Search Results (773)

Conventional antibiotic susceptibility testing (AST) primarily assesses planktonic bacteria. However, the three-dimensional architecture and barrier properties of biofilms mean that the minimum inhibitory concentration (MIC) for planktonic cells is typically far lower than the antibiotic exposure required for biofilm eradication. In this study, gelatin methacryloyl (GelMA) microspheres were used to create a three-dimensional biofilm microenvironment for the quantitative evaluation of biofilm tolerance. Escherichia coli K-12 MG1655 was immersed in GelMA microspheres and subjected to a series of antibiotic concentration gradients. Bacterial viability was inferred from time-dependent changes in microsphere diameter. The results demonstrated substantial tolerance of the resulting biofilms to ampicillin, ciprofloxacin, and ceftriaxone, with biofilm antibiotic tolerance values exceeding 200 μg/mL, 10–50 μg/mL, and 20–50 μg/mL, respectively. Relative to planktonic MICs, these tolerance levels are elevated by one to two orders of magnitude and surpass the standard clinical breakpoint thresholds. This methodology includes a high-throughput platform, involving only several hundred microspheres and achieving completion within 24 h, thereby offering a useful platform for investigating biofilm resistance mechanisms and guiding antibiotic treatment strategies. Full article

Streptococcus uberis is a bovine mastitis pathogen with a demonstrated ability to form biofilms. However, the dynamics of this process remain poorly characterized. This study aimed to comprehensively characterize biofilm formation in four S. uberis strains that differed in their biofilm-forming capacity, from weak to strong producers, and in the presence of key virulence-associated genes, such as sua, hasA and hasC. To achieve this, we integrated structural, biochemical, physiological and transcriptional analyses using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), spectral flow cytometry and qRT-PCR. The multi-faceted analysis revealed a coordinated maturation peak at 48 h, characterized by a structured architecture with water channels, a distinct biochemical signature rich in polysaccharides and proteins, and a predominantly viable bacterial population. This peak coincided with a marked upregulation of key virulence-associated genes, with sua expression increasing 2.5-fold and hasA increasing 3-fold at 48 h. This mature biofilm conferred high tolerance to antibiotics, with eradication concentrations (>256 µg/mL) exceeding planktonic MICs, although tetracycline was notably effective. At 72 h, the biofilm entered a dispersion phase characterized by structural collapse and reduced viability. These findings establish S. uberis biofilm maturation as a highly coordinated process, providing new insights into the biofilm lifecycle of this important pathogen and identifying key temporal and molecular targets for future interventions. Full article

Background/Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays including MIC determination, biofilm eradication testing (crystal violet, colony counts, and CLSM), swimming motility, and EPS quantification were performed. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In an in vivo murine excisional wound infection model treated with the mouse cathelicidin-related antimicrobial peptide (CRAMP-34), wound closure and bacterial burden were monitored. Gene expression changes were analyzed via RT-qPCR. Results: CRAMP-34 effectively eradicated pre-formed biofilms of a clinically relevant, porcine-origin E. coli strain and promoted wound healing in the murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: This research highlights CRAMP-34 as a potent anti-biofilm agent and unveils kduD as a previously unrecognized regulator of E. coli biofilm development, which is also targeted by CRAMP-34. Full article

The growing global threat of antimicrobial resistance (AMR), particularly in cutaneous wound infections, represents a significant clinical and economic challenge. Biofilm formation by multidrug-resistant pathogens, such as Acinetobacter baumannii, often complicates healing and leads to therapeutic failure. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics due to their potent membrane-disrupting mechanism of action and lower propensity to induce resistance. Background/Objectives: This study aimed to evaluate the antibacterial, antibiofilm, and in vivo efficacy of four snake venom-derived cathelicidin-like peptides—Btn (15-34) and BotrAMP14 from Bothrops atrox, and Ctn (15-34) and CrotAMP14 from Crotalus durissus—against multidrug-resistant A. baumannii, Escherichia coli, and Pseudomonas aeruginosa clinical isolates from skin infections, with emphasis on A. baumannii, a WHO priority pathogen. Methods: Minimal Inhibitory Concentration (MIC), Minimal Bactericidal Concentration (MBC), and Minimal Biofilm Inhibitory Concentration (MBIC) were determined against A. baumannii, Escherichia coli, and Pseudomonas aeruginosa. Time-kill kinetics, hemolytic activity, and cytotoxicity assays were performed. A murine skin wound infection model was established to evaluate in vivo antibacterial efficacy and safety. Results: MIC/MBC values ranged from 0.78 to 25 µM against planktonic cells. In comparison, MBIC ranged from 1.56 to 12.5 µM against biofilms. BotrAMP14 eradicated A. baumannii within 4 min, while CrotAMP14 achieved bactericidal action in 20 min at 1.56 µM. Both peptides exhibited no hemolytic activity up to 128 µM and low cytotoxicity (IC₅₀ > 128 µM). In vivo, BotrAMP14 and CrotAMP14 demonstrated significant antibacterial activity at 24 h and 48 h post-infection, respectively, surpassing that of meropenem. Conclusions: These findings suggest that BotrAMP14 and CrotAMP14 are promising topical antimicrobial agents for managing multidrug-resistant skin infections and may help address the urgent need for alternative therapies against antibiotic-resistant pathogens. Full article

