalez-Juarbe

Background/Objectives: Serratia marcescens is an opportunistic pathogen that causes severe hospital-acquired infections, notable for its biofilm formation abilities and development of extensive antibiotic resistance. Here, we aim to evaluate the efficacy of bacteriophages, antibiotics, and antimicrobial peptides (BAP), alone and in combination, against fourteen multidrug-resistant (MDR) S. marcescens isolates sourced from hospitals and other environmental settings. Methods: S. marcescens was grown planktonically or in surface-associated biofilms, and biofilm biomass was measured via changes in absorbance and colony-forming units or live/death staining. Results: Combining bacteriophage with a low-dose cocktail of penicillin–streptomycin, kanamycin, and ciprofloxacin enhanced antimicrobial activity compared with antibiotics alone. Across the isolate panel, responses to BAP treatment varied according to determined antibiotic resistance profiles. The highly resistant AR-0517 isolate was selected for detailed mature biofilm analysis, where the BAP treatment reduced biofilm biomass by 97.8% and recoverable bacteria by 99.99%. Microscopy and viability assays further confirmed extensive biofilm disruption and bacterial killing. Conclusions: These findings demonstrate that simultaneous targeting of multiple bacterial pathways can enhance antimicrobial activity against MDR S. marcescens in vitro and support further evaluation of BAP as a potential strategy for biofilm-associated infections.

Antibiotics doi: 10.3390/antibiotics15090878

Authors: Ugonna C. Morikwe Larisa C. Kiki Franklin C. Ezeanowai Shamiah Hall Shilpi Bhatia Tinyiko Nicole Maswanganye Olusola Jeje Megan S. Hill Joseph L. Graves Dongyang Deng Liesl Jeffers-Francis

Background/Objectives: Antimicrobial resistance (AMR) and virulence represent co-evolving dimensions of microbial pathogenic potential whose ecological organization in building-scale wastewater systems remains poorly understood. Methods: Using shotgun metagenomic sequencing, we characterized the temporal dynamics and ecological associations of antimicrobial resistance genes (ARGs) and virulence factors (VFs) in 12 wastewater grab samples (2 per semester) collected from a university residence hall designated for COVID-19 quarantine between 2021 and 2023. Results: The wastewater microbiome was anchored by a stable core of gut-associated anaerobic bacteria, with community composition exhibiting significant Spring-versus-Fall structuring and a year × semester interaction that explained 60% of the community variation. A marked shift toward opportunistic taxa, particularly Acinetobacter, during Fall 2023 represented the most pronounced temporal perturbation. Total ARG abundance remained stable across semesters, while resistome composition shifted significantly, indicating that temporal dynamics were driven by compositional turnover rather than changes in overall resistance burden. VF functional categories were broadly conserved across sampling periods, consistent with their structural embedding within the persistent fecal core microbiome. Correlation and network analyses revealed modular ecological coupling between resistance and virulence functional categories, with metal/co-resistance and fosfomycin classes showing the strongest associations with virulence functions. At the community level, a Benjamini–Hochberg–corrected co-occurrence network resolved into taxa-anchored resistance modules and separate virulence-function clusters, with Acinetobacter and fluoroquinolone resistance as the principal connectors. Conclusions: These findings indicate that building-scale wastewater metagenomics can capture ecologically structured functional gene dynamics, highlighting its potential as a surveillance tool for monitoring AMR and virulence in built environments.

Antibiotics doi: 10.3390/antibiotics15090877

Authors: Orit Wolfovitz Barchad Ido Weinstock Yonit Wiener-Well Eli Ben-Chetrit

Background: Adults who undergo blood-culture sampling in the emergency department (ED) may be discharged before results are finalized and later found bacteremic. Factors identifying which patients require readmission are not well defined. We sought predictors of return and hospitalization available at the index ED visit. Methods: In this retrospective study at a 1000-bed university-affiliated hospital (September 2021–March 2026), we included adults discharged from the ED with a positive blood culture identified post-discharge. The primary outcome was ED return with hospitalization within 30 days. Multivariable logistic regression was restricted to variables available at the index ED visit. Results: Of 197 eligible patients, 16 were excluded, leaving 181 analyzed: 78 (43.1%) were managed as outpatients, 36 (19.9%) returned and were re-discharged, and 67 (37.0%) returned and were hospitalized. Median age was 73 (IQR 61–83) years and 105 (58.0%) were male. Index-visit vital signs and laboratory values did not differ across trajectories. The only independent predictor of return and hospitalization was a non-UTI working discharge diagnosis (aOR 2.53, 95% CI 1.3–5.0). Creatinine ≥ 2 mg/dL (aOR 2.65, 95% CI 0.9–7.5) and previous hospitalization within 6 months (aOR 1.82, 95% CI 0.8–4.1) were significant per univariate analysis but attenuated after adjustment, and age was not predictive. Enterobacterales predominated (n = 111, 61.3%). All nine endovascular-infection cases were hospitalized. Overall mortality was 1.1% (2/181). Conclusions: Post-discharge bacteremia carried low mortality but frequent ED return and hospitalization. A non-UTI working diagnosis independently identified a higher-risk minority; renal impairment and recent hospitalization were supportive but non-independent markers.

Antibiotics doi: 10.3390/antibiotics15090876

Authors: Filippo Giorgio Di Girolamo Filippo Mearelli Donatella Denora Ludovica Ilaria Carniel Nicola Fiotti Gianni Biolo Dario Bianchini Massimiliano Fabricci Verena Zerbato Stefano Di Bella Chiara Roni

Background/Objectives: Carbapenem overuse contributes to antimicrobial resistance, but pragmatic stewardship models for high-complexity Internal Medicine wards remain underreported. We evaluated the feasibility and stewardship impact of a carbapenem-sparing intervention based on time-limited named-patient dispensing, pharmacist-supported reassessment, and mandatory infectious diseases reassessment for continuation beyond seven days, while assessing short-term clinical outcomes as exploratory safety signals. Methods: We conducted a prospective quasi-experimental before-and-after pilot implementation study in two Internal Medicine wards of a university hospital. Consecutive adult inpatients receiving a carbapenem or fluoroquinolone were enrolled during a 2-month control phase (standard care, n = 40) and a subsequent 2-month intervention phase (n = 38). Initial prescription remained at the discretion of the treating physician; targeted antibiotics were dispensed on a named-patient basis for a maximum initial duration of seven days, with pharmacist-supported reassessment on days 3 and 5 and mandatory infectious diseases reassessment for continuation beyond seven days. Results: Antibiotic therapy duration was shorter in the intervention group (median approximately 6 vs. 9 days, p = 0.0001), and antibiotic discontinuation by day 7 was more frequent than in controls (81.6% vs. 45.0%, p = 0.001). Total targeted antibiotic exposure decreased by approximately 40% (mean 5.9 vs. 10.0 Defined Daily Doses (DDD) per patient, p < 0.001), mainly driven by reduced meropenem use. No significant differences were observed in biomarker trajectories, clinical status at day 7, 30-day readmission or relapse, or 90-day mortality. Conclusions: A time-limited dispensing and reassessment model was feasible in Internal Medicine and was associated with lower carbapenem and targeted restricted-antibiotic exposure. No apparent short-term signal of clinical worsening was identified, although the study was not powered to establish safety, clinical non-inferiority, or equivalence. Larger multicentre studies using standardized stewardship metrics are needed.

Antibiotics doi: 10.3390/antibiotics15090875

Authors: Asma Mammar Hayat Trabsa Stefania Dentice Maidana Ammar Ayachi Asma Abdessemed Leonardo Albarracin Mariano Elean Kamel Boubakri Ayelen A. Baillo Haruki Kitazawa Julio Villena

Background: The vaginal microbiota of healthy women is predominantly composed of lactic acid bacteria (LAB), particularly species from the lactobacilli group, which contribute to the maintenance of vaginal health through multiple protective mechanisms. Objectives: In the present study, vaginal LAB isolated from healthy premenopausal Algerian women were evaluated for their probiotic potential. Methods: A total of 259 LAB isolates were initially screened for antimicrobial activity against vaginal pathogens, among which 164 strains exhibited inhibitory effects. Based on antimicrobial capacity and biofilm formation ability, 38 strains were selected for further characterization. A refined subset of 15 strains belonging to Lactiplantibacillus (Lpb.) plantarum, Limosilactobacillus (Lmb.) fermentum, and Ligilactobacillus (Lgb.) salivarius were subsequently identified according to their superior antimicrobial and probiotic-related properties. The selected strains were evaluated for several functional and safety characteristics, including antimicrobial activity, production of lactic acid and hydrogen peroxide (H2O2), biofilm formation, auto-aggregation, cell surface hydrophobicity, antibiotic susceptibility, hemolytic activity, and tolerance to acidic conditions. These studies were complemented with comparative genomics using the complete genome sequences of selected strains. Results: The isolates demonstrated significant inhibitory activity against vaginal pathogens associated with aerobic vaginitis, and vulvovaginal candidiasis. In addition, strong correlations were observed between metabolite production and antimicrobial activity. Several strains also exhibited enhanced biofilm formation, aggregation capacity, and favorable safety profiles. Among the tested isolates, Lmb. fermentum 70BJ2, Lpb. plantarum 87JG8, and Lpb.plantarum N17 emerged as particularly promising probiotic candidates due to their combined antimicrobial, metabolic, and adhesion-related properties. Conclusions: Overall, these findings demonstrate that the vaginal microbiota of healthy Algerian women represents a valuable source of potential probiotic strains and provide a strong basis for future in vivo validation and clinical application in the prevention and management of vaginal infections.

Antibiotics doi: 10.3390/antibiotics15090874

Authors: Nazia Tabassum Aqib Javaid Abirami Karthikeyan Minji Kim Young-Mog Kim Fazlurrahman Khan

Vibriosis causes substantial losses in marine and brackish-water aquaculture, while increasing antimicrobial resistance limits the effectiveness of conventional antibiotic treatment. Biofilm formation and quorum-sensing-controlled virulence further contribute to the persistence of pathogenic Vibrio species. Prophages integrated into Vibrio genomes encode proteins and regulatory elements that may provide alternative approaches for controlling these pathogens. This review evaluates endolysins, polysaccharide depolymerases, nucleases, holins, spanins, tailocins, regulatory proteins, and small RNAs associated with prophages and related phages. The available evidence was classified according to molecular origin to distinguish validated prophage-derived molecules from those obtained from temperate or lytic phages and from molecules characterized in non-Vibrio bacteria. Endolysins and depolymerases can disrupt bacterial cells and biofilm matrices, whereas prophage regulatory elements may influence quorum sensing, adhesion, motility, toxin production, and secretion systems. However, the evidence directly supporting prophage-derived anti-Vibrio agents remains limited. Most experimentally demonstrated activity has been reported for endolysins and one validated depolymerase, and many of these molecules originated from lytic rather than temperate phages. No direct Vibrio-specific evidence is currently available for phage-derived nucleases, holins, spanins, or tailocins as isolated control agents. Prophage genomes nevertheless provide an extensive source of regulatory and antimicrobial candidates for further investigation. Progress toward aquaculture application will require experimental validation, effective delivery methods, safety assessment, scalable production, and clear regulatory standards.

Antibiotics doi: 10.3390/antibiotics15090873

Authors: Yangyang Chen Yuxiu Ye Qiurong Wu Guolian Xu Tingting Wu Meiling Tang Sulan Luo Yong Wu

Background: Dental caries is a biofilm-associated disease in which Streptococcus mutans plays a central role. This study evaluated HT-2-1-3, a Vespidae venom-derived antimicrobial peptide, as a potential topical anti-caries lead. Methods: Candidate peptides were synthesized by Fmoc solid-phase peptide synthesis and characterized by RP-HPLC and ESI-MS. Antibacterial activity against S. mutans UA159 was assessed using MIC, MBC, growth-curve, and time-kill assays. Membrane damage, antibiofilm activity, cytocompatibility, hemolysis, and preliminary oral safety in mice were further evaluated. Results: HT-2-1-3 showed the strongest activity among the tested peptides, with MIC and MBC values of 2 µg/mL. It inhibited bacterial growth in a concentration-dependent manner and achieved complete killing at 4×MIC (8 µg/mL) within 2 h. Mechanistic assays showed rapid membrane depolarization, increased PI uptake, and concentration-dependent release of extracellular DNA and proteins. MD simulations further indicated that HT-2-1-3 entered the model S. mutans UA159 membrane at 1857 ns, supporting membrane permeabilization as a central component of its bactericidal activity. Scanning electron microscopy revealed dose-dependent membrane disruption, cell collapse, and rupture. HT-2-1-3 inhibited biofilm formation by approximately 58–63% and eradicated up to 89.0% of mature biofilms. Hemolysis remained below 4% at concentrations up to 64×MIC (128 µg/mL), and mammalian-cell viability exceeded 95% at concentrations up to 16×MIC (32 µg/mL). Repeated oral administration caused no obvious gingival irritation in mice. Conclusions: HT-2-1-3 showed strong bactericidal and antibiofilm activity against S. mutans UA159 and favorable preliminary biocompatibility. Its mode of action may involve membrane disruption, and it showed favorable preliminary biosafety. These findings support further optimization of HT-2-1-3 as a topical antimicrobial candidate for caries prevention and control.

Antibiotics doi: 10.3390/antibiotics15090872

Authors: France Laurent Jean Cyr Yombi

Background. Osteoarticular infections (OAI)—including prosthetic joint infections (PJI), fracture-related infections (FRI), and osteomyelitis—are challenging to cure because bacterial biofilm on implant surfaces confers up to 1000-fold antibiotic tolerance, driving chronicity and relapse. Rifampicin, through its bactericidal activity against quiescent, biofilm-embedded bacteria and excellent oral bioavailability, occupies a pivotal role in managing Gram-positive OAI. Methods. This comprehensive narrative review synthesises mechanistic, preclinical, and clinical evidence on rifampicin across the three major osteoarticular infection types and by causative pathogen. PubMed/MEDLINE, Embase, Scopus, and the Cochrane Library were searched (January 1990–June 2026); of several hundred candidate records identified, the 101 most relevant were selected for narrative synthesis. Results. The strongest evidence supports rifampicin in staphylococcal PJI managed with debridement, antibiotics, and implant retention (DAIR), with fluoroquinolones as optimal companions; monotherapy invariably selects resistant mutants. This benefit is expected to extend beyond PJI to other osteoarticular infections managed with hardware retention—fracture-related infection and implant-associated osteomyelitis—given the shared biofilm target on retained metalwork. Benefit is attenuated in implant-exchange procedures, and evidence for streptococci, Cutibacterium acnes, and enterococci remains insufficient for firm recommendations. Conclusions. Rifampicin remains a valuable, difficult-to-replace agent in staphylococcal PJI treated with DAIR, provided it is introduced after surgical source control and combined with a fluoroquinolone. Observational data suggest possible benefit from introduction beyond 5 days post-debridement and courses of at least 14 days, but each threshold rests on a single study and requires confirmation. High-quality randomised trials targeting others OAI with implant retention, non-staphylococcal pathogens, optimal dosing, and treatment duration are needed.

Antibiotics doi: 10.3390/antibiotics15090871

Authors: Bindu Mayi Dhanya Dharmapalan Anita Shet

Antimicrobial stewardship (AMS) is often framed as a set of prescribing-focused interventions aimed at optimizing antibiotic selection, dose, route of administration, and duration. In practice, AMS also functions as a systems discipline. Embedded within governance and regulatory frameworks, diagnostic capacity, antimicrobial consumption and use patterns, and surveillance infrastructure, AMS operates within real-world structural and economic constraints, such as resource limitations, conflicting pharmaceutical market incentives, and health system fragmentation. In lower- and middle-income countries, variation in regulatory enforcement, prescribing patterns, diagnostic limitations, and surveillance infrastructure can undermine otherwise well-designed stewardship policies. This paper frames AMS as a systems governance function and proposes the Aligned AMS Systems Governance framework, comprising four core AMS functions—governance and regulation, antimicrobial consumption and antimicrobial use measurement, diagnostic stewardship, and antimicrobial resistance (AMR) surveillance and reporting—within four broader enabling domains: infection prevention and control, public engagement and awareness, environmental stewardship, and One Health coordination. This paper is an analytical narrative review of peer-reviewed literature, global policy frameworks, and implementation guidance. Using India as an illustrative example, we show that inconsistent regulatory enforcement, non-prescription antibiotic access, diagnostic constraints, and pharmaceutical market dynamics interact to complicate stewardship implementation despite the presence of national policies. Effective AMS requires deliberate alignment of these multiple functions and domains through defined responsibilities, routine data collection and sharing across AMS functions, and continuous feedback loops linking findings to coordinated action and subsequent reassessment. Reframing AMS as systems governance clarifies actionable leverage points for implementation and supports more practical, scalable approaches to mitigating AMR across diverse health system contexts.

Antibiotics doi: 10.3390/antibiotics15090870

Authors: Patiphan Khunti Panupon Mongkolkarvin Songphon Buddhasiri Joe Pogliano Poochit Nonejuie Parameth Thiennimitr Vorrapon Chaikeeratisak

Background: The emergence of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) poses a significant public health challenge, and alternative treatments are urgently needed. Methods: Here, we identified clinical MDR-UPEC strains AT82 and AT84 collected from hospitalized patients that display extensive antimicrobial resistance at both genetic and phenotypic levels. Due to their high resistance profile, we systematically customized a phage cocktail from our coliphage library using hierarchical clustering based on host specificity and candidate selection through bacterial suppression profiles. Results: This pipeline yielded four lytic coliphages, designated Phi25-4, Phi25-6, Phi50-4, and Killian. Their genomes are relatively large ranging from 112–169 kbp and cluster into two distinct lineages comprising two closely related groups: Phi25-4/Phi50-4 and Phi25-6/Killian. Although each phage exhibited potent antibacterial activity, none alone sustained bacterial suppression during prolonged treatment. To overcome this limitation, we systematically compared the antibacterial activity of all possible phage combinations. Conclusions: The four-phage cocktail outperformed all two- or three-phage formulations, sustaining significant growth inhibition of AT82 and AT84 for up to 16 h and reducing area under the curve by more than 80% relative to controls. Cocktail potency was dose-dependent, with lower phage doses yielding the least viable cells at 48 h. Additionally, this cocktail exerted prophylactic action, significantly reducing UPEC invasion by several orders of magnitude, while the phage cocktail alone induced minimal proinflammatory cytokine responses in human bladder epithelium. Together, these findings provide an effective phage cocktail and a complementary framework for cocktail design against urinary tract infections caused by MDR bacteria.

Antibiotics doi: 10.3390/antibiotics15090869

Authors: Marija Radovanović Stanislava Čukić Jelena Filipović Tričković Jadranka Miletić Vukajlović Biljana Nikolić Jelena Marinković

Objectives: The study determined antimicrobial resistance profiles of surgical wound multidrug-resistant (MDR) isolates belonging to the ESKAPE group and evaluated antibacterial and antibiofilm activities of geraniol (G), carvacrol (C) and eugenol (E), individually and in the selected mixtures. The cytotoxicity of monoterpenes and combinations was also assessed. Methods: The antibacterial and antibiofilm activity against tested isolates of Enterococcus faecium, Staphylococcus aureus MRSA, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp. was assessed in microdilution and crystal violet assay, respectively, while cytotoxicity was estimated in XTT assay on human MRC-5 fibroblasts. Results: C showed the strongest antibacterial activity (MIC 0.32 ± 0.24 mg mL−1). Monoterpenes induced synergism in certain combinations (FICI 0.09–0.31). All individual monoterpenes inhibited biofilm formation, but G was the most active (38.78–84.72%, p < 0.05). The most pronounced biofilm eradication was observed for C (25.21–61.34%, p < 0.05). G-C mixtures showed notable inhibition of biofilm formation in all isolates except P. aeruginosa, but, on the contrary, induced biofilm eradication against P. aeruginosa only. Cytotoxicity was not detected with the applied concentrations of monoterpenes, while the G-C mixtures exhibited lower cytotoxicity than povidone-iodine (p < 0.05) used as the control. Conclusions: All monoterpenes and G-C mixtures proved significant antibacterial and antibiofilm potential against ESKAPE isolates and acceptable impact on cell viability. Practical significance: The results highlight the potential of plant monoterpenes and their binary combinations as complementary agents for controlling MDR ESKAPE pathogens and biofilm-associated infections following surgical procedures. Further in vivo and safety studies are needed to confirm their applicability in clinical settings.

Antibiotics doi: 10.3390/antibiotics15090868

Authors: Pedja Kovacevic Vladimir Mrdja Tijana Kovacevic Jelena Mrdja Ivan Soldatovic Zvezdana Rajkovaca

Introduction: Antibiotic-associated diarrhea (AAD) is a common complication of antibiotic therapy, while evidence supporting Saccharomyces boulardii for its prevention among hospitalized adults in resource-constrained settings remains limited. We evaluated the efficacy of Bularen® in preventing AAD among hospitalized adults receiving systemic antibiotics. Methods: This single-center, randomized, double-blind, placebo-controlled trial was conducted at a tertiary care hospital in Bosnia and Herzegovina. Participants were randomly assigned to Bularen® (S. boulardii) 250 mg twice daily or a matching placebo for 28 days. The primary outcome was at least one episode of AAD, defined as two or more loose or liquid stools within 24 h, during the 28-day follow-up. Results: Of 435 patients screened, 100 were randomized to Bularen® (n = 51) or placebo (n = 49), and all completed follow-up. AAD occurred in 4/51 participants (7.8%) receiving Bularen® and 35/49 (71.4%) receiving placebo (risk ratio, 0.11; 95% confidence interval, 0.04–0.29; p < 0.001), corresponding to an absolute risk reduction of 63.6 percentage points. During intravenous antibiotic therapy, AAD occurred in 0% versus 26.5% of participants, respectively (p < 0.001), and during oral therapy in 7.8% versus 51.0% (risk ratio, 0.15; 95% confidence interval, 0.06–0.41; p < 0.001). No toxin-positive Clostridioides difficile infections were identified. Conclusions: Bularen® substantially reduced AAD incidence in this study population. Given the high placebo event rate and single-center design, confirmation in larger multicenter trials using standardized AAD definitions is warranted.

