Search Results (608)

Background/Objectives: The WHO has identified carbapenem-resistant Acinetobacter baumannii (CRAb) and carbapenem-producing Enterobacterales (CPE) as the “critical priority” group of multidrug-resistant (MDR) organisms for which new therapeutic strategies are urgently needed. Here, we evaluated the in vitro synergistic activity of eugenol, cinnamaldehyde, and carvacrol in combination with β-lactams, gentamicin, or colistin against MDR Gram-negative bacteria (GNB). Methods: We selected seven MDR-GNB clinical isolates including CRAb, ESBL-producing and CPE clinical isolates displaying different antimicrobial susceptibility profiles. The genomes of clinical isolates were characterized by whole-genome sequencing and synergy testing was performed with checkerboard assay. Results: Our results demonstrate that eugenol, cinnamaldehyde, and carvacrol in combination with colistin exhibited synergistic activity (FICI < 0.5) against MDR-GNB clinical isolates ranging from 37.5 to 50%, while the effect was almost indifferent in combination with different β-lactam molecules or gentamicin against 87.5–100% of MDR-GNB strains. The synergistic interaction of eugenol, cinnamaldehyde, and carvacrol with colistin induced a statistically significant reduction (p < 0.05) in the MIC values compared with the molecules tested alone. Conclusions: Our data demonstrate that this synergistic interaction was not affected by different antimicrobial resistance genes and/or different antimicrobial susceptibility profiles. In conclusion, our results suggest that eugenol, cinnamaldehyde, and carvacrol in combination with colistin represent a potential strategy for the treatment of MDR-GNB pathogens and limit their diffusion. Full article

Cosmological filaments, galaxy clusters, and galaxies are magnetized reservoirs of cosmic rays (CRs). The exchange of CRs across these structures is usually modeled assuming that they remain charged and magnetically confined. At high energies, hadronic interactions can convert CR protons to neutrons. This physics is routinely included in air-shower and ultra-high-energy (UHE) CR propagation Monte Carlo simulations used for composition studies but is rarely treated explicitly in propagation models of CR transport and exchange between magnetized reservoirs. CR neutrons are not affected by magnetic fields and can propagate ballistically over kpc-Mpc distances before decaying back into protons, with relativistic time dilation extending their effective decay length. We show how such charged–neutral switching modifies CR confinement and escape in four representative environments: a Milky Way-like galaxy, a starburst galaxy, a galaxy cluster, and a cosmological filament. By solving the transport of a confined CR proton population in each structure using a diffusion/streaming propagation approach with hadronic pp and p$\gamma$ interactions, and treating neutron production and decay as a stochastic Poisson “jump” process, we find that neutron-mediated steps can allow additional CR escape from large-scale cosmological structures at energies where charged-particle transport alone would predict strong CR confinement and attenuation in ambient radiation fields. These effects imply a qualitative shift in how ultra-high-energy CRs are transferred from embedded sources into filaments and voids once intermediate neutron propagation is considered, with consequences for the partitioning of CRs across the large-scale structure of the Universe. Full article

(1) Background: Chronic rhinosinusitis (CRS) with recurrent symptoms despite therapy raises concern for underlying antimicrobial resistance. While inflammation is central to disease pathophysiology, increasing evidence suggests that resistant bacterial populations within the sinonasal niche may contribute to treatment failure. This study aimed to characterize the molecular resistance profiles of bacterial isolates from refractory CRS patients and evaluate genotype–phenotype concordance and clinical resistance burden. (2) Methods: Our observational study includes 99 bacterial isolates obtained by endoscopically guided nasal swabs from adult CRS patients with recurrent disease. Species identification and antimicrobial susceptibility testing were performed using the VITEK^®2 system. Resistance genes were detected using multiplex-PCR. Statistical analyses included Mann–Whitney U tests for genotype–phenotype associations, Kruskal–Wallis testing across MDR categories, Spearman correlation between gene burden and clinical risk, and concordance metrics. (3) Results: Recognized sinonasal pathogens accounted for 46.5% of isolates, predominantly Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. β-lactamase genes (tem 25.3%, shv 9.1%, ctxM 8.1%) and macrolide resistance markers (ermB 20.2%) were most prevalent, while carbapenemase genes remained infrequent. Significant phenotype–genotype correlations were observed for mecA–oxacillin, sul1–TMP-SMX, KPC–meropenem, and tem–β-lactams (p < 0.01). Gene burden increased progressively across clinical risk categories (p < 0.001), with MDR/XDR isolates concentrated in patients with repeated antibiotic exposure. Molecular and phenotypic analyses demonstrated high concordance for selected gene–antibiotic pairs, supporting targeted molecular screening as an adjunct to culture-based diagnostics in refractory CRS. Full article