Pseudomonas aeruginosa (P. aeruginosa) forms biofilms that are difficult to eliminate. The matrix protects the cells, efflux pumps reduce intracellular drug levels, and dormant subpopulations survive treatment. Routine minimum inhibitory concentration (MIC) testing does not account for these features, which helps explain why infections often continue even when therapy appears appropriate. This review describes how quorum-sensing (QS) and cyclic di-guanosine monophosphate (c-di-GMP) regulate matrix production, efflux activity, and dormancy within P. aeruginosa biofilms. Important matrix components, including Psl, Pel, alginate, and extracellular DNA, slow the movement of antimicrobial agents. Regulatory proteins such as sagS and brlR increase the activity of the MexAB-OprM and MexEF-OprN efflux systems, further reducing intracellular drug concentrations. Oxygen and nutrient limitation promote persister cells and viable but nonculturable cells, with both having the ability to survive antibiotic levels that would normally be lethal. These defenses explain the gap between MIC values and biofilm-specific measurements, such as the minimum biofilm inhibitory concentration and the minimum biofilm eradication concentration. This review also summarizes emerging antibiofilm strategies. These include QS inhibitors, compounds that lower c-di-GMP, such as nitric oxide donors, nanoparticles, depolymerases, bacteriophages, and therapies that are directed at host targets. Modern diagnostic tools, such as confocal laser scanning microscopy, optical coherence tomography, and Raman spectroscopy, improve detection and guide treatment planning. A staged therapeutic approach is presented that begins with the dispersal or loosening of the matrix, continues with targeted antibiotics, and concludes with support for immune clearance. Viewing these strategies within a One Health framework highlights the role of biofilms in clinical disease and in environmental reservoirs and supports more effective surveillance and prevention. Full article

Background: Stenotrophomonas maltophilia is an intrinsically multidrug-resistant, biofilm- forming, non-fermenter increasingly implicated in hospital-acquired infections. Evidence from non-cystic fibrosis populations, especially in the Middle East, remains sparse. Methods: We conducted a retrospective observational cohort study at a tertiary academic center (Al-Khobar, Saudi Arabia) spanning 1 May 2001–30 April 2023. Hospitalized adults (≥18 years) with culture-confirmed, clinically diagnosed S. maltophilia infection and ≥72 h of antibiotic therapy were included. The primary outcome was all-cause mortality (14-day, 30-day, 1-year). Secondary outcomes were clinical response, microbiological eradication, and infection recurrence. Predictors of 30-day mortality were assessed using multivariable logistic regression; 14-day mortality was analyzed by Kaplan–Meier/log-rank according to susceptibility-guided versus alternative therapy. Results: Of 539 patients with positive cultures, 436 met the inclusion criteria. Mean age was 60.5 ± 19.3 years; 62.2% were male. Most infections were hospital-acquired (92.9%); pneumonia composed 64.7% and bloodstream infection 15.4%. Polymicrobial growth occurred in 55.5% (predominantly Gram-negative co-isolation). Susceptibility was 95.1% to trimethoprim–sulfamethoxazole, 76.4% to levofloxacin, and 43.6% to ceftazidime. Mortality at 14 days, 30 days, and 1 year was 22.8%, 37.9%, and 57.2%, respectively. On multivariable modelling, intensive care unit (ICU) admission, leukocytosis, neutrophilia, anemia, and thrombocytopenia independently predicted 30-day mortality. Susceptibility-guided therapy was associated with improved 14-day survival (log-rank p = 0.033). Conclusions: In this large, long-running non-cystic fibrosis cohort, host acuity and early alignment of treatment to susceptibility data were dominant drivers of outcome. High polymicrobial burden and limited reliably active agents underscore the need for meticulous stewardship, robust infection prevention, and cautious interpretation of S. maltophilia antimicrobial susceptibility testing. Full article