Antibiotics doi: 10.3390/antibiotics15090867

Authors: Wajeeorn Ouancharee Sareenee Saleah Rattanaruji Pomwised Arnon Chukamnerd Komwit Surachat Rosesathorn Soontarach Kamonnut Singkhamanan Sarunyou Chusri

Background/Objectives: Enterococcus species are important causes of healthcare-associated infections, particularly multidrug-resistant (MDR) and vancomycin-resistant enterococci (VRE). This study characterized antimicrobial susceptibility, biofilm formation, genomic features, and molecular epidemiology of clinical Enterococcus isolates from southern Thailand. Methods: Forty clinical isolates, comprising E. faecalis (n = 22) and E. faecium (n = 18), were characterized by antimicrobial susceptibility testing, biofilm assays, and whole-genome sequencing. Results: All isolates were susceptible to linezolid and oritavancin, whereas non-susceptibility to ampicillin, fosfomycin, vancomycin, and daptomycin was 60.0%, 57.5%, 12.5%, and 5.0%, respectively. High-level gentamicin resistance and MDR occurred in 37.5% and 22.5%, respectively. Strong biofilm formation predominated in E. faecalis (86.4%), whereas 94.4% of E. faecium were weak producers. ST6 and ST17 predominated in E. faecalis and E. faecium, respectively. All five VRE isolates were E. faecium ST17 or ST262 and carried vanA. High-level ampicillin-resistant E. faecium harbored the PBP5 S466′ insertion and multiple C-terminal substitutions. To our knowledge, this is the first report in Thailand of both daptomycin-resistant E. faecium and resistance-associated LiaS T120A and LiaR W73C substitutions. Closely related isolates within E. faecalis ST6 and E. faecium VRE ST17, ST117, and ST262 showed spatiotemporal patterns consistent with potential epidemiological linkage or lineage persistence. Three novel STs were identified, with E. faecium ST3096 exhibiting high-level ampicillin resistance, fosfomycin resistance, and reduced daptomycin susceptibility. Conclusions: These findings reveal emerging resistant lineages and spatiotemporal patterns of genomic relatedness, supporting continued genomic surveillance of clinical enterococci in Thailand.

Antibiotics doi: 10.3390/antibiotics15090866

Authors: Viorel Dragos Radu Ana-Maria Raluca Pauna Rodica Radu Liliana Mititelu-Tartau Alin Mihai Vasilescu Theodor Florin Pantilimonescu Adriana Grigoraș Laura Florea Marius Constantin Moraru Liliana Chelaru Liviu Ciprian Gavril Roxana Florentina Gavril Beatrice Rozalina Buca Angy Abu Koush Irina Luanda Mititiuc

Background/Objectives: Colistin is a last-resort agent against multidrug-resistant Gram-negative infections and is administered predominantly to complex, comorbid inpatients in whom mortality is high. We aimed to identify independent baseline predictors of in-hospital mortality in colistin-treated patients and to characterize temporal changes in mortality over 15 years. Methods: We conducted a retrospective cohort study of 1225 consecutive patients receiving systemic colistin between November 2009 and November 2024 at Parhon Hospital, a tertiary urology–nephrology center in Iași, Romania; independent predictors of death were identified using multivariable logistic regression restricted to variables fixed at the time of colistin initiation, reported as adjusted odds ratios (aOR) with 95% confidence intervals (CI). Results: Patients were predominantly male (60.2%), with a mean age of 65.1 ± 14.4 years; 85.6% were admitted to urology or nephrology. In-hospital mortality was 27.6%. Mortality was independently associated with acute kidney injury (aOR 3.72, 95% CI 2.76–5.01), sepsis (aOR 2.79, 95% CI 2.11–3.70), emergency admission (aOR 1.91, 95% CI 1.40–2.61), Charlson comorbidity index (aOR 1.13 per point, 95% CI 1.05–1.22), and older age (aOR 1.03 per year, 95% CI 1.02–1.04); sex was not associated. Crude mortality increased across the study period (odds ratio 1.16 per year, 95% CI 1.13–1.20) and remained elevated after adjustment for case-mix (adjusted odds ratio 1.13 per year, 95% CI 1.09–1.17). Conclusions: Acute kidney injury, sepsis, emergency admission, comorbidity burden, and age were independently associated with in-hospital death. Mortality rose progressively over 15 years, only partly explained by an increasingly severe case-mix, supporting early risk stratification of colistin recipients.

Antibiotics doi: 10.3390/antibiotics15090865

Authors: Noelia García-Criado Juan Luis Santos Julia Martín Irene Aparicio Esteban Alonso

Background/Objectives: The application of organic soil amendments constitutes an important source of antibiotic residues in agricultural soils. However, studies have mainly focused on a few parent tetracyclines, whereas information on their transformation products (TPs) in organic amendments remains scarce. Therefore, this study assessed the occurrence and environmental risk of six tetracyclines and seven TPs in organic amendments. Methods: Processed livestock manures applied in the European Union (horse, poultry, and bovine manure), as well as fresh and treated sewage sludge, were analyzed using matrix solid-phase dispersion (MSPD) combined with online SPE-LC-MS/MS. Environmental risk was assessed using risk quotients (RQs) based on predicted environmental concentrations in soil (PECsoil) calculated from the maximum measured concentrations and predicted no-effect concentrations in soil (PNECsoil) obtained either directly from terrestrial ecotoxicity data or derived from aquatic ecotoxicity data using the equilibrium partitioning method and soil-water distribution coefficients. Results: Tetracyclines and their TPs were widely detected in both sample types, although concentrations varied according to matrix type and treatment. Overall, sludge showed higher detection frequencies and concentrations than manure. Doxycycline and tetracycline were the predominant parent compounds, reaching concentrations up to 2838 ng g−1 dry weight (dw) in manure and 2892 ng g−1 dw in sludge. Epimerized TPs were frequently detected and sometimes exceeded the concentrations of their parent compounds, especially epitetracycline and epioxytetracycline. Among the sludge types and treatment conditions investigated, anaerobically digested sludge showed the highest tetracycline concentrations, whereas the composted sludge sample presented the lowest concentrations. Individual RQs indicated insignificant to low ecotoxicological risk, whereas cumulative RQ (ΣRQ) values, used as conservative estimates of co-exposure to all evaluated tetracyclines and their TPs, fell within the medium-risk category for bovine manure and anaerobically digested sludge, with the composted sludge sample showing the lowest ΣRQ. Conclusions: These findings highlight the importance of including TPs in environmental monitoring and the differences in tetracycline occurrence and environmental risk to soil among the processed manure types and sludge treatment conditions investigated.

Antibiotics doi: 10.3390/antibiotics15090863

Authors: Khadijah M. Dashti Leila Vali Nawal Mohammed Ali A. Dashti

Background/Objectives: Bacillus spp. are often isolated from airborne microbiota. Five Bacillus strains (AD12–16) isolated from dust during a sandstorm in Kuwait were characterized for their potential to produce antimicrobial compounds, biosurfactants and siderophores by genotypic and phenotypic approaches. Methods: Whole-genome sequencing was performed, and biosynthetic gene clusters (BGCs) were predicted using antiSMASH. Antimicrobial activities were determined by agar-well diffusion assays. Siderophore and biosurfactant production were determined by Arnow’s and drop-collapse assays, respectively. The active metabolites were separated, fractionated, and analyzed by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and LC–MS/MS. Results: Genome mining identified many BGCs associated with known secondary metabolites such as bacillibactin and bacilysin, as well as a number of unassigned clusters. The isolates exhibited distinct antimicrobial profiles, with Bacillus subtilis AD16 displaying the highest antimicrobial activity against multidrug-resistant pathogens. Biosurfactant- and siderophore-associated activities varied among the isolates. Active fractions demonstrated bactericidal activity, while LC–MS/MS analysis revealed molecular features tentatively assigned to siderophore- and lipopeptide-associated metabolite classes based on accurate mass and MS/MS fragmentation patterns. Conclusions: The combination of unassigned BGCs, antimicrobial activity, and incompletely characterized molecular features highlights unexplored biosynthetic potential in sandstorm-derived Bacillus. Further purification and structural characterization are required to establish the identities and biological functions of the active metabolites.

Antibiotics doi: 10.3390/antibiotics15090864

Authors: Ruei-Sen Jiang Li-Kuang Chen Chun-Chieh Tseng

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major nosocomial pathogen; bacteriophage therapy is a promising alternative, but its principal challenge is the rapid emergence of phage resistance. Cocktails counter this yet they are usually assembled by simply pooling phages active against the bacterium, not designed against the resistant mutants that arise. Methods: Next evolutionary phage typing (NEPT) addresses this by inducing resistance to a primary phage and then selecting secondary phages that lyse the resulting resistant mutant. Because choosing effective secondary phages still relies on qualitative visual reading of plaques (size and clarity), this study aimed to identify a rapid quantitative criterion. Results: Using the clinical strain CRAB 43895 and primary phage ϕ8, we obtained 65 NEPT-derived secondary phages that lyse the ϕ8-resistant mutant (ϕ8R) and combined four quantitative indicators—relative bacterial growth, lytic capability, coverage rate, and plaque morphology—with binary logistic regression to predict effective inhibition by the ϕ8 + secondary-phage cocktail (96-h OD600 < 0.1). Of the four, only lytic capability (the titer after 3 h of co-culture) independently predicted effective inhibition (ROC AUC = 0.76; at ≥107 plaque-forming units (PFU)/mL, sensitivity 0.83, specificity 0.69). Conclusions: Lytic capability thus provides a rapid (~8–12 h), quantitative criterion for selecting effective secondary phages within the NEPT framework, improving on conventional qualitative typing.

Antibiotics doi: 10.3390/antibiotics15090862

Authors: Sergio Sánchez Bruni Laura Galotta María Guadalupe de Yaniz Laureano Schofs Mónica Sparo Lucila Cantón Laura Moreno Peers Davies

Background/Objectives: This prospective cross-sectional study quantified veterinary antimicrobial residues in soils from 27 commercial beef feedlots across multiple regions of Argentina and production scales. Methods: Using a validated Ultra-Fast Liquid Chromatography–tandem mass spectrometry (UFLC-MS/MS) method, we measured 15 target antimicrobials in pooled corral soil samples. Results: Monensin was detected in all feedlots and showed the highest detection frequency and widest concentration range; peak concentrations exceeded 15,000 ppb in some sites. Oxytetracycline presented pronounced, site-specific peaks (approaching 14,000 ppb), consistent with its strong adsorption to soil organic matter and limited biodegradation. These results indicate localised hotspots of antimicrobial loading in feedlot environments. These elevated concentrations signify substantial antimicrobial loading in confined animal environments and potential hotspots for environmental selection pressure. Compounds absent from farm treatment records were detected in 40.7% of feedlots, correlating with antimicrobial residues not matching available AMU records, including oxytetracycline, tulathromycin, cefquinome, enrofloxacin, and ciprofloxacin among others. Multiple non-exclusive explanations are plausible (historical persistence, manure redistribution, incomplete records, off-label use, or cross-contamination). The detection of critically important antimicrobials (e.g., fluoroquinolones and advanced cephalosporins) in soils raises One Health concerns about environmental selection pressure and the potential dissemination of resistance determinants. Conclusions: These findings underscore gaps in stewardship and the need for integrated environmental monitoring and improved antimicrobial usage traceability. These data provide a baseline for ecological risk assessment and resistome studies and can inform evidence-based policy and stewardship interventions in Argentine feedlot systems.

Antibiotics doi: 10.3390/antibiotics15090861

Authors: Rui Malheiro André Amaral Gomes Cristina Nunes Paulo Sousa Paulo Borem José-Artur Paiva Ana Lebre

Objectives: The collaborative “Stop Hospital Infection 2.0”, implemented in Portugal between 2023 and 2025, aimed to reduce the incidence of catheter-associated urinary tract infection (CAUTI), ventilator-associated pneumonia (VAP) and central-line associated bloodstream infection (CLABSI) by 50.0% in 21 hospitals. Methods: The project followed the Breakthrough Series methodology. Each team was to audit the national preventive bundles, using a Kamishibai card, and drive changes using plan–do–study–act cycles. Before the project, teams reported their 6-month baseline incidence levels. The main outcome was the incidence of infection. The secondary outcome was the proportion of teams that achieved a ≥50.0% reduction in incidence. The number of infections, deaths and bed-days potentially prevented, and costs saved during the project were estimated, along with the potential gains in the future. Results: Incidence rate for teams reporting at least 36 months decreased 47.6% in CAUTI (4.8 to 2.5 infections/1000 device-days), 66.1% in CLABSI (1.5 to 0.5 infections/1000 device-days) and 50.0% in VAP (9.9 to 5.0 infections/1000 intubation-days). The proportion of teams achieving a reduction of at least 50% in infection incidence was 60.0%, 100% and 57.1%, respectively. Teams reporting ≥24 months may have prevented 465 HAI, 108 deaths, 4526 bed-days and saved 8,375,841 euros. In the future, the teams who reported for at least 36 months may avoid 150 cases, 36 deaths, 1538 bed-days and 2,693,546 euros, yearly. Conclusions: A national collaborative focused on evidence-based bundles may be effective in decreasing the incidence of CAUTI, CLABSI and VAP in adult intensive care units.

Antibiotics doi: 10.3390/antibiotics15090860

Authors: Hongyu Wang Yasuhiko Irie

Background/Objectives: Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium and a major cause of healthcare-associated infections worldwide, exhibiting intrinsic antimicrobial resistance and readily acquiring novel resistance mechanisms. Divalent cations such as Mg2+ and Ca2+ in the culture environment can influence bacterial antibiotic susceptibility. This study evaluated the impact of varying concentrations of Mg2+ and Ca2+ on the antibiotic susceptibility of P. aeruginosa to tobramycin and ciprofloxacin under both planktonic and biofilm growth modes. Methods: The study determined the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) to compare alterations in antibiotic susceptibility across different ion conditions and bacterial growth patterns. Results: Our findings suggest that elevated concentrations of Mg2+ and Ca2+ significantly modulate bacterial antibiotic susceptibility. Conclusions: The results of our study has implications for limitations in standardised antibiotic susceptibility tests and to therapeutic strategies in cation-rich environments such as cystic fibrosis airways.

Antibiotics doi: 10.3390/antibiotics15090859

Authors: Jack H. Lambert Rayven Todd Thomas W. Bagwell Damani Andre Raybun Spelts Fantasia Gorham Jamie Woods Ayomide H. Adeyemi Shondia Evans Rafael Ponce-Terashima Kenneth I. Onyedibe

Background/Objectives: Antimicrobial resistance threatens public health globally, and critical access hospitals (CAHs) serving rural communities face structural barriers to implementing antibiotic stewardship programs (ASPs). This study evaluated whether a regional educational intervention could improve antimicrobial stewardship knowledge and implementation among rural healthcare professionals. Methods: A quantitative, quasi-experimental repeated cross-sectional evaluation was conducted following Antibiotic Stewardship conferences in 2023 and 2024. Participants from 38 rural and critical access hospitals completed pre-conference, post-conference, 6-month follow-up, and implementation surveys. Quantitative data were analyzed using descriptive statistics and unpaired chi-square tests, with Fisher’s exact test substituted where expected cell counts were below 5, with significance set at p < 0.05. Results: Seventy healthcare professionals participated across both years. Correct identification of the use of an antibiogram to guide empiric (rather than definitive) therapy improved from 27% pre-conference to 58% post-conference (p = 0.029). Confidence in antibiogram interpretation increased from 67% to 100% post-intervention (p = 0.004, Fisher’s exact) but declined to 50% (5 of 10) at 6-month follow-up. Knowledge of the minimum isolate threshold required to construct an antibiogram improved from 23% (7 of 30) pre-conference to 64% (7 of 11) at 6-month follow-up (p = 0.026, Fisher’s exact). Despite this, prescribing intent for conditions where antibiotics are typically unnecessary showed minimal improvement: recommendations for antibiotic use in middle ear infections decreased from 50% to 36.84%, and for mpox from 20% to 15.79%. All participants reported intent to modify clinical practice; however, time constraints, technology limitations, and lack of resources were consistently cited as primary barriers for implementation across both survey years. Conclusions: A regional educational intervention improved short-term antimicrobial stewardship knowledge and confidence among rural healthcare professionals, but knowledge retention declined over time and structural barriers persisted. Education alone is insufficient; sustained reinforcement and rural-adapted ASP models incorporating protected ASP time, decision-support tools and external partnerships are needed to translate knowledge gains into consistent stewardship practice.

Antibiotics doi: 10.3390/antibiotics15090858

Authors: Miriam Tenuzzo Federico Scali Claudia Romeo Flavia Guarneri Lucia Cioli Silvia Filippi Chiara Boifava Chiara Galizia Laura Birbes Giovanni Parisio Valentina Carta Marta Masserdotti Antonio Marco Maisano Giovanni Loris Alborali Benedetta Cappelletti Lisa Guardone Nicoletta Formenti

Background/Objectives: Antimicrobial resistance (AMR) can be influenced by the interconnection of human, animal and environmental compartments, making wildlife-based surveillance an important component of One Health surveillance. The red fox (Vulpes vulpes), due to its synanthropic behaviour and wide distribution, can be a valuable indicator of environmental AMR. Mobile colistin resistance (mcr) genes, which encode resistance to a highest-priority critically important antimicrobial (HPCIA), have never been identified in free-ranging foxes. Case presentation: A red fox faecal strain isolated using selective media was PCR-screened for major AMR genes, underwent antimicrobial susceptibility testing and whole-genome sequencing (WGS). This led to the identification of the first mcr-1-positive Escherichia coli from a wild red fox. The fox inhabited an area with a small human population but one of the highest cattle densities in Italy and high antimicrobial use (AMU), as established by comparing local livestock density and AMU with surrounding areas. Despite being borderline phenotypically susceptible to colistin, the isolate displayed resistance to other HPCIAs and antimicrobial classes commonly used in livestock. WGS revealed that mcr-1 was chromosomally integrated. Discussion: The study area showed near-zero colistin use in livestock alongside high overall AMU, suggesting that the mcr-1-positive isolate may have evolved through co-selection rather than direct colistin pressure. mcr-1 can persist and circulate in the environment, even where colistin use is minimal, and foxes may represent effective sentinels for tracking such events. Our results emphasise the importance of including wildlife in One Health surveillance frameworks to monitor AMR at the human–animal–environment interface.

Antibiotics doi: 10.3390/antibiotics15090857

Authors: Łukasz Grabarczyk Julia Oszytko Maria Derkaczew Kamil Sobolewski Paweł Radkowski

Background/Objectives: Antibiotics are widely used in clinical practice, yet their potential effects on endocrine homeostasis remain insufficiently recognized. This review summarizes endocrine adverse effects associated with selected antibacterial agents, their proposed mechanisms, and clinical relevance. Methods: A narrative literature search was conducted in PubMed/MEDLINE, Scopus, and Web of Science from database inception to 10 August 2026. Clinical, pharmacovigilance, case-based, animal, in vitro, and ex vivo studies were considered. Results: Reported disturbances include SIADH-related hyponatremia, hypoglycemia, altered thyroid hormone metabolism, changes in hypothalamic–pituitary–adrenal axis activity, aldosterone dysregulation, and reproductive hormone disturbances. Selected cephalosporins and carbapenems have been associated with changes in prolactin secretion, thyroid-related parameters, and gonadal function. Fluoroquinolones have been associated with dysglycemia, while effects on reproductive hormone pathways have been reported predominantly in experimental models. Selected macrolides may influence glucocorticoid metabolism and KCNJ5-dependent aldosterone production. Linezolid has been linked to hyponatremia and hypoglycemia, while metronidazole may affect pituitary hormone secretion. Trimethoprim–sulfamethoxazole may contribute to hyponatremia, hyperkalemia, hypoglycemia, and thyroid dysfunction. The supporting evidence is heterogeneous and ranges from case reports and pharmacovigilance analyses to experimental studies. Human clinical evidence is strongest for selected disturbances of glucose and electrolyte homeostasis, whereas many reported reproductive, pituitary, thyroid, and adrenal effects remain supported predominantly by experimental or case-based evidence. Conclusions: Many proposed mechanisms remain incompletely established and cannot be directly extrapolated to routine practice. Awareness of these complications may support earlier recognition, targeted biochemical monitoring, and safer antibiotic therapy, particularly in older, critically ill, or renally impaired patients and those exposed to polypharmacy.

Antibiotics doi: 10.3390/antibiotics15090856

Authors: Francisco Arnalich-Montiel David J. Galarreta María Nieves Díez García

Background/Objectives: Infectious keratitis and eye injuries, despite being well-recognized ophthalmological diseases, continue to pose diagnostic and therapeutic challenges in clinical practice; therefore, this study was conducted to assess the salient features of care provided by Spanish ophthalmologists working in emergency and ocular surface consultations to patients presenting with keratitis and ocular trauma. Methods: Nationwide cross-sectional survey with the participation of 100 ophthalmologists, including 50 ophthalmologists in emergency care and 50 ocular surface specialists. The study questionnaire assessed diagnostic approaches, treatment strategies, and factors influencing therapeutic decision making. Results: The mean age of participants (51% women) was 37.9 years, with 10.2 years of professional activity. External eye examination, slit lamp examination, fluorescein staining, computed tomography (CT) in penetrating injuries, and microbiological cultures in corneal ulcers were rated as important diagnostic methods. Topical antibiotics were especially recommended for keratitis in general (mean 80.6%) and for contact lens-related keratitis (85.6%). The use of antiseptics was limited. Topical corticosteroids were most commonly selected for viral keratitis with stromal and endothelial involvement (between 24.3% and 50.4%); chlorhexidine (52.0%) and propamidine (26.8%) for Acanthamoeba keratitis; re-epithelizing compounds (20.4%) and artificial tears (29.1%) for actinic keratitis; and topical antifungals for fungal keratitis (77%). Conclusions: This study highlights a limited availability of some specific diagnostic methods and an extensive use of topical antibiotics in the management of keratitis and ocular surface injuries, with relatively limited use of antiseptics. Areas of improvement in therapeutic approach would be awareness of a potential overuse of topical antibiotics with the consequences of increase in antimicrobial resistance.