Background: Carbapenem-resistant Enterobacterales (CRE) are a global public health concern, with carbapenem-resistant Klebsiella pneumoniae (CR-Kp) recognised as the highest-priority pathogen. This study aimed to investigate the epidemiological features of CRE isolates throughout the COVID-19 pandemic in Buenos Aires, Argentina. Methods: A prospective study was conducted in two hospitals from 2019 to 2022, recovering all CRE from inpatients. Antimicrobial susceptibility was performed by automated and/or manual tests, according to CLSI. β-lactamases detection was performed using Multiplex PCR and MALDI-TOF MS. Kp typing was assessed by multiplex PCR and/or MLST based on WGS. Results: 22% (359/1594) were CRE, predominantly CR-Kp. Overall, high non-susceptibility (NS) rates were observed in both centres. NS remained largely stable in HA, except for a significant increase in colistin NS, whereas HB showed a rise in NS to multiple antimicrobials over time. A significant shift from multidrug-resistant to extensively drug-resistant and difficult-to-treat phenotypes was observed across the study periods. Out of 359 CRE, bla_KPC was confirmed in 141, bla_NDM in 170, and bla_KPC + bla_NDM in 20 isolates. Before the COVID-19 pandemic, KPC was the main carbapenemase in HB, while NDM was already the prevalent one in HA. In 2022, both enzymes showed similar prevalence. bla_KPC-2 and bla_NDM-5 were the prevalent alleles in K. pneumoniae. Before the COVID-19 pandemic, K. pneumoniae epidemiology varied by hospital, characterised by clonal diversity; however, in 2022, CG258-tonB79 drove the epidemiology in both hospitals. Conclusions: A more extensive resistance phenotype among CRE was evidenced throughout the COVID-19 pandemic, driven by carbapenemase-producing K. pneumoniae. NDM-5 and KPC-2 were the main carbapenemases identified. A temporal shift in carbapenemase prevalence was observed in each hospital, converging in similar frequencies of KPC and NDM by 2022 across both centres. This scenario was driven by the active dissemination of K. pneumoniae ST258. Full article

Carbapenem-resistant Enterobacter cloacae Complex (CRECC) has emerged as an important multidrug-resistant pathogen in healthcare settings, although it has historically received less attention than carbapenem-resistant Klebsiella pneumoniae and other major carbapenem-resistant Enterobacterales (CRE). Recent epidemiological reports from several regions indicate increasing detection rates of CRECC in tertiary hospitals, where it is associated with bloodstream infections, pneumonia, urinary tract infections, and prolonged hospitalization. The dissemination of carbapenemase genes, particularly bla_NDM, bla_KPC, and bla_OXA-48-like, carried predominantly on conjugative plasmids (e.g., IncFII, IncX3, IncL), represents the primary resistance mechanism, often accompanied by porin loss and efflux pump overexpression. High-risk clones such as ST171 and ST78 contribute to nosocomial persistence and outbreak potential. Beyond clinical settings, CRECC and related resistance determinants have been reported in companion animals, livestock, food products, wastewater systems, and natural aquatic environments. Although most available studies examine these sectors separately, the recurring detection of genetically related resistance genes and plasmid types suggests potential epidemiological links that warrant integrated surveillance. Environmental reservoirs, particularly hospital effluents and wastewater treatment systems, may facilitate the maintenance and dissemination of resistance genes. This review synthesizes current evidence on the epidemiology, resistance mechanisms, and evolutionary dynamics of CRECC in human, animal, and environmental contexts under a One Health framework. A better understanding of its ecological distribution and genetic plasticity is essential to inform coordinated surveillance strategies and mitigate the public health risks associated with the continued spread of carbapenem resistance. Full article