Drug-resistant Klebsiella pneumoniae and related Enterobacterales represent an escalating global public health threat, increasingly limiting therapeutic options in both healthcare- and community-associated infections. This review summarizes how resistance in K. pneumoniae emerges from the synergy of intrinsic barriers and acquired determinants. Key molecular mechanisms include reduced permeability via porin remodeling (notably OmpK35/OmpK36), multidrug efflux (e.g., AcrAB-TolC and OqxAB), and enzymatic drug inactivation driven by extended-spectrum beta-lactamases and carbapenemases (e.g., KPC, OXA-48-like enzymes, and metallo-beta-lactamases). We also highlight clinically meaningful pathways underlying polymyxin/colistin resistance, including mgrB inactivation and PhoPQ/PmrAB-mediated lipid A modification. In addition to stable genetic resistance, adaptive programs can shape transient tolerance and persistence, including stress responses that modulate gene expression under antibiotic and host-imposed pressures. The ability of these organisms to form biofilms, particularly on medical devices, further complicates treatment and eradication. Finally, we discuss therapeutic implications and current options and limitations—including novel beta-lactam/beta-lactamase inhibitor combinations and siderophore cephalosporins—and emphasize the importance of aligning therapy and surveillance with the underlying resistance mechanisms and circulating high-risk lineages. Full article

Non-typhoidal Salmonella (NTS) are globally distributed zoonotic pathogens of major concern within the One Health–One Biofilm framework. Fluoroquinolone-resistant Salmonella strains are included by the World Health Organization (WHO) in the Bacterial Priority Pathogens List as high-risk agents. A key virulence determinant of Salmonella is its ability to form biofilms, which may display multidrug-resistant (MDR) characteristics and contribute to bacterial persistence and treatment failure. Animals, particularly poultry and reptiles, represent important reservoirs of Salmonella, and reptile-associated salmonellosis (RAS) may manifest as extraintestinal infections in humans. In the post-antibiotic era, there is an urgent need to identify effective alternatives to conventional therapies. This review summarizes current knowledge on Salmonella biofilms, with particular attention to their MDR potential, and discusses possible strategies for their prevention and eradication, including specific immunoprophylaxis, bacteriophage therapy, and alternative antimicrobials. The promising antimicrobials include plant-based compounds/extracts, bacteriocins, fatty acids, and synthetic/semi-synthetic substances. The integration of vaccination, phage therapy, and novel anti-biofilm compounds may provide a sustainable alternative to antibiotics in controlling Salmonella infections and aligns with the principles of the One Health approach. Full article

Background/Objectives: Boron-dipyrromethene (BODIPY) derivatives are a superior class of fluorophores prized for their exceptional photostability and tunable photophysical properties. While ideal for imaging, their translation to photodynamic therapy (PDT) has been hampered by excitation in the visible range, leading to poor tissue penetration. To overcome this, intense research has focused on developing near-infrared (NIR)-absorbing BODIPY photosensitizers (PS). This review aims to systematically summarize the hierarchical design strategies, from molecular engineering to advanced nanoplatform construction, that underpin the recent progress of NIR-BODIPY PS in therapeutic applications. Methods: We conducted a comprehensive literature review using PubMed, Scopus, and Web of Science databases. The search focused on keywords such as “BODIPY”, “aza-BODIPY”, “near-infrared”, “photodynamic therapy”, “photothermal therapy”, “nanocarriers”, “hypoxia”, “immuno-phototherapy”, and “antibacterial.” This review analyzes key studies describing molecular design, chemical modification strategies (e.g., heavy-atom effect, π-extension), nanoplatform formulation, and therapeutic applications in vitro and in vivo. Results: Our analysis reveals a clear progression in design complexity. At the molecular level, we summarize strategies to enhance selectivity, including active targeting, designing “smart” PS responsive to the tumor microenvironment (TME) (e.g., hypoxia or low pH), and precise subcellular localization (e.g., mitochondria, lysosomes). We then detail the core chemical strategies for achieving NIR absorption and high singlet oxygen yield, including π-extension, the internal heavy-atom effect, and heavy-atom-free mechanisms (e.g., dimerization). The main body of the review categorizes the evolution of advanced theranostic nanoplatforms, including targeted systems, stimuli-responsive ‘smart’ systems, photo-immunotherapy (PIT) platforms inducing immunogenic cell death (ICD), hypoxia-overcoming systems, and synergistic chemo-phototherapy carriers. Finally, we highlight emerging applications beyond oncology, focusing on the use of NIR-BODIPY PS for antibacterial therapy and biofilm eradication. Conclusions: NIR-BODIPY photosensitizers are a highly versatile and powerful class of theranostic agents. The field is rapidly moving from simple molecules to sophisticated, multifunctional nanoplatforms designed to overcome key clinical hurdles like hypoxia, poor selectivity, and drug resistance. While challenges in scalability and clinical translation remain, the rational design strategies and expanding applications, including in infectious diseases, confirm that NIR-BODIPY derivatives will be foundational to the next generation of precision photomedicine. Full article