Antibiotics doi: 10.3390/antibiotics15090855

Authors: Ayman El-Menyar Ibrahim Al-Hassani Mashhood Naduvilekandy Rafael Consunji Fernando Netto Airton Leonardo de Oliveira Manoel Prabath Nanayakkara Sagar Galwankar Łukasz Szarpak Basar Cander Eman Elmenyar Sandro Rizoli Hassan Al-Thani

Background: Sepsis is a time-sensitive, serious condition; therefore, early recognition and prompt initiation of therapy are critical to improving outcomes in patients with infection. We sought to evaluate whether prehospital antibiotic administration (PHAA) is associated with improved outcomes in patients with suspected severe sepsis. Methods: We conducted a systematic review and meta-analysis. This review was conducted in accordance with the PRISMA guidelines, and the protocol was developed a priori and registered with the International Prospective Register of Systematic Reviews (PROSPERO). PubMed, Embase, Cochrane, and Scopus were searched from inception through January 2026. Randomized controlled trials (RCTs) and observational studies evaluating empirical PHAA compared with the standard of care were included. The risk of bias and certainty of evidence were assessed. Outcomes included short- and long-term mortality, hospital length of stay (HLOS), and intensive care unit (ICU) admission. Sensitivity analyses were performed. Results: Eleven studies recruiting 23,146 patients were included (three RCTs and eight observational studies). Restricting the short-term mortality analysis to RCTs alone showed no mortality benefit with PHAA (RR 0.96, 95% CI 0.74–1.23), whereas the broader, observational-dominated pooled analysis suggested a possible reduction in short-term mortality (RR 0.84, 95% CI 0.68–1.02, I2 = 51.0%), reaching statistical significance when restricted to the adult sepsis/septic shock population (RR 0.82, 95% CI 0.69–0.99, I2 = 18.5%). No significant difference in short-term mortality was observed in the exploratory subgroup analysis of two high-severity studies (RR 0.89, 95% CI 0.67–1.18). There were no significant long-term mortality differences in the observational and RCT studies (RR 0.86, 95% CI 0.71–1.04 and RR 1.09, 95% CI 0.76–1.54, respectively). Patients who received PHAA had no significantly different ICU admissions (RR 1.12, 95% CI 0.95–1.33) or HLOS (MD −1.79 days, 95% CI −6.05 to 2.47). Even though individual studies showed no significant difference, pooled HLOS was significantly higher with PHAA for the RCT subgroup (MD 0.66, 95% CI 0.42–0.90, I2 = 0.00%) after conversion of median to mean values (for better statistical compatibility). Conclusions: PHAA was not associated with a significant reduction in short-term mortality, though a significant reduction was observed when restricted to adult sepsis/septic shock population. No mortality benefit was found in pooled RCTs, mixed-age population studies, or long-term mortality. Observational studies suggest a possible mortality benefit, whereas randomized evidence does not confirm it. This discrepancy may reflect confounding by indication, patient selection, severity, and differences in EMS systems rather than a true treatment effect. The certainty of evidence remains very low, and data on prehospital antibiotic type, blood culture, and infection severity are lacking. Therefore, high-quality prospective RCTs are warranted.

Antibiotics doi: 10.3390/antibiotics15090854

Authors: Liat Wulffhart Elizabeth Temkin Yehuda Carmeli

Background/Objectives: Screening for carriage of antibiotic-resistant bacteria (ARB) upon hospital admission and isolating carriers can prevent nosocomial spread of ARB. Screening typically targets patients at high risk of ARB carriage because of recent hospitalization or travel. For ARB with community spread among non-high-risk patients, screening only high-risk patients may be insufficient to control nosocomial transmission. We used an agent-based model to examine the impact of screening patients without recognized risk factors for ARB carriage on the acquisition and prevalence of ARB carriage in hospitals. Methods: The agents in our model of an 800-bed hospital were patients and healthcare workers (HCW). Contacts between HCW and patients or between patients may result in ARB transmission. We modelled scenarios of 1%, 5% or 20% ARB carriage prevalence upon admission among high-risk patients and ARB carriage prevalence ratios between high-risk and non-high-risk patients of 1.11, 2, 5 and 10. At baseline, screening test sensitivity was 85%, compliance with screening of high-risk patients was 80%, and isolation effectiveness was 70%. Results: Expanding screening to non-high-risk patients decreased the incidence of nosocomial ARB acquisitions. The impact of this screening on nosocomial acquisitions increased as greater community spread occurred. Carriage prevalence in the hospital was mainly influenced by imported cases; nosocomial transmission accounted for a minority of prevalent cases. Improving isolation effectiveness to 95% had a greater impact on ARB acquisitions than expanded screening when carriage prevalence among non-high-risk patients was low. Conclusions: The impact of expanded screening was small when carriage prevalence among high-risk patients was low or when the carriage prevalence ratio was high. In the scenarios modelled here, improving isolation effectiveness prevented more acquisitions than expanded screening.

Antibiotics doi: 10.3390/antibiotics15090853

Authors: Agata Paneth Izabela Korona-Głowniak Agnieszka Głogowska Jacek Szczepański Dominik Włodarczyk Katarzyna Suśniak Ewa Augustynowicz-Kopeć Nazar Trotsko

Background/Objectives: The rapid emergence of antimicrobial resistance has created an urgent need for new chemotypes with activity against bacterial, fungal, and mycobacterial pathogens. Rhodanine-based compounds are recognized as privileged scaffolds in medicinal chemistry because of their broad spectrum of biological activities, making them attractive candidates for the development of novel antimicrobial agents. Methods: A library of thirty-two novel rhodanine-based hydrazide derivatives (21–52) was synthesized by condensation of 5-ethoxymethylidenerhodanine derivatives with aromatic acid hydrazides and characterized by IR, 1H NMR, and 13C NMR spectroscopy. Their drug-likeness and pharmacokinetics properties were evaluated using SwissADME, while antimicrobial activity was assessed against Gram-positive and Gram-negative bacteria, Candida spp., and both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains using minimum inhibitory concentration (MIC) assays. Results: The synthesized compounds demonstrated selective activity against Gram-positive bacteria, whereas only limited activity was observed against Gram-negative species. The most potent derivatives (36, 40, 46, and 50) inhibited Micrococcus luteus with MIC values as low as 31.3 mg/L. Several compounds also demonstrated noteworthy antifungal activity, particularly against Candida parapsilosis (MIC = 15.6–31.3 µg/mL). Pyridyl-containing analogues displayed moderate antitubercular activity and, importantly, compounds 49 and 52 retaining activity against both drug-susceptible and drug-resistant M. tuberculosis strains. In silico analysis indicated favorable drug-like properties for most compounds, with good compliance with Lipinski’s rule of five and predicted oral bioavailability. Preliminary structure–activity relationship analysis revealed that a 3-phenylrhodanine core combined with para-halogen or para-nitro substituents significantly enhanced antimicrobial potency. Conclusions: The present study identifies rhodanine-based hydrazide derivatives as promising multifunctional antimicrobial scaffolds with activity against Gram-positive bacteria, pathogenic yeasts, and M. tuberculosis. The identified structure–activity relationships provide a rational basis for further lead optimization and support continued development of this chemotype as a potential source of new antimicrobial agents.

Antibiotics doi: 10.3390/antibiotics15090852

Authors: Rosa Kalliainen Marjo Renko Ulla Sankilampi Merja Kokki Veli-Pekka Ranta

Objectives: This retrospective study aimed to estimate vancomycin area under the curve over 24 h (AUC24) at steady state for pediatric patients from 1 month to 18 years to update the dose recommendations in a tertiary hospital. Methods: A total of 46 vancomycin treatments from 42 patients in the years 2014–2024 were analyzed. AUC24 was estimated for the initial dose regimen using Bayesian forecasting and a trough concentration (n = 32) or first-order pharmacokinetic (PK) equations and a pair of practical peak and trough concentrations at steady state (n = 14). Three published population PK models (Colin, Kloprogge, and Le models) were compared using typical parameter values (a priori predictions) to find a suitable model for Bayesian estimation. For the Le model, the effect of converting plasma creatinine (PCr) measured with IDMS-traceable enzymatic assay to Jaffe method value was evaluated using published equations. Results: Le model without enzymatic-to-Jaffe PCr conversion and the Colin model were suitable for Bayesian forecasting. These models gave similar Bayesian AUC24 estimates (a posteriori). In all 46 treatments, estimated AUC24 was within the currently recommended range of 400–600 mg·h/L in 8 (17%) treatments, below this range in 36 (78%) treatments, and above this range in 2 (4%) treatments, respectively. Estimated AUC24 was below 300 mg·h/L in 46% of treatments. Conclusions: Le and Colin models were suitable for our pediatric patients for Bayesian forecasting. AUC24 estimates for the initial dose regimen were lower than commonly recommended. A more uniform dosing policy is needed in our hospital, and higher initial doses should be considered.

Antibiotics doi: 10.3390/antibiotics15090851

Authors: Ziya İlhan Reyda Şıklaroğlu Murad Gürses Murat Bayezit Taha Gürsoy Yavuz Musabeşeoğlu Özgür Erkan Şefika Musabeşeoğlu Mehmet Karaca Hasan Altan Akkan

Background/Objectives: The increasing prevalence of antimicrobial resistance has reduced the effectiveness of conventional antibiotics, highlighting the need for alternative or adjunct antimicrobial agents. This study aimed to investigate the antibacterial, synergistic, and antibiofilm activities of Liquidambar orientalis Mill. essential oil against clinically relevant bacterial pathogens, using 24 bacterial strains representing 12 bacterial species. Methods: The volatile constituents of L. orientalis essential oil obtained by hydrodistillation of storax balsam were characterized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). Antibacterial activity was evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), disk diffusion, and agar well diffusion assays against a panel of Gram-positive and Gram-negative bacterial pathogens. Synergistic interactions with erythromycin, amoxicillin, gentamicin, and enrofloxacin were assessed using fractional inhibitory concentration index (FICI) analysis. Antibiofilm activity was also evaluated against representative biofilm-forming strains. Results: A total of 90 volatile constituents were identified by GC-MS, of which styrene was the most abundant (68.6%), followed by (E)-ethyl cinnamate (6.8%), α-pinene (5.9%), and β-pinene (2.9%). The essential oil exhibited broad-spectrum antibacterial activity, with an overall inhibition rate of 60.4%. Activity was higher against Gram-positive (77.77%) than Gram-negative (73.33%) bacteria. MIC and MBC values ranged from 15.62 to 250 µg/mL and 31.25 to 250 µg/mL, respectively. FICI analysis revealed strain- and antibiotic-dependent interactions, ranging from synergistic to antagonistic effects. Variable antibiofilm activity was observed, with the strongest inhibition against Brucella melitensis and the weakest against Pseudomonas aeruginosa. No significant differences in MIC or MBC values were observed between bacterial groups (p > 0.05). Conclusions: L. orientalis essential oil demonstrated in vitro antibacterial activity against several clinically relevant pathogens, with particularly notable activity against B. melitensis. The essential oil also showed synergistic interactions with selected antibiotics and variable antibiofilm effects. These findings suggest that L. orientalis essential oil may have potential as an antibacterial agent or adjunct to antibiotic therapy; however, further in vivo and clinical studies are required to confirm its therapeutic applicability.

Antibiotics doi: 10.3390/antibiotics15090850

Authors: Federica Di Timoteo Emanuela Di Giulio Marco Di Domenico Barbara Secondini Francesca Marotta Giusy Matteucci Katiuscia Zilli Teresa Romualdi Dalia Palmieri Giuliano Garofolo Anna Janowicz

Background/Objectives: Wastewater-based epidemiology (WBE) has emerged as a valuable One Health approach for monitoring antimicrobial resistance (AMR) at the population level. Although quantitative PCR (qPCR) and shotgun (SG) metagenomics are widely used for wastewater surveillance, studies integrating these complementary approaches remain limited. This study aimed to investigate the occurrence, seasonal dynamics, and diversity of antimicrobial resistance genes (ARGs) in municipal wastewater from Central Italy by combining targeted qPCR and SG metagenomic sequencing. Methods: Influent wastewater samples were collected monthly from eight municipal wastewater treatment plants in Central Italy between April 2025 and March 2026. Clinically relevant antimicrobial resistance genes were quantified by quantitative real-time PCR, while SG metagenomic sequencing was used to characterize resistome composition, resistance gene families, and ARG sequence diversity using bioinformatic pipelines. Results: All investigated ARGs were detected in every sample. Significant seasonal variation was observed for all investigated markers, including qnrS, blaKPC, blaCTX-M and intI1. Metagenomic analysis revealed broadly similar resistome profiles across sampling sites and time points, dominated by resistance genes to macrolide–lincosamide–streptogramin, aminoglycosides, β-lactams, and fluoroquinolones. High sequence diversity was observed within the dominant ARG families, highlighting the complementary value of SG metagenomics for comprehensive resistome characterization. Conclusions: The integration of targeted qPCR and SG metagenomics provided a comprehensive characterization of antimicrobial resistance in municipal wastewater. While qPCR enabled sensitive quantification of clinically relevant ARGs and revealed seasonal trends, metagenomics expanded resistome characterization by identifying dominant resistance classes, gene families, and sequence variants. These findings support the implementation of integrated molecular approaches for routine wastewater-based AMR surveillance within a One Health framework.

Antibiotics doi: 10.3390/antibiotics15090849

Authors: Takashi Azuma

Antimicrobial resistance (AMR) has emerged as one of the most pressing global public health challenges of the twenty-first century [...]

Antibiotics doi: 10.3390/antibiotics15090848

Authors: Natalie J. Moss Stephanie A. Marsh Colin H. Cortie Mary A. Burns Andrew Bonney Judy Mullan

Background/Objectives: C-reactive protein point-of-care tests (CRP POCTs) have become a useful tool in reducing inappropriate prescribing of antibiotics in the management of respiratory tract infections (RTIs). This review examined the barriers and enablers of CRP POCT implementation in primary care using the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework. Methods: A search was conducted in six electronic databases (PubMed, Scopus, CINAHL (EBSCOhost), Medline (EBSCOhost), ProQuest and CENTRAL) for studies up to December 2025 on CRP POCT implementation for RTIs in primary care. Eligible studies included quantitative randomised controlled trials (RCTs), qualitative studies, cost-effectiveness studies, mixed-methods and observational studies. Only English-language studies were included. Results: The review included 35 studies. CRP POCT was generally found to be an effective and acceptable tool due to reductions in antibiotic prescribing, playing a role in supporting clinical decision-making and patient communication. However, widespread adoption and usage were inconsistent across studies. Further, qualitative findings raised operational and financial constraints as key barriers to sustained implementation. Reporting of RE-AIM domains varied across study type. Notable gaps were identified in reporting of the maintenance domain along with population representativeness, non-participation, and device uptake. Of the 35 studies, 13 included ‘operational’ and/or ‘financial constraints’ as identified themes or concerns about wider implementation. Conclusions: Although CRP POCT has routinely been found to be effective, a wider comprehensive assessment of implementation is restricted due to gaps in reporting. Key barriers to sustained and scalable global implementation of CRP POCT in primary care are predominantly financial and operational.

Antibiotics doi: 10.3390/antibiotics15090847

Authors: A. L. Sreelekshmi Abhijith Pulimoottil Jaykumar Pradeesh Babu Aravind Madhavan Bipin G. Nair Geetha B. Kumar

Uropathogenic Escherichia coli (UPEC) remains the principal driver of urinary tract infections (UTIs), utilizing sophisticated immunoevasive strategies that consistently outmaneuver host defenses and conventional clinical management. This review synthesizes current evidence highlighting the development of recurrent UTIs (rUTIs). Particular emphasis is given to the transition of UPEC into intracellular bacterial communities (IBCs) and quiescent intracellular reservoirs (QIRs)—survival phenomena primarily characterized across specialized mammalian and cellular models. By highlighting the translational evidence gaps surrounding these intracellular sanctuaries, and the challenge of directly extrapolating these model-derived dynamics to human recurrent UTIs (rUTIs), alongside the role of extra-urinary niches, we provide a comprehensive view of the mechanisms driving recurrence. A critical assessment of the UPEC virulence arsenal, involving molecular mechanisms that regulate fimbrial phase variation resulting in evading host immune surveillance, to specialized adhesins, like FimH, which tightly bind uroplakin receptors, triggering host actin rearrangements via rho-GTPase signaling and facilitating bacterial internalization, is also addressed. Additionally, we integrate the roles of cytotoxic necrotizing factor-1 (CNF1) and hemolysin A (HlyA) in promoting cytosolic escape, host cell survival, and long-term persistence. Finally, this review presents a multidisciplinary framework evaluating next-generation non-antibiotic strategies, including bacteriophage therapy, vaccines, phytotherapy, probiotics, and estrogen therapy. By critically assessing their efficacy and clinical maturity across a spectrum of preclinical models and human trials, we evaluate the potential to navigate translational limitations to effectively disrupt the infection cycle.

Antibiotics doi: 10.3390/antibiotics15090846

Authors: MuthuLakshmi BackiaSubramanian Madhumala Shanmugasundaram Shanthi Mariappan Uma Sekar Renuka M K Thyagarajan Ravinder

Background/Objectives: Acinetobacter baumannii is a multidrug-resistant (MDR) nosocomial pathogen associated with significant morbidity and mortality. The emergence of strains resistant to multiple antibiotics has markedly reduced available treatment options. A. baumannii now demonstrates resistance to most first-line antibiotics, resulting in the extensive use of colistin, which in turn has led to the emergence of colistin-resistant strains. There are very limited studies from India on the mechanisms of colistin resistance. In this study, we focused on the molecular mechanisms leading to colistin resistance. Methods: A total of 225 clinical isolates of Acinetobacter baumannii were analyzed in this study. Antimicrobial susceptibility testing was done using the disk diffusion method for various classes of antimicrobial agents as per the Clinical Laboratory Standards Institute (CLSI M100 Ed 35,2025). Susceptibility to colistin was tested by the Microbroth dilution method. Colistin resistance mediated by the mcr gene and alterations in the pmrAB and lpxACD genes were investigated using conventional PCR followed by mutational analysis. Results: Among the 225 isolates, 4 isolates were found to be colistin resistant. All the resistant isolates had an MIC ≥ 16 mcg/mL. Mutations were found in lpxA and pmrB (c.391T > C, c.495T > C, c.516A > G, c.732A > G and c.966T > C). Mutations in pmrA, lpxC, and lpxD were not detected in this study. Mcr-1 gene was not detected in this study. Conclusions: Colistin is the last resort for the treatment of MDR strains, and the increase in resistance to colistin has become a huge concern among the medical community as it reduces the options available for treatment. This is the first report on the molecular mechanisms of colistin resistance in South India using PCR. The study is also the first to report four novel mutations in the genes that may be responsible for resistance. Early detection of resistance and swift control measures can help curb the morbidity and mortality rate among such A. baumannii strains.

Antibiotics doi: 10.3390/antibiotics15090845

Authors: Benard Mware Gilbert Kibet-Rono Kennedy Mwangi Doreen Lugano Shebbar Osiany Edward Kiritu Paul O. Dobi Collins Muli John Juma Regina Njeru Eunice M. Machuka Bernard Bett Ahmed Ogwell Edward O. Abworo Tulio de Oliveira Dishon M. Muloi Sofonías K. Tessema Samuel O. Oyola

Background: Wastewater and Environmental Surveillance (WES) has become a useful public health tool as an early-warning system revealing emergence/re-emergence of pathogenic diseases and spread of antimicrobial resistance (AMR). We characterized the taxonomic composition, relative abundance, and antibiotic resistance genes (ARGs) of wastewater-identified bacterial pathogens across socioeconomically and epidemiologically diverse sewerage catchments in Nairobi, Kenya—a key East African urban city representing a low- and middle-income country (LMIC). Results: Metagenomic analysis of Nairobi’s wastewater revealed distinct bacterial and antimicrobial resistance (AMR) profiles. Campylobacteraceae (52.5% ± 17.9%) and Bacteroidaceae (19.8% ± 9.9%) dominated the communities. While Arcobacter cryaerophilus was ubiquitous, Bacteroides fragilis and A. suis abundances varied by neighborhood socioeconomic status. We detected critical clinical pathogens—including Escherichia coli, Vibrio cholerae, Mycobacterium tuberculosis, and the ESKAPE species—alongside 207 distinct ARGs conferring resistance to 11 antibiotic classes. Both taxonomic and ARG compositions showed high spatial heterogeneity, with maximum variation in low-income areas. Temporal analysis captured shifting pathogen dynamics, and metagenomic abundances for Vibrio cholerae and Klebsiella pneumoniae were validated via qPCR. Conclusions: Our findings underscore the utility of WES as a scalable, non-invasive public health tool. By capturing community-level pathogen composition and AMR dynamics, WES bypasses the limitations of clinical diagnostic access, providing a vital early-warning system for underserved urban populations. To maximize its public health utility, environmental genomic signals must serve as actionable triggers for coordinated One Health responses, including targeted clinical diagnostics, localized antimicrobial stewardship reviews, proactive risk communication, and prioritized sanitation infrastructure upgrades.

Antibiotics doi: 10.3390/antibiotics15090844

Authors: Arwa Al Rujaibi Zaaima Al Jabri Azza Mohammed Al Mamari Amira ElBaradei Hafidha Al-Hattali Faiza Syed Zakariya Al Muharrmi Amina Al-Jardani Meher Rizvi

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) increasingly combines high-risk clonal expansion, mobile resistance platforms and limited treatment options. We investigated the genomic epidemiology, resistance and virulence architecture, plasmid backbones, and therapeutic vulnerabilities of CRKP circulating in Oman. Methods: Between 2021 and 2024, 135 non-duplicate CRKP isolates were recovered from diverse clinical specimens. New antimicrobial agents were evaluated phenotypically, while 38 representative extensively drug-resistant (XDR)/pan-drug resistant (PDR) isolates underwent whole-genome sequencing (WGS) for multilocus sequence typing (MLST), capsular typing, resistome, virulome, plasmid and mobile genetic elements (MGEs) analysis. In vitro synergy of ceftazidime–avibactam/aztreonam, meropenem/fosfomycin and amikacin/fosfomycin was assessed using gradient diffusion-based FICI. Results: WGS revealed a striking shift towards OXA-232-producing ST-2096, which dominated the sequenced collection and carried KL64 with a conserved multidrug-resistant backbone. NDM-5/ST147 and NDM-1 + KPC-2/ST11 formed distinct high-risk lineages with broader extended-spectrum β-lactamase (ESBL) repertoires, greater plasmid heterogeneity and, in co-producers, the highest MGE burden. Across isolates, resistance was reinforced by widespread blaCTX-M variants, armA, aac(6′)-Ib-cr, fosA, porin alterations and fluoroquinolone-resistance mutations, while core virulence and fitness loci including fimH, mrkA, iutA, fyuA and irp2 were widely retained. Cefiderocol showed the most consistent in vitro activity across carbapenemase groups, and eravacycline remained active, whereas plazomicin and fosfomycin activity was compromised by methyltransferase and fos genes. Ceftazidime–avibactam/aztreonam demonstrated universal synergy, while meropenem/fosfomycin and amikacin/fosfomycin showed limited, carbapenemase-dependent activity. Conclusions: CRKP in Oman is characterised by convergent clonal expansion, plasmid-mediated resistance, retained virulence potential and narrowing therapeutic options. Integrated genomic surveillance with carbapenemase-directed susceptibility and synergy testing is essential to guide precision antimicrobial stewardship in high-risk healthcare settings and inform early infection prevention responses to emerging regional CRKP lineages.