Carbapenems are essential for the treatment of severe infections caused by Gram-negative bacteria, particularly in critically ill and immunocompromised patients. However, the global rise of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa, and Acinetobacter baumannii has significantly eroded their effectiveness, and the phenomenon is now recognized as a major public health threat. Resistance is driven by the complex and evolving interplay of enzymatic and non-enzymatic mechanisms, occurring within highly successful clonal lineages and mobile genetic platforms. This review summarizes advances since 2020 in the molecular basis of carbapenem resistance, integrating enzymatic mechanisms across Ambler classes A, B, C, and D with emerging non-enzymatic contributors, including porin remodeling, efflux pump upregulation, target-site alterations, and outer-membrane adaptations. Particular attention is given to adaptive genome dynamics, such as IS26-mediated gene amplification, plasmid multimerization, and heteroresistance, that generate unstable resistance phenotypes and complicate routine susceptibility testing. Newly introduced β-lactam/β-lactamase inhibitor combinations exert distinct selective pressures: ceftazidime–avibactam favors KPC Ω-loop variants and permeability defects, often restoring carbapenem susceptibility, whereas meropenem–vaborbactam and imipenem–relebactam resistance is driven mainly by porin loss and β-lactamase gene amplification. Cefiderocol resistance is multifactorial, frequently involving impaired siderophore uptake and heteroresistance, while sulbactam–durlobactam remains active against OXA-producing A. baumannii but is compromised by metallo-β-lactamases and PBP3 alterations. Carbapenem resistance is increasingly characterized by convergent, multi-layered adaptations that undermine both established and novel therapies. While high-level randomized evidence remains limited for some resistance mechanisms, emerging mechanistic, microbiological, and clinical data support the need for mechanism-aware diagnostics, repeated susceptibility assessment during therapy, and stewardship strategies informed by resistance biology. Integrating molecular context into routine practice will be critical to preserving emerging treatment options and limiting the global impact of carbapenem resistance. Full article

Cefepime combined with late-generation β-lactamase inhibitors—enmetazobactam, zidebactam, and taniborbactam—represents a promising strategy to treat multidrug-resistant Gram-negative infections. These combinations expand the therapeutic armamentarium beyond established β-lactam/β-lactamase inhibitor regimens, offering targeted activity against ESBL-, AmpC-, and carbapenemase-producing Enterobacterales, as well as multidrug-resistant Pseudomonas aeruginosa. In vitro studies highlight potent and broad activity, with mechanisms including β-lactamase inhibition and, in the case of zidebactam, dual β-lactam enhancement through PBP2 binding. Clinical evidence demonstrates efficacy in complicated urinary tract infections and suggests potential for treating extensively drug-resistant infections, including those unresponsive to conventional β-lactam/β-lactamase inhibitors. Emerging resistance mechanisms—such as PBP alterations, porin loss, efflux pump overexpression, and evolving KPC or NDM variants—underscore the need for ongoing surveillance and robust susceptibility testing. This review provides a comprehensive overview of the mechanisms of action, in vitro activity, pharmacokinetic/pharmacodynamic properties, clinical outcomes, and resistance patterns of these cefepime-based combinations. It also highlights future directions, including the establishment of clinical breakpoints, evaluation in severe infections, and exploration of combination strategies to counteract complex resistance. Overall, these agents exemplify a strategic evolution in β-lactam therapy, offering versatile options to reduce carbapenem reliance while maintaining high efficacy against multidrug-resistant Gram-negative pathogens. Full article