Background/Objectives: Deep eutectic solvents (DESs) have recently gained attention for their antimicrobial properties, particularly because they target both planktonic bacteria and biofilms. Among these, DESs based on α-hydroxy acids (αHAs) are of interest due to their inherent antibacterial properties and favorable biocompatibility. However, effects of the αHA molecular structure and hydrogen bonding ability within a DES formulation on biological activity has not yet been thoroughly investigated. Methods: This study systematically investigates DESs formed by combining glycolic acid, lactic acid or tartaric acid with either choline chloride or tetraethylammonium chloride. Results: All DESs demonstrate broad-spectrum antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa and effectively inhibit biofilm formation while exhibiting low cytotoxicity toward 3T3-L1 fibroblasts. Conclusions: DES formation enhances antibacterial efficacy while attenuating cytotoxicity compared to the individual components, thereby decoupling bactericidal activity from toxicity. Physicochemical characterization confirms the formation of a eutectic phase and reveals that biological activity is primarily governed by acidity rather than by the specific αHA structure or eutectic strength. These results provide new insights into structure-function relationships in DESs and establish a design strategy for biocompatible, non-cytotoxic antimicrobial agents. Full article

This study presents a comprehensive evaluation of the antimicrobial activities of silver nanoparticles (AgNPs) synthesized using three distinct methods: plant extracts, bacterial supernatant, and a conventional chemical method. AgNPs were synthesized from Crassula ovata (Jade) leaf extract, Bacillus licheniformis bacterial supernatant, and a standard chemical reduction method using trisodium citrate. The synthesized AgNPs were characterized using UV–Vis spectroscopy, Transmission Electron Microscopy (TEM), and Dynamic Light Scattering (DLS). The antimicrobial efficacy of the AgNPs was tested against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Methicillin-resistant Staphylococcus aureus (MRSA). Our findings reveal significant differences in the biological activities of the AgNPs depending on the synthesis method. The MBC values for the plant extract-synthesized AgNPs were 10 µg/mL for E. coli, 12.5 µg/mL for P. aeruginosa, 10 µg/mL for S. epidermidis, and 15 µg/mL for MRSA. The bacterial supernatant-synthesized AgNPs showed MBC values of 10 µg/mL for E. coli, 12.5 µg/mL for P. aeruginosa, 7.5 µg/mL for S. epidermidis, and 12.5 µg/mL for MRSA. In contrast, citrate-reduced AgNPs exhibited higher MBCs: 60 µg/mL for E. coli and P. aeruginosa, 40 µg/mL for S. epidermidis, and 80 µg/mL for MRSA. Notably, the AgNPs synthesized using plant and bacterial supernatant demonstrated superior antimicrobial activity compared to those synthesized chemically. This comparative study highlights the potential of eco-friendly synthesis routes for producing AgNPs with enhanced biological activities. The findings suggest that plant extract and bacterial supernatant-mediated synthesis of AgNPs could serve as a viable and sustainable alternative to conventional chemical methods, offering promising applications in medical and pharmaceutical fields. Full article

Background: the primary challenge with implant infections is the formation of biofilm, which harbors dormant bacteria that reduce the effectiveness of antibiotics and amplify antibiotic resistance, exacerbating the global antimicrobial resistance crisis. A potential novel treatment strategy is radioimmunotherapy, which uses antibodies linked to radioisotopes to deliver targeted radiation to the bacteria and biofilm. We describe the first in vivo use of targeted radiation therapy, employing Actinium-225 (α-radiation) and Lutetium-177 (β-radiation) labeled antibodies to treat a Staphylococcus aureus biofilm-associated intramedullary implant infection. Untargeted radiation in the form of unbound radionuclide treatment was also evaluated. Methods: to assess therapeutic efficacy, bacterial counts were performed on implant and surrounding bone after seven days of follow-up. Biodistribution was evaluated using SPECT/CT and ex vivo gamma counting. Results: radioimmunotherapy using an antibody against wall teichoic acid which was labeled with Actinium-225 and Lutetium-177 achieved bacterial reductions between 45% and 93% on the implant and surrounding bone. Surprisingly, a similar antimicrobial effect was observed with unbound Actinium-225 treatment reducing the bacterial load by 80% on the implant and 98% in the surrounding bone. Indications of maximum tolerated dose (MTD) with Lutetium-177 labeled antibodies were observed through hepatic and renal function evaluations. Conclusions: These results should be interpreted in the context of the study’s constraints, particularly the limited animal sample size. Nonetheless, the results suggest that in vivo applied radiation may help reduce a biofilm-associated infection at the implant site as well as in the surrounding bone. These findings encourage further investigation into the use of targeted and non-targeted radiation, potentially combined with antibiotics, to develop effective strategies for eradicating biofilm-associated implant infections. Full article