Antibiotics doi: 10.3390/antibiotics15090843

Authors: Shahd Mohammad Mosab Albalas Mohammad M. Al-Ahmad

Background/Objectives: Bacteremia poses a significant clinical challenge, often necessitating the off-label use of novel β-lactam/β-lactamase inhibitor (BL/BLI) combinations such as Ceftolozane/Tazobactam (C/T), Ceftazidime/Avibactam (C/A), and Meropenem/Vaborbactam (M/V). Despite their widespread use, no conclusive data support their effectiveness in bacteremia. This meta-analysis aims to evaluate the clinical effectiveness of C/T, C/A, and M/V in treating bacteremia, with a focus on survival outcomes and clinical cure rates. Methods: A systematic review and meta-analysis were conducted following a comprehensive search of Scopus and PubMed databases. Studies reporting survival and clinical cure rates in patients treated with C/T, C/A, or M/V for bacteremia were included. Data extraction followed predefined criteria, and a random-effects meta-analysis was performed to synthesize the findings. Results: Twenty studies met the inclusion criteria, with 18 reporting survival outcomes and 10 evaluating clinical cure rates. The pooled survival and clinical cure rates were 73.6% (95% CI: 67.5–79.8%, p < 0.001) and 76.2% (95% CI: 69.2–83.3%, p < 0.001), respectively, both exhibiting moderate heterogeneity. Subgroup analyses revealed consistent survival trends across study quality, with low- and high-risk-of-bias studies demonstrating comparable estimates. Given that C/A was the most frequently studied agent, its dedicated subgroup analysis yielded a pooled survival estimate of 73.4% (95% CI: 66.4–80.3%, p < 0.001). Conclusions: This meta-analysis provides supporting evidence for the clinical effectiveness of C/T, C/A, and M/V in bacteremia reinforcing the role of BL/BLIs in antimicrobial stewardship as viable options for resistant infections. However, further randomized controlled trials (RCTs) are warranted to validate these results and refine treatment strategies for bacteremia.

Antibiotics doi: 10.3390/antibiotics15090842

Authors: Patrícia Bernardo Susana M. Fernandes Ana S. Nepomuceno Denise Pinto João M. Ribeiro João Gouveia

Background/Objectives: The clinical impact of empirical antifungal therapy in patients with secondary peritonitis and sepsis remains incompletely established. This study aimed to identify risk factors for fungal infection in this population and the impact of empirical antifungal therapy on clinical outcomes. Methods: This was a retrospective cohort study of critically ill patients who underwent abdominal surgery for visceral lesions. Demographic and microbiological data were extracted from electronic health records. Results: A total of 411 patients, 56.9% male and with an average age of 68.8 ± 15.8 years, admitted following urgent gastrointestinal surgery were included. Fungi were isolated from peritoneal fluid or blood in 13.9% of patients during their ICU stay, with a median hospital length of stay until identification of 10 days (interquartile range 1–19 days). Candida albicans was isolated in 63.2% of the positive samples. Empirical antifungal was provided in 27.3% (n = 112). Each additional surgical procedure (OR: 1.47; p < 0.001) was the only risk factor identified for fungal infection in multivariate logistic regression. ICU mortality among patients with and without fungal documentation was similar, as was ICU mortality among patients with and without empirical antifungal therapy. Conclusions: The mortality impact of fungal isolation remains uncertain. Our results suggest that routine empirical antifungal therapy should be approached with caution, prioritizing targeted therapy for patients with confirmed positive cultures or a persistent, refractory clinical course despite adequate source control. These findings should be interpreted as hypothesis-generating and warrant confirmation in future prospective studies before informing changes in clinical practice.

Antibiotics doi: 10.3390/antibiotics15090841

Authors: Madhumala Shanmugasundaram MuthuLakshmi BackiaSubramanian Shanthi Mariappan Uma Sekar Kennedy Kumar Palraj Binesh Lal Yesudhason Rhea Michelle J. Khodabux

Background: Enterococcus species have emerged as significant multidrug-resistant nosocomial pathogens. Linezolid remains a vital last-resort therapeutic option for the management of severe infections caused by vancomycin-resistant Enterococci. However, resistance to linezolid occurs through several mechanisms, including the presence of the cfr, cfr(D), and optrA genes, as well as mutations in domain V region of the 23S ribosomal RNA gene. This study aimed to detect and characterize linezolid resistance in clinical Enterococcus species. Methodology: A total of 266 clinical isolates belonging to the Enterococcus species were analyzed in this study. Antimicrobial susceptibility testing was performed using the disc diffusion method. The Minimum Inhibitory Concentration (MIC) of linezolid was determined by the agar dilution technique. PCR was performed to detect the presence of cfr, cfr(D), and optrA genes, along with mutations in domain V of the 23S rRNA gene. Results: Among the 266 isolates analyzed, 25 (9.4%) were found to be resistant to linezolid, with MIC values ≥ 8 µg/mL. Of these resistant isolates, the cfr gene was detected in one isolate, cfr(D) in sixteen isolates, and optrA in nine isolates. Notably, four isolates carried mutations in the domain V region of the 23S rRNA gene, including a novel C2626T mutation (in India) along with the previously reported G2592T mutation. Conclusions: This study reports the detection of cfr- and cfr(D)-mediated linezolid resistance among Enterococcus species in India. Furthermore, the presence of optrA and a novel C2626T mutation, alongside the G2592T mutation, was identified among resistant isolates. These findings underscore the urgent need for molecular surveillance to prevent further dissemination of these multidrug-resistant pathogens.

Antibiotics doi: 10.3390/antibiotics15090840

Authors: Gaia Casalino Davide Messina Roberta Tardugno Giuseppe Fracchiolla Giancarlo Bozzo Dalila Salierno Francesco D’Amico Martina Canale Antonella Bove Antonio Camarda Elena Circella

Background/Objectives: Colibacillosis, caused by enteropathogenic strains of Escherichia coli (EPEC), is one of the most common infectious diseases in rabbit farms. In the past, antimicrobials were widely used to control colibacillosis, but their use in prevention is currently prohibited. Therefore, the adoption of new prevention strategies as alternatives to the use of antibiotics is very important. The aim of this study was to evaluate the antimicrobial efficacy of cinnamon essential oil (Cinnamomum zeylanicum) against EPEC strains responsible for colibacillosis in rabbits. Methods: For the experiment, 124 strains of E. coli isolated from rabbits that had died of colibacillosis in 10 industrial farms were used. Suspensions of each strain prepared according to CLSI standards with a bacterial density of 1 × 108 CFU/mL were tested using 100% pure commercial cinnamon essential oil (CEO) at concentrations ranging from 0.2 to 0.8 μL/mL. Minimum inhibitory concentrations MIC50 and MIC90 were determined according to a protocol described previously. Results: MIC50 and MIC90 were 0.5 μL/mL and 0.6 μL/mL, inhibiting 64.5% and 94.3% of strains, respectively. Furthermore, strains exposed to 0.4 μL/mL were 12.9 times more likely to be inhibited than those exposed to 0.3 μL/mL (p = 0.0001). Conclusions: CEO was found to be effective against E. coli strains tested at high bacterial densities that can usually be found per gram of faeces in rabbits affected by colibacillosis. Consequently, cinnamon should be useful in preventing colibacillosis by limiting the replication of E. coli, as it is usually found in lower densities in the intestines of healthy rabbits, preventing the onset of colibacillosis and reducing the use of antibiotics in rabbit farms.

Antibiotics doi: 10.3390/antibiotics15090839

Authors: Adela Baca-García Pilar Baca Adela Abellán María Teresa Arias-Moliz Pilar Valderrama

Objective: This study aims to provide a global landscape of research into oral hygiene products with antimicrobial or microbiome-modulating activity through a comprehensive bibliometric analysis to identify trends and hotspots that may influence future research frontiers. Methods: A structured bibliographic search was conducted within the Web of Science Core Collection database from 2006 to 2025. Manual screening was performed to exclude duplicate records, studies that did not align with the core topic, and those failing to meet the predefined inclusion criteria. Bibliometric and visual analyses were performed using VOSviewer, CiteSpace, and the R package ‘bibliometrix’ to evaluate production metrics, citation networks, and multi-level collaboration patterns. Results: The analysis included 1007 publications. Sreenivasan PK was the most productive author, and Lundberg JO was the most cited. The United States, followed by India, Brazil, and China, led global research volume, while the United Kingdom and the Netherlands led in total citations. The International Journal of Dental Hygiene was the most productive journal (n = 48), and the Journal of Dentistry was the most cited (n = 1356). Burgeoning research hotspots include the impact of mouthwashes on the oral microbiome and systemic disorders, the controlled clinical use of chlorhexidine, and alternative formulations incorporating probiotics, herbal extracts, or hyaluronic acid. Conclusions: This study underscores a global shift in dental research priorities from traditional bacterial elimination toward preserving oral microbiota eubiosis. While chlorhexidine remains a subject of research due to its widespread use for therapeutic benefits, bibliometric research highlights its potential systemic consequences as a hotspot. Therefore, future research should focus on innovative antimicrobial formulations for mouthwashes and toothpastes that maintain oral health without causing dysbiosis.

Antibiotics doi: 10.3390/antibiotics15090838

Authors: Chileshe Lukwesa-Musyani Shadrick M. Ngosa Mwelwa Chikombola Davis Sondashi Nayuda Kaonga Evans Mwila Mpabalwani

Background/objectives: Antimicrobial stewardship (AMS) is essential to optimize antibiotic use and limit antimicrobial resistance (AMR), particularly in pediatric populations where diagnostic uncertainty and system constraints complicate care. This study investigated critical AMS indicators, namely, missed antibiotic doses, microbiological diagnostics, and antibiotic prescribing at hospital discharge, which are key challenges that may affect treatment outcomes in hospitalized children under five years old. Methods: A prospective descriptive study was conducted in two tertiary pediatric hospitals in Zambia among children aged 29 days to 59 months. Information on missed antibiotic doses, microbiological investigations, and antibiotic prescribing at discharge from hospitalization was obtained from medical records and medication charts. Results: Patients experienced 1–8 missed doses, most commonly involving benzylpenicillin and ceftriaxone. Documentation of reasons for missed doses was largely absent. Microbiological testing was requested in 36.6% of patients, but only 14.8% of antibiotic prescriptions were supported by culture and susceptibility testing. Of requested tests, 80.9% of results were unavailable at discharge, limiting clinical utility. Only 19.1% of results were available, with a low culture positivity rate (27.3%). Overall, 41.8% of patients were discharged on antibiotics, predominantly from the WHO “Access” group (78%), though 22% were “Watch” antibiotics, and none from the “Reserve” group. Shorter hospital stay was significantly associated with discharge antibiotic prescribing (p = 0.0079; χ2 = 78.577, p < 0.001). Conclusions: Significant AMS gaps exist, including frequent missed doses, limited diagnostic support, and high discharge antibiotic use. Strengthening medication administration systems, improving laboratory capacity and turnaround times, and optimizing discharge prescribing are critical for enhancing pediatric AMS and reducing AMR.

Antibiotics doi: 10.3390/antibiotics15090837

Authors: Lidvine Godaert Claire Roubaud-Baudron Emmanuel Forestier Moustapha Dramé the ENDOLA Study Group the ENDOLA Study Group

Background/Objectives: Infective endocarditis (IE) is a condition with complex management, and several guidelines exist to guide clinicians. Adherence to these recommendations varies and could even be described as poor. The aim of this study was to determine whether the medical specialty of the unit that initiated first-line treatment for IE was associated with risk factors of non-adherence to the 2015 ESC treatment guidelines in a population of patients aged 75 years and over. Method: We analysed data from patients with possible or definite IE included in the ENDOLA cohort, an observational, longitudinal, retrospective, nationwide, real-life cohort study involving 34 French hospitals. Logistic regression was performed using a manual stepwise method to identify variables independently associated with non-compliance of antibiotic treatment of IE with the recommendations of the 2015 ESC guidelines. Results: In total, 899 patients were included (Mean age: 83 ± 5 years, mean Charlson comorbidity score: 3 ± 2). The distribution across the medical units was 51.9% infectious disease (n = 467), 19.8% geriatric ward (n = 178), 18.5% cardiology (n = 166), and 9.8% other wards (n = 88). Patients admitted to geriatric wards were older (p < 0.0001) and were more likely to be dependent for activities of daily living (p < 0.0001). Admission to a geriatric unit was associated with a higher rate of non-compliance with the ESC guidelines (OR 1.6 (1.1–2.4); p: 0.03); prescriptions by cardiologists were more often in compliance with the guidelines (OR 0.6 (0.4–1.0); p: 0.04). Conclusions: The first-line antibiotic treatment prescribed for IE varies considerably depending on the unit responsible for providing care at the time of initial admission.

Antibiotics doi: 10.3390/antibiotics15090836

Authors: Ibrahim Idris Babafela Awosile

Background/Objectives: Beta-lactam antimicrobials are among the most prescribed drugs in veterinary medicine, yet comprehensive epidemiological assessments of resistance across animal species, bacterial taxa, and clinical specimen types remain limited. This study aimed to characterize the epidemiology of phenotypic beta-lactam resistance among bacterial isolates submitted to veterinary diagnostic laboratories in Texas and Oklahoma from 2018 to 2024 and to identify factors and organism–specimen–antimicrobial combinations associated with the overall resistance burden. Methods: A retrospective analysis was conducted using antimicrobial susceptibility testing data from 166,686 bacterial isolates collected between 2018 and 2024. Following data cleaning and quality assessment, 789,871 susceptibility observations across seven beta-lactam antimicrobials were analyzed. Descriptive analyses, univariable and multivariable logistic regression, bacterial diversity analysis, and resistance burden ranking were performed to characterize resistance patterns and identify factors associated with resistance. Results: Resistance varied markedly among the seven beta-lactam antimicrobials. Amoxicillin showed the highest resistance prevalence (56.3%), whereas ceftazidime (11.2%) and imipenem (17.2%) had the lowest. Resistance differed substantially across animal categories, bacterial taxa, and clinical specimen groups. Multivariable analyses identified state, animal category, bacterial group, and clinical specimen group as factors independently associated with resistance, while no consistent temporal differences were detected across 2018–2024 after multivariable adjustment. After sample-size standardization, bacterial richness was highest among zoo/wildlife/aquatic animals, while diversity and evenness varied across animal categories and antimicrobials. Staphylococcus pseudintermedius isolated from skin specimens and tested against amoxicillin contributed the greatest overall resistance burden. Conclusions: Beta-lactam resistance exhibited substantial epidemiological heterogeneity across host species, bacterial taxa, clinical specimens, and states. These findings provide an evidence base for antimicrobial stewardship, resistance surveillance, and empirical antimicrobial therapy in veterinary medicine.

Antibiotics doi: 10.3390/antibiotics15090835

Authors: Jiankang Zhao Yanyan Fan Bin Cao

Background: β-lactam/β-lactamase inhibitor combinations (BL/BLIs) and siderophore cephalosporin serve as critical polymyxin alternatives for carbapenem-resistant Pseudomonas aeruginosa (CRPA). However, systematic multicenter clinical data from China and the resistance mechanisms underlying non-susceptibility to these agents remain poorly defined. Methods: We collected 442 CRPA isolates from 10 hospitals across eight Chinese provinces (2017–2025), determined minimum inhibitory concentrations (MICs) for cefiderocol (FDC), ceftazidime–avibactam (CZA), imipenem–relebactam (IMR), and ceftolozane–tazobactam (CT) through broth microdilution, and performed whole-genome sequencing. Resistance genes, virulence factors, and sequence types (STs) were identified. A chromosomal co-selection score (eight non-mobile resistance genes) was constructed. Multivariable regression and receiver operating characteristic (ROC) analysis identified predictors of non-susceptibility. Results: Carbapenemase genes were detected in only 33 isolates (7.5%): metallo β lactamase (MBL) genes in 28, blaKPC-2 in 2, and blaGES 5/14 in 3; the ST463 clone was absent. MBL producers were resistant to CZA, IMR, and CT (0–4% susceptible) but 100% susceptible to FDC. KPC 2 and GES producers were CZA-susceptible (100%) but IMR- and CT-resistant. Among 127 CZA non-susceptible isolates, 99 (78%) lacked carbapenemase genes; ST270 (from a single center) accounted for 64.6% of these. The chromosomal score independently predicted MIC (ρ: CT 0.64, FDC 0.59, IMR 0.50, CZA 0.25—largely attributable to ST270; all p < 0.001). ExoU positive isolates had lower MICs (FDC median 0.12 vs. 0.25 mg/L, p < 0.001). FDC remained active against 97.3% of all isolates and 100% of carbapenemase-positive ones. Genotype-based prediction yielded area under the ROC curve (AUC) values of 0.86 (CT) and 0.80 (CZA). Conclusions: Mechanisms other than acquired carbapenemases—consistent with chromosomal determinants—accounted for most non-susceptibility in this predominantly northern Chinese respiratory tract CRPA collection. Carbapenemase types determine distinct drug susceptibility patterns. Routine carbapenemase detection fails to identify most CZA-resistant strains. FDC retained the broadest in vitro activity and is a reliable therapeutic option pending clinical outcome data, and ST270-associated resistance deserves continuous surveillance.

Antibiotics doi: 10.3390/antibiotics15090834

Authors: Xiaoju Ma Zhanjie Li Zhaofan Luo Yan Lu Mingming Chen Runhan Huang Xinhai Zhao Li Du Huiping Huang Youpeng Chen

Background: Antimicrobial resistance is a major global health challenge, with multidrug-resistant organisms (MDROs) emerging faster than new antimicrobial agents are being developed. The epidemiology of MDROs varies substantially across healthcare settings; however, newly established hospitals remain understudied. This study aimed to characterise the epidemiology and temporal trends of MDROs in a newly established tertiary hospital. Methods: A five-year retrospective longitudinal surveillance study was conducted from 2019 to 2023 in a newly established tertiary hospital in Southern China. Clinical isolates were collected from intensive care unit (ICU) and non-ICU wards. Nine major MDROs were investigated, including extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) and Escherichia coli (ESBL-ECO); carbapenem-resistant Klebsiella pneumoniae (CRKP), Escherichia coli (CRECO), Acinetobacter baumannii (CRAB), and Pseudomonas aeruginosa (CRPA); methicillin-resistant Staphylococcus aureus (MRSA); and vancomycin-resistant Enterococcus faecium (VREfm) and Enterococcus faecalis (VREfa). Results: A total of 1533 MDRO isolates were identified during the study period. The predominant pathogens were ESBL-ECO (48.1%), ESBL-KP (16.4%), MRSA (11.4%), and CRPA (11.1%). Most isolates were recovered from non-ICU wards (85.4%), and more than half (51.5%) were community-onset. Isolation rates of ESBL-ECO, CRECO, CRAB, and CRPA were significantly higher in the ICU than in non-ICU wards (all p < 0.05). The incidence density rate of hospital-acquired infections caused by ESBL-KP, CRAB, and CRPA was also significantly higher in the ICU (all p < 0.05). Longitudinal analysis revealed divergent trends among MDROs. While most MDRO isolation rates remained below provincial and national benchmark levels, CRKP and CRAB showed significant increasing trends. CRPA exhibited a distinct pattern, increasing rapidly during the early years after hospital opening, exceeding both provincial and national benchmarks by the second year and remaining elevated thereafter. Conclusions: MDRO epidemiology during the first five complete calendar years after hospital opening was characterised by a predominance of ESBL-ECO, ESBL-KP, MRSA, and CRPA, together with substantial non-ICU and community-onset burdens and heterogeneous temporal patterns across organisms. CRPA increased rapidly and remained above the available external benchmark thereafter. These findings highlight the need for hospital-wide, organism-specific MDRO surveillance, while maintaining intensified infection prevention and control in ICUs and continued vigilance for carbapenem-resistant pathogens, particularly CRPA.

Antibiotics doi: 10.3390/antibiotics15090833

Authors: Siân Price Yen Truong Paria Sanaty Zadeh Gloria Tumukunde Claire Brandish Edwin Panford-Quainoo Sarah Cavanagh Esther Obiri-Yeboah Maxencia Nabiryo Elizabeth Ward Gizem Gülpinar Victoria Rutter Helena Rosado Chikondi Savieli

Background: Antimicrobial resistance is a growing global health challenge, particularly in low- and middle-income countries where antimicrobial stewardship (AMS) implementation and leadership capacity remain limited. Pharmacists play a central role in AMS but often lack access to structured leadership development. To address this gap, the Commonwealth Pharmacists Association developed the UK–Africa Leadership Fellowship in Antimicrobial Stewardship (UK-ALF-A), a 12-month bidirectional leadership development programme combining online learning modules, live webinars, peer-learning sessions, dual mentorship from UK- and Africa-based mentors, and workplace-based AMS continuous quality improvement projects. UK-ALF-A was designed to strengthen leadership capability among pharmacists in the United Kingdom and sub-Saharan Africa. Methods: This study evaluated changes in leadership capability among pharmacists participating in the programme and explored how fellows applied these capabilities within AMS-related practice. A mixed-methods evaluation was conducted with 40 fellows (20 UK, 20 Africa), using pre- and post-programme self-assessments and 360-degree colleague feedback based on the International Pharmaceutical Federation Global Advanced Development Framework (FIP GADF), alongside post-fellowship surveys and semi-structured interviews. Results: The proportion of fellows self-assessing at the highest competency level increased from 10% pre-programme to 39% post-programme, with corresponding increases in colleague feedback ratings from 17% to 49%. Qualitative data from open-ended survey responses and semi-structured interviews suggested improvements in leadership confidence, communication, and ability to influence AMS practice. Fellows reported leading prescribing improvements and multidisciplinary engagement, often without formal authority, with greater relative gains observed among Africa-based participants. Conclusions: UK-ALF-A was associated with enhanced leadership capability and contributions to AMS practice, alongside reported changes in individual and organisational practices that support broader health system strengthening, highlighting its value as a workforce development model.