Carbapenemase-producing bacteria, particularly those expressing the KPC-3 variant, pose a critical global health threat due to their resistance to nearly all β-lactam antibiotics, including carbapenems. Rapid and reliable detection tools are urgently needed to improve infection control and guide patient management. Nanobodies (VHHs) present a promising alternative to conventional antibodies thanks to their high stability, small size, and capacity to access cryptic epitopes. Here, we report the generation and characterization of a nanobody specifically targeting KPC-3. An immune VHH phage display library was constructed, with over 90% of clones containing correctly sized inserts. After three rounds of biopanning, high-specificity binders were identified by ELISA screening. Sequencing identified a nanobody with hallmark VHH features, which was expressed and validated by ELISA and Western blot. Although kinetic assays showed no inhibition of KPC-3 enzymatic activity, interestingly, the nanobody demonstrated high-binding recognition of both KPC-2 and KPC-3 in periplasmic extracts from clinical strains. Structural modeling further supported these results, highlighting favorable interaction surfaces. This study provides the first evidence of a nanobody raised against KPC-3 that recognizes a conserved epitope shared by KPC-3 and KPC-2, underscoring its promising use as a molecular tool for detecting KPC variants and establishing a basis for future affinity maturation toward therapeutic applications. Full article

Antimicrobial resistance (AMR) is recognised as a major global public health threat, with the environment increasingly acknowledged as a key reservoir and dissemination pathway for resistant bacteria and resistance genes. In this study, 148 surface water samples were collected between 2023 and 2024 from six rivers and three canals discharging wastewater into two lake waters in Southern Italy to assess the occurrence and genomic features of extended-spectrum β-lactamase (ESBL)-, AmpC-, and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae. Relevant isolates were obtained using selective culturing, and tested for antimicrobial susceptibility by broth microdilution. Major β-lactam resistance genes were detected by real-time PCR. Whole-genome sequencing (WGS) was performed on presumptive carbapenemase-producing isolates. ESBL- and/or carbapenemase-producing Enterobacterales were detected in 67.6% of samples, yielding a total of 176 non-duplicate isolates. The most prevalent gene was bla_CTX-M, detected in 79.3% of positive isolates (96/121), while carbapenemase genes were detected in 20.6% (25/121) of isolates, mainly bla_OXA-48 and bla_VIM. WGS analysis of carbapenemase PCR-positive isolates revealed occurrence of clinically relevant high-risk clones, such as K. pneumoniae ST512/ST307 carrying bla_KPC-3 and E. coli ST10 harbouring bla_OXA-244. These findings highlight a potential risk to public health and underscore the importance of integrating environmental compartments into One Health surveillance frameworks for AMR. Full article

Antimicrobial resistance (AMR) in poultry-associated Escherichia coli (E. coli) is a persistent One Health concern, particularly when ESBL/pAmpC determinants co-occur with resistance to multiple antimicrobial classes. Between March and October 2024, we investigated commensal E. coli from three interconnected compartments of the poultry production chain in Bosnia and Herzegovina (parent-breeder flocks, commercial broiler farms, hatchery-associated material). A total of 333 samples were examined, and 99 E. coli isolates were recovered (29.7%). Phenotypic characterization included ESBL confirmation, disk diffusion susceptibility testing, and EUVSEC broth microdilution. Targeted real-time PCR assays were used to screen key ESBL/pAmpC-associated genes and selected carbapenemase and plasmid-mediated colistin resistance targets within the targeted panel. ESBL phenotypes were detected in 52/99 isolates (52.5%), and multidrug resistance was highly prevalent across compartments (93/99; 93.9%). ESBL/pAmpC-associated genes were detected in 91/99 isolates (91.9%), with bla_TEM predominating. Gene pattern analysis indicated that bla_TEM occurred most frequently as a single determinant and as part of the predominant multi-gene combinations, most notably bla_TEM + bla_CMY and bla_TEM + bla_CTX-M, while bla_SHV was sporadic. Carbapenemase genes (bla_KPC, bla_NDM, bla_GES, bla_OXA-48) and mcr-1 to mcr-9 were not detected. Overall, our findings indicate a substantial ESBL/MDR burden throughout the poultry production chain, supporting the need for strengthening antimicrobial stewardship and biosecurity measures across both farms and hatcheries. Full article