Bacteriophage therapy is regarded as a promising alternative for treating and preventing antibiotic-resistant bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent and difficult-to-treat pathogens. S. aureus also contributes to the formation of both single- and mixed-species biofilms. Treating biofilms remains a major challenge for antibiotic-based eradication of pathogens, as the biofilm matrix provides a protective barrier for bacteria. The selection of highly active phages targeting S. aureus is therefore crucial for medical applications, given the high prevalence and drug resistance of this pathogen. In this study, S. aureus phage vB_SaS_GE1 (GE1) was isolated and characterized as a potential therapeutic agent. The phage was isolated and propagated, and its host range was determined using standard methods. Whole-genome sequencing and annotation of the phage DNA were performed. A time–kill assay and evaluation of the anti-biofilm activity of the Staphylococcus phage, both alone and in combination with Pseudomonas phage GEC_PNG3 (PNG3) on mixed-species biofilms, were conducted. The results indicated that GE1 is a lytic phage that does not carry virulence-determining genes. The time–kill assay demonstrated sustained lytic activity of GE1 without the emergence of phage-resistant mutants in the tested MRSA strains. Although phage treatment increased biofilm matrix production compared to the control, the viable cell count within the biofilms was reduced. Overall, the characteristics assessed indicate that vB_SaS_GE1 is safe and exhibits strong antibacterial activity against MRSA strains. Full article

Human infections caused by pathogenic bacteria remain a major global health concern. Among them, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi are particularly prevalent and associated with significant morbidity and mortality. While antibiotics have long been the cornerstone of bacterial infection treatment, the widespread and often inappropriate use of these drugs has led to the emergence of multidrug-resistant (MDR) strains. This escalating resistance crisis underscores the urgent need for alternative therapeutic strategies. Amid the escalating global antimicrobial-resistance crisis, a genome-wide screen of 1800 Saccharomyces cerevisiae knockouts identified a TAD1-deficient mutant whose cell-free supernatant (CFS) rapidly eradicates multidrug-resistant E. coli, S. aureus, K. pneumoniae, and S. typhi in vitro. CFS disrupts pathogenic biofilms, downregulates biofilm-associated genes, and exerts bactericidal activity by triggering intracellular reactive oxygen species (ROS) accumulation and compromising envelope integrity. Probiotic profiling revealed robust tolerance to an acidic pH and physiological bile, high auto-aggregation, and efficient co-aggregation with target pathogens. In both Galleria mellonella and murine infectious models, administration of CFS or live yeast significantly increased survival, attenuated intestinal histopathology, and reduced inflammatory infiltration. These data establish the TAD1-knockout strain and its secreted metabolites as dual-function antimicrobial-probiotic entities, offering a sustainable therapeutic alternative to conventional antibiotics against multidrug-resistant bacterial infections. Full article

This study synthesizes novel photosensitizer calcination betaine hydrochloride carbon dots (CBCDs) to address the critical challenge of Enterococcus faecalis (E. faecalis) biofilms, a major cause of root canal treatment failure. To this end, this study investigates the effective elimination via reactive oxygen species (ROS) mediated by these CBCDs. CBCDs were prepared by calcining betaine hydrochloride and rigorously characterized for their structural and chemical properties using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). Their optical characteristics were also thoroughly analyzed through UV-Vis and fluorescence spectroscopy. The RNO-ID assay was performed to explicitly confirm ROS production, particularly verifying significant singlet oxygen (¹O₂) generation. Bactericidal efficacy of the CBCDs was comprehensively evaluated against planktonic E. faecalis and its formed biofilms. Live/dead staining was subsequently performed to observe their state after treatment. As a result, TEM confirmed nanosized CBCDs, and FTIR/XPS analyses identified crucial functional groups. Colony Forming Unit (CFU) assays revealed a dose-dependent reduction in E. faecalis viability, achieving complete eradication at 200 mg/L under light irradiation. Complete cell death and inactivation of the formed biofilms with increasing CBCD concentrations were also strongly evidenced by red fluorescence. The obtained results underscore CBCDs as highly effective photodynamic agents for the robust elimination of E. faecalis biofilms, offering a promising new strategy to combat persistent oral infections. Full article