Antibiotics doi: 10.3390/antibiotics15090832

Authors: Mehtap Usta Kübra Zengin Yakup Kara Remziye Nalçacıoğlu Zihni Demirbağ Oktay Yıldız Sevgi Kolaylı

Background/Objectives: Despite its hostile physicochemical environment characterized by high osmotic pressure, acidic pH, and low water activity, honey serves as a unique ecological niche for resilient microorganisms. This study aimed to recover bacterial isolates from honeys of different botanical origins and to characterize the probiotic-associated properties of a honey-derived Bacillus licheniformis isolate. Methods: A total of 20 bacterial isolates were recovered from four honey samples, including two blossoms and two honeydew honeys. 16S rRNA gene sequence analysis showed that the recovered isolates had taxonomic assignments consistent with Bacillus licheniformis. Because 16S rRNA sequencing does not provide sufficient resolution to establish strain-level diversity or clonality, one isolate, designated Blhoney, was selected for subsequent molecular and functional characterization. Results: In vitro assays revealed that Blhoney exhibits robust tolerance to simulated gastrointestinal stress, including extreme acidity (pH 1–3), bile salts (0.3–1%), pepsin, and pancreatin, maintaining viability for up to 24 h. Phenotypic antimicrobial testing demonstrated measurable inhibition zones for erythromycin, kanamycin, chloramphenicol, ampicillin, tetracycline, gentamicin, streptomycin, and spectinomycin, whereas no measurable inhibition zones were observed for penicillin G, rifampicin, ciprofloxacin, or ceftazidime. However, MIC determination and standardized breakpoint interpretation were not performed; therefore, these findings were interpreted solely as preliminary phenotypic inhibition-zone observations. Although the isolate did not display direct antagonistic activity against the tested bacterial pathogens under experimental conditions, its persistence under the tested gastrointestinal stress conditions supports further investigation of its probiotic-associated characteristics. Conclusions: Overall, these findings indicate that the honey-derived Blhoney isolate exhibits preliminary probiotic-associated characteristics under the tested in vitro conditions. However, further strain-level genomic and safety characterization is required before its potential probiotic application can be established.

Antibiotics doi: 10.3390/antibiotics15090831

Authors: Sotiris K. Hadjikakou Christina N. Banti Kristi Gjerazi

Tetracycline (TC), a widely used broad-spectrum antibiotic, was experimentally and theoretically investigated for its potential effect on thyroid hormone biosynthesis through interaction with thyroid peroxidase (TPO). Horseradish peroxidase (HRP) was used as a model of TPO, while FeTPPCl was employed as a model of the active site of the enzyme. The inhibitory effect of TC on the catalytic oxidation of iodide to active iodine was studied spectrophotometrically through the formation of triiodide (I3−), and the IC50 value was determined. Kinetic studies using Michaelis–Menten and Lineweaver–Burk analysis demonstrated that TC acts as a reversible non-competitive inhibitor of HRP, leading to decreased Km and Vmax values and the formation of the ESI complex. Molecular docking calculations further supported the experimental findings, indicating that TC binds at a site different from the active center of the enzyme. The results suggest that tetracycline may interfere with thyroid hormone biosynthesis by inhibiting TPO-mediated iodide oxidation, revealing a possible mechanism associated with thyroid-related side effects during prolonged antibiotic administration.

Antibiotics doi: 10.3390/antibiotics15090830

Authors: Minchae Kang Jinkyung Lee Hyokeun Song

Campylobacter jejuni and Campylobacter coli account for most recognized cases of human campylobacteriosis. For C. coli, gene-carriage surveys often stand in for direct tests of function; this distinction is maintained throughout the review. Virulence is discussed as a sequence of events rather than as a catalogue of factors. Motility and adhesion have the most reproducible support during colonization and epithelial contact, whereas biofilm formation, quorum sensing, and T6SS-associated phenotypes vary substantially across strains and models. We then consider host–cell injury, persistence, transmission, and the uncommon post-infectious outcomes linked to sialylated LOS. Antimicrobial resistance is treated in parallel, with emphasis on target-site change, ribosomal protection or methylation, enzymatic inactivation, and CmeABC-mediated efflux. The One Health model places both species within overlapping animal, food, and environmental reservoirs rather than assigning C. jejuni exclusively to poultry or C. coli to swine. A narrower conclusion emerges from the resistance-fitness literature: gyrA Thr-86-Ile and CmeABC can alter colonization or competitive fitness, but the available data do not show that resistance generally predicts more severe human disease. This distinction frames the proposed priorities for surveillance and intervention.

Antibiotics doi: 10.3390/antibiotics15090829

Authors: Dan-Dan Li Tong Xia Xin-Yu Zhang Huiyan Li Wen-Xin Niu Tie Yao Joon-Hee Lee Li-Li Wang

Background/Objectives: Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that causes serious acute and chronic infections by employing quorum sensing (QS)-regulated virulence, biofilm formation, and host-damaging inflammation. To overcome the yield limitation of two previously identified marine secondary metabolites with dual QS inhibitory and PPAR-γ agonistic activities, we further screened marine-derived natural products for more abundant candidates with similar anti-virulence and anti-inflammatory properties. Methods: In this study, isoflavones were evaluated for anti-QS and PPAR-γ transactivation activities using reporter gene assays, and for antibacterial, anti-virulence, and anti-inflammatory effects via broth microdilution, biofilm, G. mellonella infection, and ELISA cytokine assays. Results: Daidzein and genistein were selected for their optimal anti-QS and PPAR-γ activation activities. They inhibited a key QS regulator and suppressed pyocyanin production and biofilm formation in P. aeruginosa without affecting bacterial growth, indicating minimal selective pressure for resistance. In addition, daidzein and genistein were found to synergistically sensitize the wild-type P. aeruginosa strain to gentamicin, carbenicillin, tobramycin, ampicillin, and polymyxin B, and synergistically or partially synergistically sensitize a multidrug-resistant strain to gentamicin, tobramycin, and ampicillin. Moreover, a Galleria mellonella infection model confirmed that daidzein and genistein significantly enhance the efficacy of gentamicin against P. aeruginosa infection in vivo. Furthermore, in host macrophages, daidzein and genistein significantly inhibited LPS-induced production of NO, IL-6, and IL-1β when combined with an RXR agonist, implying a protective effect on host tissues through PPAR-γ activation. Conclusions: These findings demonstrate that daidzein and genistein serve as effective adjuncts to conventional antibiotics, exerting multifaceted actions against P. aeruginosa infection.

Antibiotics doi: 10.3390/antibiotics15090828

Authors: Ploychomphoo Poollak Nattita Srichomthong Kittaporn Yartirat Phuchit Pooruk Rachanon Kiewdee Nitchawat Paiyabhroma Sattaporn Weawsiangsang Sittichai Urtgam Nontaporn Rattanachak Touchkanin Jongjitvimol Jirapas Jongjitwimol

Background/Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a WHO critical-priority pathogen, and variations in the outer membrane porin CarO are one mechanism implicated in carbapenem resistance. No prior study has characterized carO mutation patterns among A. baumannii in Tak, Thailand. This study aimed to characterize carO mutation patterns in clinical isolates, evaluate their association with CRAB, reconstruct phylogeny, and predict the structural consequences of CarO representatives relative to a crystallographic reference structure (PDB 4RL9). Seventy-seven A. baumannii isolates were recovered from Mae Sot Hospital, and re-confirmed by PCR based on the presence of blaOXA-51-like gene. Results: The carO gene was sequenced and mutations compared between CRAB and carbapenem-susceptible (CSAB) isolates. Of the 77 isolates, 70 yielded successfully carO sequence data by next-generation sequencing (NGS), including 64 CRAB (91.43%) and 6 CSAB isolates. Two overlapping mutation clusters were significantly associated among CRAB isolates (73.44% and 75.00%; p = 0.0008 and p = 0.0006) and were absent from all 6 CSAB isolates (8.57%). Phylogenetic analysis resolved 3 clinical lineages, with variant III predominating. AlphaFold2 models predicted structural divergence among CarO variants with the single variant IV isolate showing the most extensive predicted remodeling of the CarO barrel. Conclusions: carO mutation was strongly associated with carbapenem resistance in this Thai cohort. However, these findings do not establish a direct causal role of carO mutation in carbapenem resistance. Predicted structures were consistent with potential alterations of porin function. These findings provide a regional characterization of carO-mediated resistance and support further functional validation of CarO as a resistance marker.

Antibiotics doi: 10.3390/antibiotics15090827

Authors: Peter Erdelyi Ria Benko Laszlo Papp Mario Gajdacs Maria Matuz Dorottya Gyarfas Edit Hajdu Laszlo Orosz Adam Visnyovszki Zoltan Peto

Background/Objectives: Surveillance of local antibiotic resistance plays a critical role in guiding empirical antibiotic therapies in emergency departments (EDs). This study aimed to collect and analyze a five-year period of prevalence and AMR rates among “ESKAPE” pathogens in a tertiary-care ED setting. Methods: This retrospective observational study included a complete census of microbiological specimens collected from patients presenting to the emergency department between 1 January 2019 and 31 December 2023. Data was retrieved from the MedBakter laboratory information system. Non relevant isolated and duplications were excluded. Only the first isolates per patient per phenotype were included in the final dataset. Results: The final dataset contained 6510 isolates. The most frequent isolates were E. coli (2717, 41.7%), K. pneumoniae (715, 11.0%), and P. mirabilis (696, 10.7%) followed by E. faecalis (662, 10.2%). Prevalence of multi-drug resistance (MDR) was 23.26% among “ESKAPE” pathogens, with highest prevalence in M. morganii (79.6%), P. stuartii (78.5%) and S. marcescens (77.1%). Difficult-to-treat resistance (DTR) was found in 32 isolates, while extensive drug resistance (XDR) was found in 5 isolates. The prevalence of other high-priority MDR bacteria was 12.4% for MRSA and 4.7% for VRE, while among Gram-negative bacteria the prevalence of carbapenem-resistant Enterobacterales was 0.52%. The prevalence of cephalosporin-resistant Enterobacterales varied greatly between 0 and 33%. Conclusions: Despite these important AMR trends, the cumulative antibiogram, according to the World Health Organization (WHO)-issued Access, Watch and Reserve (AWaRe) classification, revealed room for empirical antibiotic choices that spare Watch and Reserve agents. The results will aid the revision of the local antibiotic guidelines.

Antibiotics doi: 10.3390/antibiotics15090826

Authors: Monica A. Stefaniak Vijay Singh Gondil Jingdong Yang Adil Omar Allen G. Oliver Ansley M. Nemeth Roberta J. Melander Mayland Chang Paul M. Dunman Christian Melander

Background/Objectives: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for new antibacterial agents. Here, we report the identification and evaluation of a novel Gram-positive selective antibacterial: NDM-552. Methods: Antibacterial activity was determined by broth microdilution against a panel of Gram-positive and Gram-negative pathogens. Mechanistic studies included BOCILLIN-FL competition assays, microscale thermophoresis (MST), and antibiotic interaction with oxacillin and moenomycin. Additionally, time–kill analyses, frequency-of-resistance measurements, cytotoxicity assays, plasma stability studies, and a murine wound infection model were performed to characterize NDM-552. Results: NDM-552 exhibits activity against Gram-positive pathogens including S. aureus, Enterococcus faecium, and Staphylococcus epidermidis, with minimum inhibitory concentrations (MICs) of 1–2 µg/mL. NDM-552 displays no activity against Gram-negative bacteria. NDM-552 displays bacteriostatic activity against MRSA and additive interactions with oxacillin and moenomycin. The frequency of resistance in MRSA is low (2.39 × 10−9). BOCILLIN-FL competition assays and MST binding analysis identify the essential penicillin-binding protein 2 (PBP2) as the potential target of NDM-552. NDM-552 demonstrates acceptable cytotoxicity, favorable plasma stability, and reduces bacterial burden in a murine wound infection model. Conclusions: NDM-552 is a potent Gram-positive selective antibacterial agent that potentially targets PBP2-associated cell wall biosynthesis and exhibits in vivo efficacy against MRSA. Findings establish NDM-552 as a promising scaffold for the development of new antibacterial agents.

Antibiotics doi: 10.3390/antibiotics15090824

Authors: Begüm Nalça Erdin İrfan Aydın Yüksel Akkaya Meziyet Zeliha Gürlek Ustabaşı Zeliha Alıcıkuş Can Turan İbrahim Halil Kılıç

Background: Candidozyma auris (C. auris) is an emerging multidrug-resistant yeast associated with healthcare-related transmission, difficult laboratory identification, antifungal resistance, and high mortality in critically ill patients. This study aimed to describe the clinical and microbiological characteristics of early institutional C. auris cases detected in a tertiary hospital in Istanbul, Türkiye. Methods: Adult patients hospitalized between January 2021 and December 2022 with C. auris isolation were retrospectively reviewed. Demographic data, underlying conditions, intensive care unit (ICU) exposure, invasive procedures, antimicrobial and antifungal use, culture sources, antifungal susceptibility testing (AFST) results, and clinical outcomes were evaluated. C. auris isolates were identified using VITEK MS v.3.2, and AFST was performed using C. auris isolates were identified using VITEK MS v.3.2, and AFST was performed using Sensititre YeastOne YO10 (Thermo Fisher Scientific, Waltham, MA, USA) panels. Results were interpreted according to CDC interim breakpoints. Multiple isolates from an individual patient were retained when they represented different specimen types or sampling events. Results: Thirty-three adult patients and 62 clinical isolates were included. The mean age was 67.8 years, and 20 patients (60.6%) were aged 65 years or older. Most patients had prior ICU admission (93.9%), urinary catheterization (97.0%), central venous catheterization (90.9%), mechanical ventilation (81.8%), and broad-spectrum antimicrobial exposure. Blood cultures represented the most frequent source of isolation (56.5%), followed by urine cultures (27.4%). Resistance rates were 98.4% for amphotericin B, 88.7% for fluconazole, 6.6% for micafungin, and 6.5% for caspofungin. Overall mortality was 72.7%. While mortality was numerically higher among patients aged 65 years or older and those requiring inotropic support, only the need for mechanical ventilation was statistically significantly associated with mortality. The cohort-level APACHE II-based expected mortality was 48.0%, whereas observed mortality was 72.7%; this comparison was descriptive and no formal calibration analysis was performed. Conclusions: Early institutional C. auris cases in this tertiary hospital occurred predominantly among elderly, critically ill patients with prolonged healthcare exposure, invasive devices, and extensive antimicrobial use. The findings support the need for early laboratory recognition, cautious interpretation of commercial AFST results, particularly given the propensity of Sensititre YeastOne to overestimate amphotericin B resistance, strengthened infection control practices, and multicenter studies incorporating molecular typing to clarify transmission dynamics and antifungal resistance patterns.

Antibiotics doi: 10.3390/antibiotics15090825

Authors: Kübra Can Kurt Liam F. Katzin Landon Tamaddon Ali Arslan Alexander G. Lucaci Christopher E. Mason

Background/Objectives: Antimicrobial peptides (AMPs) are considered alternatives to classical antibiotics due to limited resistance development in bacteria. However, bacteria can develop resistance to AMPs through evolutionary adaptation, including oligosaccharide modifications and multidrug efflux pumps. Further research is needed to elucidate the defense mechanisms employed against AMPs to address the emerging resistance problem. Pexiganan is a cationic peptide with effective broad-spectrum antimicrobial activity. The aim of this study is to elucidate the genomic and transcriptomic basis of E. coli’s evolutionary adaptation to pexiganan. Methods: The E. coli ATCC BAA-2523 strain became resistant to pexiganan via evolutionary adaptation methodologies. Whole-genome and transcriptome analyses of resistant and susceptible populations were conducted using Nanopore sequencing and Illumina RNA sequencing, respectively. Results: Resistance development became particularly evident after 15 µg/mL, and the bacteria demonstrated the ability to grow even at high doses (up to 1000 µg/mL). Increases in expression levels of classical ARGs such as emrB, acrF, OXA, sul2, and dfrA14 in the pexiganan-resistant strain indicate that bacteria have the potential for broad-spectrum resistance to other antibiotics alongside pexiganan. Missense mutations have been identified in the phosphatidylserine synthase and cardiolipin synthase genes, which are involved in membrane biosynthesis. Increased expression was observed in the membrane-bound lytic murein transglucosylase (mltF), the murein hydrolase activator (EnvC), and the Antigen 43 (Ag43) gene. Conclusions: Pexiganan may not only target the cell membrane but also trigger the bacterium’s overall transcriptional response and cross-resistance and MDR systems. Upregulation of multidrug efflux pumps, lytic murein transglucosylase, the murein hydrolase activator and the Antigen 43 gene might be associated with resistance. In addition, the missense mutation was detected in the membrane biosynthesis genes pssA and clsB. In vivo infection models, targeted functional genomics, and comprehensive phenotypic cross-resistance testing will be required to validate our results.

Antibiotics doi: 10.3390/antibiotics15090823

Authors: Yasmin Abdelkhalek Dina H. Amin Hassan M. Gebreel Hayam A. E. Sayed Ahmed M. H. Kamel

Background/Objectives: Bacterial colonization is an under-recognized component of antimicrobial resistance (AMR), particularly among cardiovascular outpatients who frequently interact with healthcare services. While surveillance has traditionally focused on multidrug-resistant (MDR) pathogens causing clinical infection, the ecological characteristics of colonization including multisite carriage, polymicrobial communities, and commensal or low-virulence organisms recovered on antimicrobial-containing media remain poorly defined. This study investigated the burden and ecological patterns of colonization by organisms with reduced antimicrobial susceptibility in cardiovascular outpatients. Methods: In this cross-sectional study, 36 adult cardiovascular outpatients were screened using sputum, nasal, and skin specimens (108 samples) on antimicrobial-supplemented selective media. Isolates were identified by MALDI-TOF mass spectrometry, susceptibility was determined using the VITEK 2 system, and colonization patterns were analyzed using exact statistical methods. Results: Colonization by organisms with reduced antimicrobial susceptibility was detected in 61.1% of patients (95% CI, 44.9–75.2), and at least one validated MDR pathogen in 52.8% (95% CI, 37.0–68.0). Sputum showed the highest colonization prevalence. Multisite colonization occurred in 72.7% of colonized patients, with 40.9% positive at all three anatomical sites. Polymicrobial colonization was common (mean 1.9 species per colonized patient). Resistant commensal bacteria including Neisseria flavescens, Neisseria mucosa, Moraxella catarrhalis, and Lacticaseibacillus casei were recovered alongside recognized pathogens and showed reduced susceptibility across multiple antibiotic classes. Colonization showed significant concordance across anatomical sites, indicating a whole-body colonization state rather than a series of independent site events. Prior hospitalization and concurrent antibiotic therapy were significantly associated with colonization, whereas prior percutaneous coronary intervention and individual comorbidities were not. Conclusions: Cardiovascular outpatients frequently harbour complex bacterial communities characterized by multisite colonization, polymicrobial carriage, commensal and low-virulence organisms recovered on selective media, and MDR pathogens. These findings highlight bacterial colonization as a potential community reservoir of AMR and support targeted pre-procedural screening together with community antimicrobial stewardship. As a single-centre, cross-sectional study of 36 patients, these results are exploratory and hypothesis-generating and require confirmation in larger, multicentre cohorts.

Antibiotics doi: 10.3390/antibiotics15090822

Authors: Pablo Pacheco Lucía Sánchez José Luis Píriz María Gil-Molino Joaquín Rey Pedro Fernández-Llario María Jesús Palacios Jorge Peña María Isabel Igeño Alberto Quesada Alejandro Gallardo-Soler

Background: Plasmid-mediated colistin resistance threatens the effectiveness of one of the last-line antibiotics for treating multidrug-resistant Gram-negative infections. Although wildlife is increasingly recognized as a reservoir of mobile antimicrobial resistance genes, its role in the emergence and dissemination of novel plasmid-mediated colistin resistance (mcr) genes remains poorly understood. Objectives: This study aimed to characterize two distinct mcr-carrying E. coli isolates identified in Spanish wildlife. Methods: Among 807 faecal samples from healthy wild animals screened, two mcr-positive E. coli isolates were identified. Genomic characterization included whole-genome sequencing, plasmid analysis, and functional assays, followed by heterologous expression and lipid A characterization by Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC-HRMS). Results: Analysis of the two mcr-positive E. coli isolates revealed the first mcr-1-positive isolate from a wild boar (Sus scrofa) in Extremadura, Spain, carrying mcr-1 on a 33.3-kb conjugative IncX4 plasmid, while the isolate recovered from an Iberian lynx harbored mcr-13, a novel plasmid-borne mcr-like gene encoding a putative phosphoethanolamine (pEtN) transferase on a 163-kb non-conjugative plasmid. MCR-13 showed 82% amino acid identity with MCR-10, increased colistin MIC two-fold in a susceptible E. coli host, and mediated phosphoethanolamine modification of lipid A, although to a lower extent than MCR-1. Conclusions: The co-occurrence of established (mcr-1) and a novel mcr-like gene mcr-13, further supports the role of wildlife as a potential reservoir of emerging resistance genes and highlights the value of including wildlife in One Health surveillance programs.