Objectives: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) merges multidrug resistance with hypervirulence, posing unprecedented therapeutic challenges. This study aimed to evaluate the efficacy of a recombinant fusion protein vaccine, KPC-Pal, designed to target both the carbapenemase KPC-2 and the virulence-associated peptidoglycan-associated lipoprotein Pal. Methods: The KPC-Pal fusion protein was constructed, expressed, and purified. Its protective efficacy was systematically assessed in a murine pneumonia model by measuring antigen-specific antibodies, cytokine profiles, and memory cell populations. The synergistic effect with the antibiotic meropenem was evaluated both in vitro and in vivo. Furthermore, the interaction with innate immune signaling via TLR2 was investigated. Results: Immunization with KPC-Pal conferred superior protection, resulting in significantly higher survival rates and reduced bacterial burdens in the lungs compared to immunization with either KPC-2 or Pal alone. It induced a robust Th2-biased humoral response and a mixed Th1/Th2/Th17 cellular immune profile, along with enhanced formation of tissue-resident memory T cells. Antibodies generated against KPC-Pal enhanced the efficacy of meropenem in vitro and in animal models, demonstrating a synergistic effect. While Pal alone strongly activated TLR2-driven inflammatory pathways, the KPC-Pal fusion selectively modulated MAPK signaling, mitigating excessive cytokine production. Additionally, KPC-Pal vaccination elicited cross-reactive antibodies against KPC-3 and KPC-33 variants. Conclusions: KPC-Pal functions as both an antigen and a self-adjuvant, offering a promising dual-target strategy for combating K. pneumoniae infections. Full article

Background: Pancreatic Ductal Adenocarcinoma (PDAC) has a dismal five-year survival rate of 13% and is closely associated with cachexia. Cancer cachexia is a multifactorial syndrome characterized by irreversible wasting of skeletal muscles, fat loss and systemic inflammation. While cachexia is known to confer resistance to immune checkpoint inhibition in several cancers, the bidirectional relationship between cachexia and the immune system in PDAC remains unclear, necessitating the development of novel preclinical models. Our laboratory has characterized a novel pancreatic cancer cachexia model in C57BL/6J mice by utilizing the pancreatic cancer cell line called KPCL-4 derived from KPC-LSIY mice (Kras^LSL-G12D/+Tp53^LSL-R172H/+Pdx1-Cre/R26^LSL-LSIY). Methods: KPCL-4 cells were orthotopically injected into the pancreas of male and female C57BL/6J mice and hallmarks of cachexia were assessed at endpoint by measurement of tumor weight, terminal tumor-adjusted body weight, skeletal muscle, adipose tissue, liver and spleen masses, proteolytic markers and grip strength. Plasma cytokine and chemokine concentrations were quantified by Luminex assay and high-dimensional flow cytometry was used to investigate changes in tumor-infiltrating immune populations. Results: We observed a sex bias in cachexia presentation despite similar tumor weights in male and female mice, whereby males exhibited a >5% decrease in terminal tumor-adjusted body weight (p < 0.001), >50% fat loss (p < 0.001), upregulation of proteolytic markers in skeletal muscles (p < 0.01) and reduction in skeletal muscle mass (p < 0.05), function (p < 0.01) and cross-sectional area (p < 0.0001) whereas females demonstrated conserved skeletal muscle mass with 33% fat loss (p < 0.05), reduction in muscle cross-sectional area (p < 0.0001) and splenomegaly (p < 0.01). While intra-tumoral immune populations did not exhibit sex-specific differences, plasma cytokine concentrations were differentially upregulated in males and females, suggesting functional differences in immune cells as potent drivers of sex bias in KPCL-4-driven cachexia. Conclusions: The KPCL-4 orthotopic PDAC model exhibits prominent hallmarks of cachexia and serves as a novel platform for investigating the complex interplay between cancer cachexia and immunomodulation. Full article