Antibiotics doi: 10.3390/antibiotics15090821

Authors: Han Wang Keqiang Zhang Muheng Liu Shenwei Cheng Cheryl Marie Cordeiro Erik Sindhøj Junfeng Liang Yuanfang Zeng Shizhou Shen Suli Zhi

Background: Manure-amended agricultural soil is a critical reservoir of antibiotic resistance genes (ARGs), posing escalating threats to environmental health and food safety. However, the temporal trajectories of ARG prevalence throughout the complete reproductive cycle of cash crops, and their mechanistic linkages with fertilization regimes and microbial community succession, remain inadequately understood. Methods: To bridge this knowledge gap, we conducted an in situ field experiment over the entire growth period of Chinese cabbage at a long-term manure-amended farm in Tianjin, China. Six contrasting fertilization strategies were evaluated: unfertilized control (CK1), unfertilized baseline control (CK2), traditional full-rate combined manure–chemical fertilization (TF), traditional half-rate combined manure–chemical fertilization (T1), half-dose sole manure fertilizer (T2), and half-dose sole chemical fertilizer only (T3). Results: Our results demonstrated that ARG abundance and associated mobile genetic elements (MGEs) exhibited a pronounced transient surge immediately post-fertilization, yet reverted to baseline levels by harvest, revealing a tangible resilience of the soil resistome. Notably, the optimized half-organic fertilization (T2) effectively curtailed the proliferation of manure-derived pathogenic taxa while preserving beneficial keystone phyla (e.g., Acidobacteria and Proteobacteria), indicating a trade-off between nutrient provisioning and ecological filtering. Co-occurrence network analysis further identified MB-A2-108, Saccharimonadales, and Rokubacteriales as pivotal hosts for multidrug-resistant ARGs, underscoring that microbial interspecific interactions—rather than taxonomic richness alone—are the primary drivers of resistome succession. Quantitative risk assessment confirmed that the T2 regimen reduced the composite ARG contamination index (CFzone) by 25% relative to conventional full fertilization (TF), while maintaining comparable cabbage yields. Conclusions: Collectively, our findings advocate for precision organic fertilization as a nature-based solution that synchronizes nutrient supply with crop demand, curtails ARG propagation, and mitigates long-term agroecological risks.

Antibiotics doi: 10.3390/antibiotics15090820

Authors: Florian Wagenlehner Heidrun Taeschner Horst Lorenz Anja Berwanger Nacera Infed Oda Ewald Peter Gerke

Background/Objectives: Antibiotics are the recommended first-line therapy for acute uncomplicated lower urinary tract infections (cystitis), but antibiotic resistance and tolerability concerns require antibiotic-sparing strategies. This study evaluated D-mannose as a stand-alone alternative. Methods: This multicenter, randomized, controlled, double-blind study compared D-mannose to fosfomycin in women aged 18–70 with cystitis. Clinical cure (CC) was assessed using the Acute Cystitis Symptom Score (ACSS) up to day 8, with non-inferiority tested at the 15% margin. Post hoc analytical approaches accounted for potential bias due to low sample sizes and missing values. Results: The study randomized 118 patients. At baseline, severe symptoms (ACSS ≥ 12) were more frequent in the D-mannose group compared to fosfomycin (32.8% vs. 22.8%). Efficacy analyses were performed on the per-protocol set (D-mannose: N = 57; fosfomycin: N = 54). Median time to CC was 5.0 days (D-mannose) and 3.0 days (fosfomycin) overall, and 4.0 days vs. 3.0 days in the subgroup of moderate disease severity. The point estimate for the CC rate difference on day 8 (−10.1%) favored fosfomycin but was within the non-inferiority margin. Due to wide confidence intervals, non-inferiority was not statistically confirmed. The post hoc analysis yielded smaller confidence intervals with estimated CC rates of 84.2% (D-mannose) vs. 83.5% (fosfomycin) on day 8 and statistically demonstrated the comparability of both treatments. Recurrence rates were similar (13.2% vs. 13.3%), and there was no statistically significant difference for additional antibiotic use (19.3% vs. 11.1%). Investigators and patients favored D-mannose over fosfomycin for tolerability, and gastrointestinal adverse events were less frequent (9.8% vs. 24.6%). Conclusions: Post hoc analysis indicated that D-mannose treatment is comparable to fosfomycin in uncomplicated cystitis in line with the prespecified non-inferiority criteria. These findings warrant confirmation in an adequately powered trial but suggest that D-mannose is a promising alternative to antibiotics given its favorable risk–benefit ratio.

Antibiotics doi: 10.3390/antibiotics15090819

Authors: Luigi Russo Sofia Mao Tindara Scirocco Michela Sabbatucci Andrea Zovi Antonio Vitiello Maria Rosaria Gualano Walter Ricciardi Leonardo Villani

Background: Antimicrobial resistance (AMR) remains a major public health threat in Italy, where high incidence and mortality persist. In this study we aimed to describe national and regional trends in AMR rates from 2015 to 2024. Methods: We conducted an ecological time-series analysis using publicly available data from the Italian AMR surveillance system (AR-ISS). We included six priority pathogens: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE-faecium), third-generation cephalosporin-resistant Escherichia coli (3GCREC), carbapenem-resistant Klebsiella pneumoniae (CRKP), Pseudomonas aeruginosa (CRPA), and Acinetobacter spp. (CRAS). We analyzed annual resistance rates at national and regional level. Temporal trends were estimated using linear regression models, with calendar year as the independent variable. Regression coefficients, 95% CIs, and p-values were used to assess direction and significance. Relative percentage changes were calculated for 2015–2024 and, when relevant, for 2023–2024. Results: Resistance declined over time for four of six pathogens, but increased markedly for VRE-faecium, from 11.1% to 34.9%. Significant downward trends were observed for MRSA (β = −0.91, 95% CI: −1.35; −0.48, p = 0.0013), 3GCREC (β = −0.53, 95% CI: −0.99; −0.06, p = 0.0304), CRKP (β = −0.94, 95% CI: −1.35; −0.53, p = 0.0008), and CRPA (β = −0.86, 95% CI: −1.44; −0.27, p = 0.0095); VRE-faecium showed a strong upward trend (β = +2.76, 95% CI: 2.58; 2.94, p < 0.0001). CRAS remained consistently high throughout the study period. Considerable regional heterogeneity was observed across pathogens and geographical areas. Conclusions: Despite progress for several pathogens, AMR remains a critical challenge in Italy. The persistent increase in VRE-faecium and the high burden of CRAS support the need for strengthened surveillance, stewardship, and region-specific infection prevention interventions.

Antibiotics doi: 10.3390/antibiotics15090818

Authors: Tina Birk Annette Nygaard Jensen Tina Beck Hansen

Background/Objectives: Rapid surveillance tools are needed to monitor antimicrobial-resistant bacteria in the food chain. This study evaluated whether qPCR-based quantification of tetracycline resistance genes could replace culture-based quantification of tetracycline-resistant Escherichia coli in pig caecal content and carcass skin swabs. Methods: Caecal content (n = 89) and skin swab samples (n = 68) were collected across four Danish abattoirs and analysed by both qPCR and Petrifilm cultivation. Method comparisons were restricted to samples within the quantifiable range of Petrifilm. E. coli concentrations (uidA gene) and tetA/tetB gene concentrations were compared with Petrifilm counts of E. coli and culturable tetracycline-resistant E. coli. Method agreement was evaluated using paired t-tests, Bland–Altman analysis, and Spearman correlation. Results: In caecal samples (n = 84), no significant difference in mean log E. coli concentration was observed between uidA qPCR and Petrifilm counts (p = 0.33). However, Bland–Altman analysis showed poor agreement at the individual-sample level, with limits of agreement ranging from −1.09 to 1.21 log10 CFU/g. In skin swab samples (n = 43), qPCR systematically overestimated E. coli counts compared with cultivation, with a significant bias of 0.75 log10 CFU/1400 cm2 (p < 0.001). No significant correlation was observed between tetA/tetB and culturable tetracycline-resistant E. coli in caecal (rs = 0.091, p = 0.44) or skin swab samples (rs = 0.13, p = 0.48). Conclusions: qPCR may be useful for population-level surveillance of tetracycline resistance genes in pig caecal samples but cannot replace culture-based methods for accurate quantification of tetracycline-resistant E. coli in individual samples.

Antibiotics doi: 10.3390/antibiotics15090817

Authors: Douha Shouqair Subham Verma Rashed Alghafri Rania Nassar Lobna Mohamed Fatima Al Dhaheri Dean Everett Ahmed A. Shibl Jorge Rodríguez Danesh Moradigaravand Mushtaq Khan Richard Goering Abiola Senok

Background/Objectives: Wastewater-based antimicrobial resistance (AMR) surveillance typically relies on treatment plant influent as a single integrated matrix, obscuring source-specific signals. In arid settings where treated effluent is reused, understanding how resistomes and virulomes are structured across wastewater compartments is essential for One Health monitoring. Methods: Shotgun metagenomic sequencing was applied to 57 wastewater samples collected in Dubai, United Arab Emirates, between October 2024 and January 2025. Samples represented nine community sewer nodes, two tertiary-care hospital outflows, and influent and effluent from two wastewater treatment plants (WWTP). Datasets were used for taxonomic, resistome, and virulome profiling. Alpha diversity was compared using Wilcoxon rank-sum tests, beta-diversity differences were assessed using permutational multivariate analysis of variance, and source-associated AMR genes were identified using linear discriminant analysis effect size analysis. Results: A total of 1470 bacterial species, 822 antimicrobial resistance genes (ARGs), and 1554 virulence factor genes were identified. Bacterial diversity was significantly lower in WWTP effluent than in other compartments. Hospital wastewater was enriched for class D β-lactamases, including multiple blaOXA variants, whereas community wastewater and WWTP influent shared dominant macrolide and aminoglycoside resistance genes including msr(E), mph(E), strB and aadA1. Despite marked reductions in bacterial diversity after treatment, no significant difference in ARG diversity was observed between WWTP influent and WWTP effluent (p = 0.558), with resistance genes such as blaVEB, msr(E), mph(E) detected in the latter. Virulome profiles shifted from fimbrial gene dominance in untreated sources toward biofilm- and persistence-associated genes in WWTP effluent. ARG alpha diversity varied over time, whereas taxonomic and virulome diversity remained stable. Conclusions: Community and influent wastewater capture population-level AMR carriage, hospital outflows concentrate clinically relevant resistance determinants, and WWTP effluent retains resistance markers despite microbial biomass reduction. Compartment-resolved metagenomic surveillance provides a practical One Health framework for identifying high-value monitoring points.

Antibiotics doi: 10.3390/antibiotics15080816

Authors: Amy McLeman Alexander D. H. Kingdon Robin Hoeven George Taylor Ellie Allman Issra Bulgasim Claudia McKeown Richard N. Goodman Sabrina Moyo Adam P. Roberts

Background: Here, we report the isolation and identification of a Paenibacillus polymyxa strain from the citizen science project, Swab and Send. P. polymyxa is well known for its production of polymyxin E (colistin), and polymyxin B. Polymyxins are ranked in the highest-priority critically important antimicrobial classification by the WHO and are of particular importance for treating Gram-negative multi-drug-resistant pathogens. Due to their clinical use, most of the literature focusses on these polymyxin variants, and there is sparse genetic research on other polymyxin variants. Methods: The Paenibacillus polymyxa 1G strain was isolated and tested for antimicrobial activity using a combination of on-agar and liquid-based inhibition assays to detect antimicrobial activity against Escherichia coli. The isolate was also tested for activity in liquid media against a panel of isolates showing various resistances to test if our strain could overcome current clinically important resistance mechanisms. Our isolate was whole-genome-sequenced, and bioinformatics was carried out on the resulting sequence to analyse the relevant biosynthetic gene cluster. The cell-free supernatant from the P. polymyxa 1G isolate was also analysed using mass spectrometry to confirm the production of the polymyxin. Results: P. polymyxa 1G was active on agar and resulted in antimicrobial activity, with a 99% reduction in area under the curve when tested in liquid media against E. coli. P. polymyxa 1G did not inhibit the growth of E. coli containing mcr-1 or mcr-4 colistin-resistance genes. Using whole genome sequencing, we are able to describe the biosynthetic gene cluster of the putative polymyxin A, compare the pmxA, pmxB, and pmxE genes to five other polymyxin genes that encode known polymyxin variants, and provide mass spectrometry data that supports the production of polymyxin A1 (1157 m/z) and A2 (1143 m/z). Conclusions: Here, we report the isolation and identification of a P. polymyxa strain producing a polymyxin A-like compound that was discovered through the citizen science project, Swab and Send. We add to the genetic and mass spectrometry data for polymyxin A, and demonstrate that this putative polymyxin A, produced naturally by our strain, is unable to overcome the current clinically relevant resistance mechanisms to colistin.

Antibiotics doi: 10.3390/antibiotics15080815

Authors: Theresa Strohhammer Alessandra M. Martorana Alessandra Polissi

The peptidoglycan (PG) sacculus of Gram-negative bacteria is continuously reorganized by a diverse enzymatic repertoire, including lytic transglycosylases, endopeptidases, carboxypeptidases, amidases and LD-transpeptidases, that operate alongside PG synthases to maintain envelope integrity throughout the cell cycle. This remodeling machinery has been extensively characterized in the context of growth and division and it is now emerging also as a key determinant of bacterial survival under non-growing and stress conditions. This review summarizes current knowledge on PG remodeling in Gram-negative bacteria, with emphasis on its regulation during stationary phase, environmental stress, and outer membrane perturbation. How these remodeling pathways, characterized by increased 3–3 cross-linking and enhanced PG–outer membrane coupling, contribute to survival under β-lactam exposure and how related enzymatic configurations can give rise to antibiotic tolerance and resistance is also discussed. Finally, recent progress in targeting PG remodeling enzymes, particularly lytic transglycosylases and peptidases, as adjuvant strategies to potentiate existing antibiotics are reviewed. In summary, PG remodeling represents a mechanistically validated but still underexploited target for addressing antibiotic tolerance and resistance in Gram-negative pathogens.

Antibiotics doi: 10.3390/antibiotics15080814

Authors: Marija Cavka Marija Kvesic Ivankovic Ana Maravic Mia Dzelalija Jelena Marinovic Ivana Goic-Barisic Marija Tonkic Toni Kljakovic Gaspic Anita Novak

Background/Objectives: Acinetobacter baumannii is a major cause of infections in Intensive Care Units (ICUs), driving mortality through high-level antimicrobial resistance. This study utilized whole-genome sequencing (WGS) to evaluate the phenotypic, genotypic and virulence features of carbapenem-resistant A. baumannii (CRAB), which has caused ventilator-associated pneumonia/tracheobronchitis (VAP/VAT) in the ICU of the University Hospital of Split, Croatia. Methods: Over 1 year, lower respiratory tract specimens from 79 VAP/VAT patients were analyzed. CRAB isolates were identified via MALDI-TOF MS and evaluated for antimicrobial susceptibility, and a representative subset underwent WGS and multilocus sequence typing (MLST). Results: Out of 106 specimens, 18 non-duplicate CRAB strains were isolated. Five isolates underwent genomic analysis, identifying two globally distributed, high-risk Pasteur lineages: ST2 and ST492. These lineages displayed distinct resistomes: ST2 carried blaOXA-23 and blaADC-73, while ST492 harbored plasmid-borne blaOXA-72 (Rep3-T1/AB082 cluster) and blaADC-30. All isolates shared aminoglycoside/macrolide-resistance genes, conserved efflux pumps, and virulence determinants (bau, bas, ent, bar) crucial for acinetobactin synthesis and respiratory colonization. Phylogenetic analysis confirmed regional circulation and genetic links to neighboring countries. Conclusions: This study highlights the evolutionary dynamics of endemic CRAB lineages in a Croatian ICU and their global dissemination, which poses a critical threat, demanding strict infection control and novel therapeutics.

Antibiotics doi: 10.3390/antibiotics15080813

Authors: Betul Zehra Temur Esma Bolat Meltem Ayas Sengül Nisa Demirci Nihan Unubol Neval Yurttutan Uyar Ozge Can Tanil Kocagoz

Objectives: The escalation of multidrug-resistant (MDR) clinically relevant bacterial isolates, including Escherichia coli and key ESKAPE pathogens, represents a critical global healthcare threat. While antimicrobial peptides (AMPs) offer promising alternatives, metabolic instability often limits their clinical use. This study investigated the therapeutic potential, antimicrobial efficacy, and resistance dynamics of peptide D-TN6 against highly resistant bacterial strains. Methods: D-TN6 efficacy was evaluated against 164 clinical isolates, including pan-drug-resistant, carbapenem-resistant, and polymyxin-resistant phenotypes. A 20-passage serial induction assay compared resistance development kinetics of D-TN6 against gentamicin over an extended period. Results: D-TN6 demonstrated potent efficacy against MRSA (minimum inhibitory concentration (MIC)90: 1 µg/mL) and remained effective against polymyxin B-resistant Klebsiella pneumoniae (MIC90: 8 µg/mL) and pan-drug-resistant Acinetobacter baumannii (MIC90: 8 µg/mL). In resistance assays, while the gentamicin MIC increased 256-fold by the 17th passage, the D-TN6 MIC remained constant at 1 µg/mL throughout the 20-passage study. Conclusions: These findings underscore D-TN6 as a robust therapeutic candidate. Its efficacy against MDR strains and its feature of low propensity for resistance development—likely due to membrane disruption—position D-TN6 as a promising solution for life-threatening infections where conventional last-resort agents fail.

Antibiotics doi: 10.3390/antibiotics15080812

Authors: Wasan Katip Puntapong Taruangsri Siriporn Okonogi Shaun Wen Huey Lee Ajaree Rayanakorn

Background: Colistin remains an important therapeutic option for complicated urinary tract infections (cUTIs) caused by multidrug-resistant Gram-negative bacteria. A loading dose is often recommended to rapidly achieve steady-state plasma concentrations, but its clinical benefit specifically for cUTIs, where local urinary drug concentrations markedly exceed plasma levels, remains unclear. Methods: We conducted a retrospective cohort study of adult patients treated with intravenous colistin for cUTIs at Nakornping Hospital, Chiang Mai, Thailand, between 2015 and 2022. Patients were classified as receiving loading-dose (LD) or non-loading-dose (non-LD) colistin. The primary outcome was 30-day all-cause mortality. Secondary outcomes included clinical response, microbiological response, and nephrotoxicity (defined by RIFLE criteria). Inverse probability weighting (IPW) using the propensity score was applied to adjust for baseline covariates, and Cox proportional hazards regression was used to estimate crude and adjusted hazard ratios (HR) using the non-LD group as the reference. Results: Among 123 patients (74 LD, 49 non-LD), unadjusted 30-day all-cause mortality was 35.14% versus 40.82% (p = 0.524), clinical response was 75.68% versus 71.43% (p = 0.599), and microbiological response was 78.38% versus 65.31% (p = 0.109) for LD versus non-LD, respectively. Nephrotoxicity occurred in 48.65% versus 38.78% (p = 0.281). After IPW adjustment, no statistically significant differences were observed between the LD and non-LD groups for 30-day all-cause mortality (aHR 0.78, 95% CI 0.43–1.42, p = 0.419), clinical response (aOR 1.48, 95% CI 0.54–4.08, p = 0.445), or microbiological response (aOR 2.10, 95% CI 0.81–5.48, p = 0.128). For the safety outcome, nephrotoxicity was analyzed using the Fine–Gray competing risk model, treating death as a competing event. The IPW-adjusted subdistribution hazard ratio was (asHR) 2.23 (95% CI 0.83–5.95, p = 0.110), indicating no statistically significant difference in renal safety between the LD and non-LD groups. Conclusions: Loading-dose colistin was not associated with a statistically significant reduction in 30-day all-cause mortality or improvements in clinical response and microbiological eradication in patients with cUTIs. Nephrotoxicity was numerically lower without a loading dose, but the wide confidence intervals in this study of 123 patients precluded a definitive conclusion regarding renal safety. Omitting the loading dose may be a reasonable option for localized urinary infections, particularly in patients at high risk for acute kidney injury, pending confirmation in larger prospective studies.

Antibiotics doi: 10.3390/antibiotics15080811

Authors: Tomáš Pagáč Ján Víglaš Petra Olejníková

Background/Objectives: Azole resistance in fungal pathogens is a growing clinical and environmental concern. However, short-term cellular responses during the first hours of azole exposure remain insufficiently characterised, particularly in filamentous fungi. This study used Neurospora crassa as a genetically tractable model to investigate early cellular responses to azole stress. Methods: Exponentially growing mycelia were exposed to conidium-derived MIC80 reference concentrations of fluconazole, voriconazole, ravuconazole, and ketoconazole. Early responses were assessed by monitoring radial growth, intracellular glycerol, H2DCF-DA microscopy, antioxidant enzyme activities, Calcofluor White staining, and RT-qPCR analysis of genes associated with osmoregulation, sterol homeostasis, oxidative stress, and cell-wall remodelling. Results: Azole exposure was associated with reduced net post-transfer radial growth and rapid treatment- and time-dependent changes in intracellular glycerol. Induction of hog1 together with treatment-dependent changes in gpd1 and glycerol accumulation was consistent with an early osmoregulatory response. Sterol-homeostasis genes showed selective feedback regulation, and several cell-wall-remodelling genes underwent treatment-dependent transcriptional changes. Qualitative H2DCF-DA microscopy showed limited oxidant-associated fluorescence during azole challenge. Catalase activity remained close to control levels, whereas representative SOD-activity experiments showed larger fold changes after voriconazole and ravuconazole exposure. Representative Calcofluor White micrographs showed irregular septation and localised regions of enhanced cell-wall-associated staining. Conclusions: Neurospora crassa shows multiple concurrent early responses to azole stress involving osmotic adjustment, sterol-pathway feedback, antioxidant responses, and cell-wall remodelling. These findings provide a framework for future studies examining how such stress responses contribute to recovery, tolerance, or longer-term adaptation.

Antibiotics doi: 10.3390/antibiotics15080809

Authors: Ilias Karaiskos George L. Daikos Sofia Michelidou Christina Mouratidou Alexandra Gavala Aikaterini Gkoufa Aikaterini Sakagianni Eleni Mouloudi Evdoxia Tsigou Christina Routsi Stamatis Karakonstantis Christina Stamatopoulou Despina Markantonaki Foteini Veroniki Maria Pirounaki Anna Kyriakoudi Sevasti Ampelioti Charalambos Anastogiannis Antonia Koutsoukou Helen Giamarellou Konstantinos Pontikis

Background: Pandrug-resistant (PDR) Acinetobacter baumannii represents a major therapeutic challenge in regions where carbapenem-resistant A. baumannii (CRAB) is endemic. Whether the PDR phenotype independently worsens clinical outcomes beyond the effects of disease severity and therapeutic limitations remains uncertain. This study compared the characteristics, management, and outcomes of severe infections caused by PDR and carbapenem-resistant, colistin-susceptible A. baumannii. Methods: We conducted a retrospective analysis of prospectively collected data across 11 tertiary-care hospitals in Greece (February 2022–June 2024). Consecutive adults with bloodstream infection or hospital-acquired/ventilator-associated pneumonia caused by CRAB or PDR A. baumannii were enrolled. The primary outcome was 14-day clinical failure; secondary outcomes included 28-day mortality, microbiological eradication, organ dysfunction, and organ-support-free days. Multivariable logistic and Cox regression analyses, before and after propensity score matching, were performed to adjust for confounding. Results: Among 142 patients, 91 (64%) had PDR and 51 (36%) had carbapenem-resistant, colistin-susceptible infections. Clinical failure occurred in 41% of patients and did not differ significantly between PDR and CRAB infections (39% vs. 45%; p = 0.440). Twenty-eight-day mortality was 33% and 22%, respectively (p = 0.139). After adjustment, the PDR phenotype was not independently associated with clinical failure or mortality. Higher APACHE II score and pneumonia independently predicted clinical failure, whereas sulbactam-containing therapy was associated with lower odds of failure (OR 0.24, 95% CI 0.07–0.79). Older age, higher SOFA score, impaired lactate clearance, and tigecycline-containing therapy independently predicted 28-day mortality. In matched analysis, PDR showed a non-significant upward trend in 28-day mortality (HR 2.36, 95% CI 0.97–5.76; p = 0.059). Conclusions: In severe A. baumannii infections, the PDR phenotype was not an independent determinant of clinical failure or short-term mortality. Patient severity and antimicrobial strategy were major outcome correlates; treatment associations should be interpreted cautiously given the observational design.

Antibiotics doi: 10.3390/antibiotics15080810

Authors: Bhuwad Chinwatanawongwan Chavarat Jarungvittayakon Siwadol Wongsak Paphon Sa-ngasoongsong Pattarana Sae-Chew Thidarat Rujirawat Penpan Payattikul

Background: Biofilm formation on polyethylene surfaces contributes significantly to treatment failure in periprosthetic joint infection (PJI). In certain clinical situations, such as discontinued implant systems, replacement polyethylene liners may be unavailable, necessitating more complex revision procedures. This study evaluated the efficacy of a low-cost ultrasonic bath sonicator for biofilm removal and bacterial reduction on polyethylene surfaces, with and without povidone–iodine. Methods: An exploratory in vitro experimental study was performed using twenty ultra-high-molecular-weight polyethylene (UHMWPE) coupons (1 × 1 cm, 3 mm thickness) allocated into five groups (n = 4/group): negative control, positive control, povidone–iodine only (PVP-I), sonication only (SON), and sonication with povidone–iodine (SON+PVP-I). Biofilm formation using Staphylococcus epidermidis was established through an optimized protocol developed from sequential pilot experiments. Residual biofilm was quantified using optical density at 600 nm (OD600), while viable bacterial burden was determined by colony-forming unit counts per milliliter (CFU/mL). Results: SON demonstrated the lowest residual biofilm burden (OD600 = 0.194), whereas no culturable bacteria were detected above the assay detection limit (<10 CFU/mL) in the SON+PVP-I group. SON alone resulted in the greatest reduction in residual biofilm biomass, while the addition of PVP-I was associated with reduced recovery of culturable bacteria. Conclusions: A low-cost ultrasonic bath sonicator effectively reduced residual biofilm biomass on polyethylene surfaces, while no culturable bacteria were detected following combined sonication and povidone–iodine treatment. These findings suggest that sonication and chemical disinfection may provide complementary roles in polyethylene decontamination strategies. However, because of the exploratory study design and non-equivalent bacterial recovery procedures across treatment groups, the bacterial recovery findings should be interpreted cautiously. Further studies are required before clinical application.

Antibiotics doi: 10.3390/antibiotics15080808

Authors: Maarten Lambert Chloé Corrie Hans Smit Katja Taxis Lisa Pont

Background: Australia has experienced high rates of antibiotic shortages, yet little is known about how Australian pharmacists experience and manage them. This study aimed to explore Australian community and hospital pharmacists’ experiences of antibiotic shortages, including their perceived clinical impact and approaches to managing shortages. Methods: An exploratory qualitative study using semi-structured interviews was conducted with Australian community and hospital pharmacists between May and August 2024. Participants were recruited via convenience sampling through professional networks and social media. Interviews were audio-recorded, transcribed verbatim, and analysed thematically using an inductive–deductive approach, with coding performed independently by two researchers. Results: Fifteen interviews were conducted before data saturation was reached. Sixty percent of participants were female, 67% worked in hospital settings, and pharmacists were located across five Australian states. Four overarching themes were identified: factors driving shortages, communication and collaboration, impact of shortages, and mitigation and prevention. Pharmacists described antibiotic shortages as frequent and unpredictable, attributing them to supply-chain vulnerabilities, limited manufacturing, and market competition. Shortages were reported to affect treatment choice, contribute to antimicrobial resistance concerns, increase pharmacist workload and stress, and negatively affect patients. Pharmacists used strategies such as stock management, collaboration, and importing alternatives, but felt that lasting solutions required coordinated national action. Conclusions: Australian pharmacists perceive antibiotic shortages as a complex challenge affecting patient care, antimicrobial stewardship, workload, and healthcare efficiency. While pharmacists have developed reactive strategies to manage shortages, stronger communication, collaboration, and national policy coordination, alongside improved supply-chain resilience, are needed to reduce the impact of future shortages.

Antibiotics doi: 10.3390/antibiotics15080807

Authors: Morana Magaš Bojana Mohar Vitezić Kata Ivanišević Maja Abram

Background/Objectives: Hospital water and wastewater environments are potential reservoirs for antimicrobial resistance genes (ARGs), particularly in high-risk clinical units. This study examined site-specific ARG DNA detection patterns in wet and waste-associated sites of a haematology ward and an intensive care unit (ICU) to identify environmental hotspots that may inform targeted infection prevention and control (IPC) interventions. Methods: A descriptive point-prevalence environmental study was conducted in May 2025 in a tertiary acute-care hospital. Samples were collected after routine cleaning and during usual clinical activity. Real-time PCR assays were used to detect blaTEM, blaSHV, blaCTX-M, blaOXA-48-like, blaVIM, blaNDM, blaIMP, blaKPC, and mcr-1. Analytical summaries included samples meeting predefined quality-control criteria. Ct signals were categorised descriptively; Ct values ≤ 40 were considered positive, with Ct values 36–40 classified as very low molecular signals, whereas Ct values > 40 were considered negative. Results: The analytical dataset comprised 60 samples: 31 from haematology and 29 from ICU. At least one included ARG signal was detected in 40/60 samples (66.7%), including 23/31 haematology samples (74.2%) and 17/29 ICU samples (58.6%), blaTEM (51.7%) and blaSHV (43.3%) were most frequent. Sink drains showed the broadest multi-gene profiles, especially combinations of ESBL- and carbapenemase-associated genes. Haematology showed broader carbapenemase-associated diversity, whereas ICU detections clustered in patient-room sink drains and sluice-room wastewater infrastructure. Conclusions: ARG DNA signals were unevenly distributed, with sink drains representing most frequently positive sampling sites for multi-gene detection. This molecular, single-timepoint environmental study does not demonstrate viable organisms, colonisation, or transmission; however, point-prevalence environmental assessments may identify wet and wastewater-associated hotspots that can inform targeted IPC interventions against the spread of multidrug-resistant microorganisms.

Antibiotics doi: 10.3390/antibiotics15080806

Authors: Martin Novotny Daniela Javorska Annamaria Toporova Nicolas Kardos Katarina Curova

Backgroud/Objectives: Clostridioides difficile infection (CDI) remains a major problem for healthcare facilities. The COVID-19 pandemic was associated with increased hospitalizations, frequent antibiotic use, and changes in infection prevention and control regulations, which may have influenced the epidemiological and clinical characteristics of CDI. The aim of this study was to evaluate whether a history of COVID-19 was associated with clinical characteristics, treatment exposure, recurrence, ribotype distribution, and mortality in patients with CDI. Methods: This retrospective study included patients diagnosed with CDI during the period 2020–2022 at the Louis Pasteur University Hospital in Košice, Slovakia. We analyzed 324 patients, of whom 172 (53.1%) had no history of COVID-19 and 144 (44.4%) had previous or concurrent COVID-19, defined as documented infection within three months before or at the time of CDI diagnosis. Eight patients (2.5%) developed COVID-19 after CDI diagnosis and were excluded from the comparative analyses. Results: Patients with previous or concurrent COVID-19 had higher exposure to PPIs (88.2% vs. 73.3%; p = 0.0009), corticosteroids (70.1% vs. 26.2%; p < 0.0001), cephalosporins (72.9% vs. 61.6%; p = 0.034) and macrolides (37.5% vs. 20.3%; p = 0.0007). Overall antibiotic exposure was high (93.8%) and did not differ significantly between patients with previous or concurrent COVID-19 and those without COVID-19 (93.8% vs. 94.2%; p = 0.871). Previous or concurrent COVID-19 was not associated with increased 90-day mortality after CDI (42.4% vs. 43.6%; p = 0.824). Conclusions: Our findings suggest that a history of COVID-19 was associated with the risk factor profile, recurrence rate and the molecular epidemiology of CDI, rather than with a more severe clinical course or worse prognosis.

Antibiotics doi: 10.3390/antibiotics15080805

Authors: Plamen Bekyarov Momchil Lambev Silviya Mihaylova

Background/Objectives: Monitoring antibiotic consumption is a central component of antimicrobial stewardship because it provides a standardized way to identify changing prescribing patterns, detect potentially excessive use, and support targeted interventions. The WHO ATC/DDD framework is widely used for hospital-based utilization studies and enables comparison across wards and time periods. Methods: This single-center observational study used antibiotic data obtained from the hospital pharmacy to compare predicted and observed consumption for 2024 and 2025. Antibiotic use was expressed as DDD per 100 bed-days and analyzed for penicillins, cephalosporins, fluoroquinolones, tetracyclines, nitroimidazole derivatives, lincosamides, and aminoglycosides. The aim of this study was to quantify antibiotic consumption using ATC/DDD methodology, evaluate how closely modeled predictions matched observed values in 2024 and 2025, and identify the antibiotic classes and wards most relevant for stewardship action. Results: Cephalosporins showed the closest agreement between forecasted and observed consumption, with absolute percentage errors of 2.16% in 2024 and 4.61% in 2025 and a two-year mean absolute percentage error of 3.39%. Consumption of fluoroquinolones exceeded predictions, particularly in gynecology, while use of nitroimidazole derivatives was lower than expected, especially in 2025. Use of tetracyclines was absent in both years, and aminoglycosides and lincosamides contributed only minimally to total consumption. Ward-level analysis showed that intensive care accounted for the highest cephalosporin exposure, whereas the largest deviations from forecast were observed in gynecology and high-risk pregnancy wards. Conclusions: Predictive modeling captured antibiotic use reasonably well for some antibiotic classes but showed lower accuracy for others. Forecast accuracy varied across antibiotic groups, indicating that the model performed differently depending on the observed consumption pattern. However, the underlying reasons for these differences cannot be determined from the present aggregated consumption analysis.

Antibiotics doi: 10.3390/antibiotics15080804

Authors: Lenin García Gutiérrez Alfonso Méndez-Tenorio Mario Ángel López-Luis Sandra Alejandra Ávila-Huerta Gloria León-Ávila Santiago R. Castaño-Valencia Gabriela Ibáñez-Cervantes

The resistome, defined as the complete set of antibiotic resistance genes (ARGs) present in the microbiota of a given environment, is a critical component for understanding the evolutionary dynamics of antimicrobial resistance (AMR) and its impact on human, animal, and environmental health. This review summarizes current methods and technological advances and offers a forward-looking perspective on resistome research. A systematic literature search was conducted. References on short-read and long-read sequencing, amplicon sequencing, shotgun metagenomics, and multi-omics integration were included, as were bioinformatics tools for the detection, quantification, and annotation of ARGs. The results indicate that next-generation sequencing (NGS) technologies have significantly improved the characterization of ARGs across ecosystems, enabling high-resolution microbial profiling and the discovery of new variants. Furthermore, integrating multi-omics approaches with computational tools improves data accuracy, reduces analysis and reporting times, and facilitates the development of predictive models. However, significant challenges remain, which will be key to strengthening epidemiological surveillance under the One Health approach.

Antibiotics doi: 10.3390/antibiotics15080803

Authors: Junjie Wang Yingxia Hou Xue Li Ran Zhao Xiaoquan Mu Yifan Liu Chengcheng Huang Frank Fu Fengxia Yang

In the original publication [...]

Antibiotics doi: 10.3390/antibiotics15080802

Authors: Nadia Jaidane Thierry Naas Souad Fayad Pierre Châtre Wejdene Mansour Aymen Bouaziz Pauline François Laetitia Du Fraysseix Bogdan I. Iorga Nahed A. Al Laham Lamia Tilouche Farouk Barguellil Marisa Haenni

Background/Objectives: The emergence of plasmid-mediated mcr genes has enabled horizontal dissemination of resistance to colistin, a last-resort antibiotic against multidrug-resistant Enterobacterales. In Tunisia, genomic data on mcr-positive Escherichia coli are still limited. This study reports the genomic characterization of human clinical mcr-positive E. coli isolates from the Military Hospital of Tunis. Methods: Between August 2023 and March 2025, seven E. coli isolates with low-level colistin-resistance (MIC = 4–8 µg/mL) were collected from six patients. They were characterized by antibiotic susceptibility testing and WGS to determine resistome, MLST, genetic relatedness, and plasmid content. Results: The E. coli isolates belonged to diverse sequence types (STs), except for two isolates collected from the same patient 2.5 months apart, which were highly related. Overall, this pattern is consistent with a polyclonal spread. The mcr-1.1 gene was located on IncI2 (n = 5) or IncX4 (n = 2) plasmids, which exhibited high similarity both among themselves and in comparison with plasmids previously reported in human and livestock isolates. Most isolates were multidrug-resistant, harboring acquired resistance genes to multiple antibiotic classes, and chromosomal mutations conferring fluoroquinolone resistance. Three isolates additionally carried chromosomal insertions of the blaCTX-M-55 gene. Resistance to cefiderocol was observed in one isolate and was associated with CirA and Fiu truncation. Conclusions: These findings highlight ongoing dissemination of mcr-1.1-positive E. coli isolates in Tunisia, primarily driven by plasmid transfer. Continuous genomic surveillance and One Health-oriented antibiotic stewardship are essential to limit the spread of colistin-resistance and the emergence of resistance to newer agents such as cefiderocol.

Antibiotics doi: 10.3390/antibiotics15080801

Authors: Alejandro Madrid Valentina Silva Katy Díaz Evelyn Muñoz David Cabezas Karel Mena-Ulecia Iván Montenegro Carmina Sirignano Enrique Werner Ximena Besoain

Background/Objectives: Postharvest fungal decay caused by Botrytis cinerea and Monilinia fructicola poses major threats to global fruit security. Driven by the need for sustainable crop protection agents, this work presents the systematic synthesis, biological evaluation, and computational modeling of a comprehensive 33-compound library of 3-acyl-bromoindole regioisomers (series 4a–k, 5a–k, and 6a–k) to establish clear structure–activity relationship (SAR) design rules. Methods: The regioisomeric library was assembled via a microwave-assisted catalytic protocol in an ionic liquid, expanding the known chemical space with seven newly synthesized 4-bromoindole derivatives (4d–f, 4h–k). Primary in vitro data were modeled using Hansch QSAR and Principal Component Analysis (PCA). Postharvest in vivo efficacy was evaluated on fresh ‘Lapins’ sweet cherries inoculated with M. fructicola. Molecular docking and 100 ns molecular dynamics (MD) simulations were performed against succinate dehydrogenase (SDH) and M. fructicola catalase 2 (MfCat2). Results: In vitro screening demonstrated marked target selectivity: parent core 4 displayed high mycelial suppression against M. fructicola (EC50 = 7.05 µg/mL), whereas C3-acylation with a four-carbon linear chain (4c) achieved optimal broad-spectrum dual action (98% and 86% spore germination inhibition). In vivo cherry bioassays proved that bromoindoles 4, 6a, and 6d significantly suppressed Brown Rot severity to 44–47% (a 20–27% reduction vs. untreated control). Docking and MD trajectories confirmed stable multi-target binding within SDH and MfCat2 active sites (RMSD < 2.0 Å). Conclusions: Bromine regiochemistry dictates pathogen selectivity and life-stage targeting. The novel 4-bromoindole derivatives and multi-target profile establish these scaffolds as promising leads for postharvest crop protection.

Antibiotics doi: 10.3390/antibiotics15080800

Authors: Otília Vágó Krisztián Laczi László Orosz Georgina Horváth Károly Péter Sárvári Diána Szabó Regina Csordás Zoltán Szabó Caleb Ardizzone Titanilla Szögi Katalin Burián Dezső Péter Virok

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major therapeutic challenge due to its extensive antibiotic resistance. This study examined how bacteriophage exposure affects the genome, proteome and antibiotic susceptibility of a clinical MRSA isolate. Methods: The MRSA isolate was exposed to the PYOFAG bacteriophage cocktail, and small colony variants (SCVs) of the surviving bacteria were compared with the untreated parental strain by phenotypic testing, whole-genome sequencing, comparative proteomics, and transmission electron microscopy. Results: Phage exposure induced marked remodeling in MRSA, including altered growth, colony morphology, and increased susceptibility to various antibiotics, especially β-lactams and aminoglycosides. Genomic analysis identified multiple mutations and the loss of two genomic regions, including changes in tarS, a gene linked to wall teichoic acid glycosylation, phage adsorption, and β-lactam resistance. Proteomic analysis revealed broad changes in metabolic, stress-response, and cell-envelope-associated pathways. Transmission electron microscopy showed a significant reduction in cell wall thickness after phage treatment. Conclusions: Bacteriophage exposure drives phenotypic and molecular adaptation in MRSA and may create evolutionary trade-offs that weaken resistance mechanisms. These findings support the potential of bacteriophages as both direct antibacterial agents and modulators of antibiotic susceptibility.

Antibiotics doi: 10.3390/antibiotics15080799

Authors: Asiya Nazir Prathap Bava Arif Hussain Touseef Amna Mohammad Chand Jamali Afsheen Raza Jayanthi Barasarathi

Chronic diseases and aging-related disorders are driven by interconnected mechanisms, including oxidative stress, low-grade inflammation, metabolic dysregulation, and glycation. Targeting these overlapping pathways remains a major challenge for conventional single-target therapeutics. In this context, endophytic fungi have emerged as a promising source of bioactive metabolites with multi-target pharmacological potential. This review provides a mechanistic overview of endophyte-derived metabolites, including alkaloids, terpenoids, polyketides, and phenolic compounds, with a focus on their ability to modulate key signaling pathways such as NF-κB, Nrf2, PI3K/Akt, AMPK, and the AGE–RAGE axis. Evidence from experimental studies suggests that these metabolites exhibit anticancer, anti-inflammatory, antioxidant, and metabolic regulatory effects through coordinated modulation of cellular signaling networks. Several endophyte-derived metabolites also possess antimicrobial activity against bacterial and fungal pathogens and may represent a promising source of novel anti-infective agents. Their ability to modulate host immune responses and microbial-associated signaling pathways further highlights their relevance for antimicrobial discovery and microbiome-based therapeutic strategies. Particular attention is given to pathway-level convergence in chronic diseases, including cancer, diabetes, and inflammation-associated disorders, as well as their relevance to aging and health span. The pharmacological potential of these compounds is discussed alongside key limitations, including issues related to bioavailability, reproducibility, and translation into clinical applications. Overall, endophytic fungal metabolites represent a structurally diverse and mechanistically rich resource for the development of multi-target therapeutic strategies. Future integration of metabolomics, genome mining, and advanced disease models will be essential to bridge the gap between experimental findings and clinical application.

Antibiotics doi: 10.3390/antibiotics15080798

Authors: Felix Werneburg Juliane Beschauner Laura Isabell Werneburg Alexander Zeh Natalia Gutteck Karl-Stefan Delank

Background/Objectives: In methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, randomized evidence indicates comparable efficacy of cefazolin and antistaphylococcal penicillins with less nephrotoxicity; whether this extends to bone and joint infection (BJI) is unknown. We compared both agents in MSSA BJI. Methods: We retrospectively analyzed all adults at a single center with culture-confirmed, monomicrobial MSSA BJI treated with inpatient intravenous cefazolin or flucloxacillin monotherapy (January 2022–January 2025)—a selected population excluding rifampicin-based combination therapy, outpatient parenteral therapy, polymicrobial infection, and concurrent bacteremia/endocarditis. Endpoints, a priori exploratory, comprised effectiveness (mortality; clinical success) and renal safety (peri-treatment acute kidney injury [AKI]); the AKI comparison was additionally adjusted for confounders. Results: Among 110 patients (64 cefazolin, 46 flucloxacillin), baseline characteristics were comparable except for more frequent nephrotoxic co-medication with cefazolin (56.2% vs. 26.1%; p = 0.002). No statistically significant differences in effectiveness were detected: 30-day clinical success was 89.1% versus 80.4% (ARD 8.6 percentage points, 95% CI −4.7 to +23.3) and one-year clinical success 79.7% versus 65.2% (ARD 14.5, 95% CI −2.2 to +31.0); these imprecise estimates are compatible with effects ranging from no difference to a clinically relevant benefit of cefazolin. Peri-treatment AKI occurred in 19.0% versus 30.4% (ARD 11.4, 95% CI −4.7 to +27.7; adjusted odds ratio 2.27, 95% CI 0.87–5.91); the difference was confined to stage 1. Conclusions: In this selected inpatient monotherapy cohort, cefazolin was associated with numerically fewer AKI events, without statistically significant differences in any comparison. These exploratory findings support cefazolin as a rational targeted option for MSSA BJI, pending prospective confirmation.