Background: The emergence of polymyxin-resistant, carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a critical challenge to clinical management. This study aimed to delineate the molecular mechanisms driving the acquisition of polymyxin resistance in CRKP. Methods: We analyzed polymyxin-susceptible and polymyxin-resistant CRKP isolates obtained from a single patient. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentrations. Whole genome sequencing was employed to identify variations in two-component systems and to screen for mcr genes, which were involved in polymyxin resistance. Differential gene expression was assessed using RNA sequencing and validated by quantitative real-time PCR. Furthermore, site-directed mutagenesis was utilized to confirm the causal role of specific mutations in conferring the resistant phenotype. Results: An L96P mutation in the PhoQ protein was found in the polymyxin-resistant CRKP isolate. Compared with the PhoQ wild-type, this mutation significantly upregulated expression of phoP/Q, pmrD, and arnBCADTEF operon-related genes. A corresponding L96P mutant was subsequently constructed in the polymyxin-susceptible ATCC 13883 strain via site-directed mutagenesis. Antimicrobial susceptibility testing confirmed that the PhoQ L96P mutation elevates the minimal inhibitory concentrations of colistin and polymyxin B to 64 mg/L and >32 mg/L, respectively, from a baseline of 0.5 mg/L. Conclusions: The PhoQ L96P mutation is a pivotal driver of polymyxin resistance in CRKP, primarily mediated through the upregulation of the arnBCADTEF operon. Full article

Background/Objectives: Rapid pathogen identification is essential to optimize antimicrobial therapy and improve patient outcomes, particularly in severe infections. Syndromic molecular diagnostics have been introduced to overcome the limitations of conventional culture-based methods. This study evaluated the diagnostic performance and real-life implementation of BioFire^® FilmArray^® syndromic panels compared with routine microbiological diagnostics. Methods: A total of 955 clinical specimens collected between 2022 and June 2025 were retrospectively analyzed, including positive blood cultures (n = 400), lower respiratory tract samples (n = 309), cerebrospinal fluid (n = 158) and stool specimens (n = 88). FilmArray^® BCID2, Pneumonia Plus, Meningitis/Encephalitis and Gastrointestinal panels were performed on the Biofire Fimarray^® instrument according to clinical indication and compared with conventional culture-based identification and phenotypic antimicrobial susceptibility testing. Results: Overall diagnostic concordance between BioFire^® FilmArray^® syndromic panels and conventional methods was high across all specimen types, with the highest positive percent agreement (PPA) observed for bloodstream infections (97.7%) and gastrointestinal pathogens (100%). In respiratory samples, the Pneumonia Plus panel detected a considerable number of microorganisms that could not be identified by culture, including viral pathogens and fastidious bacteria. Molecular detection of antimicrobial resistance markers showed excellent concordance with phenotypic profiles, with 100% agreement for CTX-M, carbapenemases (KPC, NDM, OXA-48-like, IMP), and vanA/B, while lower concordance was observed for mecA/C in staphylococci. In parallel, semi-quantitative bacterial loads provided by the Pneumonia Plus panel showed a strong essential agreement with culture-based quantification (97.4%, ±1 log₁₀). Across all panels, syndromic testing significantly reduced diagnostic turnaround time. Conclusions: Syndromic molecular panels provide rapid and reliable simultaneous detection of pathogens, as well as early resistance marker detection, thereby supporting timely antimicrobial optimization and stewardship when integrated with conventional microbiological diagnostics. Full article

Background/Objectives: Carbapenemase-producing Escherichia coli (CP-Ec) has emerged as an important contributor to the global crisis of antimicrobial resistance. Although less prevalent than carbapenemase-producing Klebsiella pneumoniae, CP-Ec exhibits marked genomic plasticity, efficient plasmid-mediated dissemination, and increasing involvement in bloodstream infections. This comprehensive review summarizes the global epidemiology, molecular features, treatment options, clonal structure and transmission dynamics of CP-Ec. Particular attention is given to the expanding repertoire of NDM, OXA-48-like, and KPC carbapenemases and their associated plasmid backbones. Key high-risk clones, including ST410, ST167 and ST131, are highlighted as drivers of international spread. Conclusions and Future Directions: CP-Ec bloodstream infections represent a growing clinical challenge, often associated with severe outcomes and limited therapeutic options, particularly for NDM producers. The emergence of treatment failures with last-resort agents further underscores the need for improved management strategies. Strengthened global surveillance, integration of genomic epidemiology, optimized antimicrobial stewardship, and targeted infection control measures are essential to limit the dissemination of CP-Ec and mitigate its impact on human health. Full article