Antibiotics doi: 10.3390/antibiotics15080797

Authors: Isabel Gloor Senta Faulhaber Madlaina Schöni Felix W. A. Waibel Javier Aragón-Sánchez Benjamin A. Lipsky Mazda Farshad Ilker Uçkay

Background/Objectives: When presented with chronic osteomyelitis of the adult diabetic foot (DFO), clinicians, patients and their families have two options: rational use of antibiotics or direct surgery. Methods: We conducted a scientific literature review of 118 different articles and administered questionnaires to eighty DFO international and Swiss experts who have already published on the specific choice between a first-line conservative, antibiotic-based therapy versus direct surgery for DFO. Results: According to this specific literature and the ranking of clinical importance, the presence of ischemia came first (91% consensus favoring surgery), followed by the presence of gangrene (94% consensus), the perceived frailty of the patient (80%), sepsis (83%), and major soft tissue loss (81% consensus). Generally, for more than 90% of all experts, gangrene, bone exposed to air, destroyed bone, and hindfoot DFO motivated for direct surgery. We obtained twenty-four questionnaires from colleagues who we addressed as experts. Their opinions aligned with the literature. Compared to the international experts, Swiss clinicians were less hesitant to amputate in case of long-lasting foot ulcers, soft tissue loss, sepsis or patients with a history of low compliance. Combining literature reviews and questionnaires, large necrotic areas and destructed bone may predict direct surgery. Alternatively, clinicians can choose a first-line antibiotic therapy with minimal soft tissue debridement, off-loading and professional wound care. Conclusions: There is no universal consensus. The decision between antibiotics and surgery remains individualized, while severe ischemia, destroyed bone and large tissue loss are predictors of direct surgery; their absence favors antibiotic treatment.

Antibiotics doi: 10.3390/antibiotics15080796

Authors: Francesca Gavaruzzi Giordano Madeddu Elena Delfina Ricci Matteo Faltoni Alessandra Bandera Francesca Colucci Andrea De Vito Angelo Maccaro Paolo Maggi Stefania Piconi Giovanni Cenderello Marco Merli Leonardo Luzi Giuseppe Vittorio De Socio Silvia Mascolo Linda Bussini Goffredo Angioni Paolo Bonfanti Stefania Cicalini

Background/Objectives: Multidrug-resistant (MDR) Pseudomonas aeruginosa pneumonia and bloodstream infections (BSI) remain challenging because of their severity, limited therapeutic options, and high mortality, despite the availability of novel β-lactam-based agents. We aimed to describe real-world treatment patterns, characteristics associated with treatment and infusion-strategy selection, and the association of these strategies with clinical outcomes in the retrospective, multicentre Italian SUSANA cohort, an observational study evaluating the use, safety, and outcomes of novel antibiotics in hospitalized patients. Methods: Adults with MDR P. aeruginosa pneumonia and/or BSI treated with novel antipseudomonal agents for ≥72 h were included. Treatment was classified as monotherapy or combination therapy, and infusion strategy as prolonged/continuous or standard. The primary outcome was 28-day all-cause mortality. A multivariable Cox regression analysis was performed. Results: Among 173 patients, pneumonia was the most frequent clinical presentation (74.0%). Monotherapy was used in 111 patients (64.2%), combination therapy in 62 (35.8%), and prolonged/continuous infusion in 120 (69.4%). Ceftolozane–tazobactam (CT) (64.7%) and ceftazidime–avibactam (CZA) (20.2%) were the most prescribed agents. Patients receiving prolonged/continuous infusion, regardless of monotherapy or combination therapy, had greater baseline clinical severity than those receiving standard infusion. Overall, 28-day mortality was 23.7%. In the multivariable analysis, neither combination therapy nor prolonged/continuous infusion was independently associated with reduced mortality, whereas septic shock (aHR 4.10, 95% CI 1.01–16.66) and ICU admission (aHR 2.53, 95% CI 1.06–6.08) increased mortality risk. Conclusions: In this multicentre real-world cohort, treatment strategies for MDR P. aeruginosa pneumonia and BSI appeared to be influenced by baseline severity and patient-related factors. Mortality was more strongly associated with markers of severe illness, including ICU admission and septic shock. No independent association with improved survival was observed for combination therapy compared with monotherapy or for prolonged/continuous compared with standard infusion. These findings highlight the prognostic role of baseline clinical severity and support the need for prospective, randomized, pharmacokinetic/pharmacodynamic (PK/PD)-guided studies to identify patients who may benefit from these strategies.

Antibiotics doi: 10.3390/antibiotics15080795

Authors: Halil Kurt

Background/Objectives: Antimicrobial resistance in microbial communities is a global health concern that leads to millions of deaths each year. Many bacterial pathogens have resistance to multiple antibiotics. Domestic wastewater treatment facilities are reservoirs for antibiotic-resistant bacteria and resistance genes. Wastewater-based epidemiology surveillance is crucial for monitoring antibiotic resistance genes (ARGs). Türkiye has one of the highest levels of antibiotic resistance with a lack of research on resistomes. This study is a focused reanalysis of publicly available wastewater metagenomes from Türkiye, comparing them to global and other country’s results. Methods: Ten metagenomic data of wastewater treatment from Türkiye were downloaded from NCBI-SRA database. Metagenome assemblies were performed and high-quality metagenome-assembled genomes (HQ-MAGs) were included in the study. Taxonomic annotations and antibiotic resistance profiles were identified in both the metagenome assemblies and HQ-MAGs. Results: A total of 401 different ARGs in 25 antibiotic classes have been identified, including Mcr (including mcr-1, mcr-2, mcr-3 and mcr-5 variants) and optrA. The vanR two-component regulatory system genes for controlling vancomycin antibiotic resistance were one of the most dominant along with other vancomycin resistance genes such as vanA and vanB. A total of 115 HQ-MAGs were obtained with at least eight ARGs. The HQ-MAG with the highest number of resistance genes (58) was found to belong to E. coli. The most frequently encountered resistance genes in HQ-MAGs were the multidrug ABC transporter, vanR, bacA and patA which confer resistance to multidrug, glycopeptide, bacitracin and fluoroquinolone antibiotic groups, respectively. Conclusions: To effectively address the problems of antibiotic resistance outbreaks, comparable AMR surveillance at national and global levels is required for the identification and prioritization of ARGs and resistance genes. This is the first report conducted in Türkiye.

Antibiotics doi: 10.3390/antibiotics15080794

Authors: Evandro Gomes da Silva Júnior Ingrid Gonçalves Pereira Dantas Matheus dos Santos Lourenço João Arthur de Oliveira Borges Isaac Moura Araújo José Thyálisson da Costa Silva Ana Carolina Ferreira Araújo Priscilla Ramos Freitas Alexandre Janaína Esmeraldo Rocha Maria Karollyna do Nascimento Silva Leandro Igor José dos Santos Nascimento João Xavier de Araújo-Júnior Edeildo Ferreira da Silva-Júnior Thiago Mendonça de Aquino Francisco Jaime Bezerra Mendonça Junior Emmanuel Silva Marinho Hélcio Silva dos Santos António Raposo Henrique Douglas Melo Coutinho

Background/Objectives: Synthetic compounds, particularly thiadiazine derivatives with antibacterial properties, have emerged as promising candidates in addressing the growing challenge of bacterial multidrug resistance. Thiadiazine derivatives are six-membered heterocyclic compounds containing two nitrogen atoms and one sulfur atom, exhibiting diverse medical and pharmacological activities. This study aimed to evaluate the potentiating activity of thiadiazine derivatives against multidrug-resistant bacteria. Methods: ADMET (absorption, distribution, metabolism, excretion, and toxicity) assays were performed to assess similarity with more than 370,000 three-dimensional structures of bioactive compounds. The multidrug-resistant bacterial strains Staphylococcus aureus 10 and Pseudomonas aeruginosa 24 were used to investigate both the direct antibacterial activity and the antibiotic-modifying activity of thiadiazine derivatives. Results: The thiadiazine analogs did not exhibit direct antibacterial activity, presenting a minimum inhibitory concentration of 1024 μg/mL. However, they demonstrated a significant antibiotic-modifying effect, potentiating the activity of conventional antibiotics, particularly norfloxacin, against the tested strains. In silico analyses indicated that the analogs predominantly exhibited affinity for G protein-coupled receptors and possessed physicochemical characteristics compatible with potential drug candidates. Conclusions: Although the evaluated thiadiazine derivatives lacked direct antibacterial activity, they significantly enhanced the efficacy of antibiotics against multidrug-resistant bacteria. Combined with their favourable in silico pharmacokinetic and physicochemical profiles, these findings suggest that thiadiazine derivatives may represent promising antibiotic adjuvants for combating multidrug-resistant bacterial infections.

Antibiotics doi: 10.3390/antibiotics15080793

Authors: Teshome Belachew Eshete Shyam Kumar Mishra Naresh Kumar Mark Willcox

Background/objectives: Multidrug-resistant and extensively drug-resistant Gram-negative pathogens are a major cause of severe ocular infections, yet treatment options are increasingly compromised by escalating antibiotic resistance. Membrane-active peptides (colistin and Mel4) offer the potential to restore antibiotic susceptibility. This study investigated the efficacy of peptide- and colistin-based adjuvant strategies in enhancing the activity of conventional antibiotics. Methods: Antibacterial activity was assessed using MIC/MBC testing, checkerboard assays, time–kill kinetics, and biofilm disruption studies, supported by confocal microscopy. Toxicity was assessed using L929 fibroblasts and red blood cells. A mechanistic study was performed with a membrane permeability assay. Results: Clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae showed high resistance to six antibiotic classes (up to MICs > 2048 mg/L), while polymyxin B and colistin remained active (MIC ≤ 2 mg/L). Mel4 displayed variable activity (MIC 7.8–500 mg/L). A total of 102 antibiotic–antibiotic, antibiotic–Mel4, and antibiotic–colistin combination tests were performed, of which 45 showed synergistic interactions. Combining traditional antimicrobials with either colistin or Mel4 restored antibiotic susceptibility, reducing MICs up to 2048-fold. Aminoglycoside–colistin combinations significantly lowered MICs, especially against K. pneumoniae. Time–kill assays confirmed rapid bactericidal effects (>3 log10 reduction in 3 h). The ciprofloxacin–Mel4 combination effectively disrupted biofilms (62–92%) with low toxicity and high cell viability. Membrane permeability assays showed that ciprofloxacin has limited activity, whereas both Mel4 and the ciprofloxacin–Mel4 combination showed enhanced activity across concentration gradients and over time. Conclusions: Membrane-active antimicrobials, colistin and Mel4, enhance conventional antibiotics against multidrug- and extensively drug-resistant ocular Gram-negative pathogens by restoring susceptibility, accelerating bactericidal effects, and disrupting biofilms. With low toxicity, these combinations represent promising therapeutic strategies for severe multidrug-resistant ocular infections.

Antibiotics doi: 10.3390/antibiotics15080792

Authors: Daniele Mengato Elisabetta Conte Giacomo Berti Roberto Brunoro Sara Lo Menzo Margherita Boschetto Elisabetta Lolli Cristina Contessa Maria Mazzitelli Stefano Carraro Ignazio Castagliuolo Daniele Donà Tiziano Martello Annamaria Cattelan Francesca Venturini Vincenzo Baldo

Background: Education is a cornerstone of antimicrobial stewardship, yet evidence on its impact across the entire hospital workforce remains limited. This study aimed to assess knowledge, attitudes, and practices regarding antibiotic use and AMR among workers employed at an Italian tertiary-care hospital, and to explore the association between questionnaire performance and participation in antibiotic-related training activities. Methods: A repeated cross-sectional survey was conducted in 2024 and 2025 at a large Italian tertiary-care hospital (Padua, Northern Italy). A structured questionnaire adapted from an ECDC-validated instrument was distributed through the institutional email system to all hospital employees, including healthcare professionals and non-healthcare workers. The questionnaire investigated demographic and professional characteristics, participation in antibiotic-related training activities, and knowledge, attitudes and practices regarding antibiotic use and AMR. Questionnaire performance was assessed using both a binary scoring system and a partial credit scoring system. Multivariable linear regression analysis was performed to identify independent predictors of performance. Results: A total of 3193 participants completed the survey (1928 and 1265 in 2024 and in 2025, respectively). The mean (SD) total score was 5.75 ± 2.25 out of 15 points, with the lowest performance observed in the practice domain. No significant differences were found between the two survey years (2024 and 2025). Participants who attended antibiotic-related training performed significantly better than untrained participants (6.23 vs. 5.01; p < 0.001), with prior training emerging as the strongest independent predictor of questionnaire performance (β = 4.8; p < 0.001). Younger age was associated with better performance, while longer seniority (>30 years) was associated with lower scores. Among healthcare professionals, modestly higher scores were observed in 2025 compared with 2024, particularly in the practice domain. Conclusions: Persistent opportunities for improvement remain in AMR-related knowledge, attitudes and particularly practices across the hospital workforce. Participation in antibiotic-related training activities was associated with improved questionnaire performance, supporting the role of structured and continuous educational interventions within hospital AMS programs. Repeated institutional educational initiatives may contribute to strengthening stewardship-related awareness and practices over time.

Antibiotics doi: 10.3390/antibiotics15080791

Authors: Nicola Logoluso Antonio Pellegrini Delia Romano’ Valerio Pascale Virginia Suardi Carlo Luca Romano’

Background/Objectives: One-stage revision has become an increasingly accepted strategy for the treatment of chronic periprosthetic joint infection (PJI) of the knee in carefully selected patients. The use of a resorbable defensive antibacterial coating (DAC® hydrogel) represents an attractive adjunct to reduce bacterial adhesion and biofilm formation on revision implants. However, evidence regarding its long-term performance is limited. Methods: We retrospectively reviewed 52 consecutive patients undergoing one-stage revision for knee PJI or an infected spacer between 2014 and 2023 using a standardized surgical and antimicrobial protocol including local application of DAC hydrogel loaded with vancomycin alone or vancomycin plus meropenem. Clinical, microbiological, and radiographic outcomes were evaluated. Infection-free survival was assessed using Kaplan–Meier analysis. Results: Forty-eight patients underwent revision for PJI and four for septic spacers. Four patients were unavailable for final evaluation. At follow-up of 7.7 ± 2.9 years (range 2.5–12.2 years), 47 of 48 clinically evaluable patients remained infection-free, corresponding to a crude infection-free proportion of 97.9%. Kaplan–Meier infection-free survivorship at 12 years was 98.1% (95% CI, 87.1–99.7%). Mean Knee Society Score improved from 47.7 to 84.4 points, while functional KSS improved from 41.9 to 82.9 points. Pain at rest decreased from 6.9 to 0.7 and pain during movement from 8.1 to 1.6 on the visual analogue scale. Conclusions: This series represents, to our knowledge, the longest reported follow-up of one-stage knee revision using the DAC hydrogel coating. The favorable outcomes observed with this hybrid fixation strategy support further investigation of this approach in larger, long-term, comparative multicenter studies.

Antibiotics doi: 10.3390/antibiotics15080790

Authors: Katarzyna Kuszewska Karolina Klesiewicz Paulina Leśniak Agnieszka Chmielarczyk

Carbapenem-resistant Enterobacterales (CRE) represent one of the most important antimicrobial resistance threats in Europe, particularly in healthcare settings. Their increasing epidemiological significance is driven by the dissemination of carbapenem-resistant Klebsiella pneumoniae, the emergence of high-risk clones, horizontal transfer of mobile genetic elements, and the convergence of antimicrobial resistance and hypervirulence. Despite the growing epidemiological importance of CRE, surveillance and preparedness remain heterogeneous across European countries, creating a need for a continent-wide assessment of existing surveillance systems, implementation gaps, and emerging challenges. This narrative review summarizes the current epidemiology of CRE in Europe and critically discusses the role of European and global surveillance systems in monitoring their spread. Particular attention is given to the European Antimicrobial Resistance Surveillance Network (EARS-Net), European Antimicrobial Resistance Genes Surveillance Network (EURGen-Net), Healthcare-Associated Infections Surveillance Network (HAI-Net), European Surveillance of Antimicrobial Consumption Network (ESAC-Net), Point Prevalence Survey (PPS), EpiPulse/TESSy, the Central Asian and European Surveillance of Antimicrobial Resistance (CAESAR) programme, and the World Health Organization Global Antimicrobial Resistance and Use Surveillance System (WHO GLASS). Although Europe has well-established surveillance infrastructures, substantial heterogeneity persists among countries with respect to microbiological and genomic diagnostic capacity, reporting practices, infection prevention and control (IPC), antimicrobial stewardship, healthcare infrastructure, and implementation of national policies. These differences limit data comparability and may delay the detection of colonization, outbreaks, and cross-border transmission. The available evidence indicates that effective CRE control requires integrated surveillance combining microbiological, genomic, epidemiological, and antimicrobial consumption data within a coordinated One Health framework. Further expansion of genomic surveillance, harmonization of surveillance indicators, strengthening of healthcare system preparedness, and wider implementation of antimicrobial stewardship programmes are identified as priority areas for improve CRE prevention and control across Europe. By integrating evidence across surveillance, diagnostics, infection prevention and control, and antimicrobial stewardship, this review highlights key gaps in current European approaches and provides a framework for prioritizing future surveillance and preparedness efforts.

Antibiotics doi: 10.3390/antibiotics15080789

Authors: Junjie Yu Shangqian Ning Yuchang Di Hongbo Feng Xiaoying Li Kun Wang Rongxuan Wang Menghan Ma Sicheng Pan Chenglin Jiang Yi Jiang Tom Hsiang Jiazhen Chen Wenhong Zhang Lixin Zhang Xueting Liu Xuelian Zhang Guoliang Zhu

Background: Endophytic fungi are important sources of structurally diverse antimicrobial natural products, but their potential to yield antitubercular scaffolds remains insufficiently explored. This study aimed to uncover new decalin polyketides from the endophytic fungus, Trichoderma longicollum 17007, and to evaluate their biological activity. Methods: Feature-based molecular networking (FBMN) analysis of the ethyl acetate extract from rice-based cultures was performed using trichoharzin as a seed compound to guide targeted isolation. Structures were established by HRMS, 1D and 2D NMR spectroscopy, NOESY analysis, and quantum chemical ECD calculations. Cytotoxicity and activity against the avirulent Mycobacterium tuberculosis H37Ra strain were evaluated, and genome mining was performed to identify a candidate biosynthetic gene cluster. Results: Six new decalin polyketides, tandyukisins K–P (1–6), and two known analogues, trichoharzin (7) and tandyukisin J (8), were isolated. Compounds 1–6 are C1-O-acetylated analogues bearing a 3-methylpentenedioate-type acyl side chain. Comparative 13C NMR analysis revealed empirical features useful for assigning side-chain attachment site, double-bond position and geometry. Tandyukisins K (1) and P (6) showed moderate activity against the avirulent M. tuberculosis H37Ra strain, with a MIC value of 12.5 μg/mL. Conclusions: These findings expand the structural diversity of decalin polyketides and provide the first evidence of antitubercular activity by this compound family.

Antibiotics doi: 10.3390/antibiotics15080788

Authors: Manar O. Lashkar Milap C. Nahata

Background/Objectives: Urinary tract infection (UTI) is among the most common serious bacterial infections occurring in the first 90 days of life and a leading indication for parenteral antibiotic therapy. Despite its clinical importance, no randomized trial has compared empiric antibiotic regimens, route of administration, or treatment duration specifically in this population. This review synthesizes current evidence to support rational antibiotic treatment decisions for neonates (ages 0–28 days) and young infants (ages 29–90 days), including those born prematurely. Methods: A targeted PubMed and MEDLINE search was conducted through July 2026. Studies reporting treatment outcomes, pharmacokinetic data, or antibiotic safety in infants aged 0–90 days were included. Broader pediatric data were incorporated where age-specific evidence was unavailable and are presented with explicit population caveats. Results:Escherichia coli is the predominant uropathogen across all age strata; extended-spectrum beta-lactamase (ESBL)-producing organisms account for a clinically important minority of community-acquired isolates. No comparative trial of empiric regimens existed for confirmed UTI in this age group. Shorter intravenous courses were not associated with higher 30-day recurrence rates for either nonbacteremic or bacteremic UTI after exclusion of meningitis. Intravenous-to-oral (IV-to-oral) transition with amoxicillin–clavulanate was pharmacokinetically supported and clinically safe in selected term neonates, based on indirect evidence from neonatal infection trials. Non-carbapenem therapy is appropriate for ESBL UTI without concomitant bacteremia in clinically improving infants. Exclusion of meningitis before shortening intravenous therapy or initiating oral step-down is essential, particularly in neonates ≤ 28 days of age. Conclusions: Short parenteral courses with timely transition to oral antibiotics are recommended in appropriately selected infants with UTI, consistent with antimicrobial stewardship principles and supported by observational evidence. The evidence base is limited, with key gaps in empiric regimen selection, management of premature infants, ESBL treatment in bacteremic patients, and optimal duration of parenteral and oral antibiotic therapy.

Antibiotics doi: 10.3390/antibiotics15080787

Authors: Hailey Dyce Paul Schweiger Robin Patel Stephen Johnson Isabelle Sharp William R. Schwan

Background: Staphylococcus aureus causes bloodstream and skin infections in humans. The prevalence of multidrug-resistant S. aureus strains means new antibiotics are needed. A novel antimicrobial drug named SK-03-92, a synthetic aromatic organic stilbenoid compound, kills S. aureus cells within 30 min, but an increase in both biofilm formation and persister cells occurs. SK-03-92 treatment downregulates transcription of the biofilm regulating protein regulator (brpR) gene and biofilm regulating protein sensor (brpS) gene in S. aureus. BrpR/BrpS system may be a LytTR regulatory system tied to biofilm formation, creation of persister cells, and late-stage competence in S. aureus. The aim of this study was to determine what biofilm, late-stage competence, and persister-associated genes were regulated in a brpR mutant compared to wild-type strains. Methods: In this study, involvement of BrpR in regulating other genes was assessed by comparing transcriptional changes in a brpR mutant strain to the S. aureus parent strain via RNA sequencing (RNA-Seq). Bioinformatic analysis was then performed on the RNA-Seq data to assess what biochemical pathways might be involved. Results: From these analyses, 440 genes were identified that had significant differences in transcript abundance when comparing the brpR mutant to wild-type strains. Quantitative reverse transcription polymerase chain reaction analysis confirmed bacA, icd, metE, and pdhA transcript levels were lower, whereas alr and mraY were higher in the brpR mutant versus wild-type strain. Furthermore, an enzymatic assay targeting NADH production from the pyruvate dehydrogenase complex showed lower levels in the mutant compared to wild-type strain. Conclusions: Overall, the study demonstrated several biosynthetic pathways tied to biofilm formation and late-stage competency may be regulated by BrpR and some potential leads for the mechanism of action of the SK-03-92 drug were uncovered.