Search Results (152)

Background: Chronic wounds are frequently associated with biofilms, in which not only bacterial but also fungal pathogens can impair wound healing. Among the most relevant opportunistic pathogens is Staphylococcus aureus; together with Candida albicans, both are part of the human skin microbiome but can also colonize chronic wounds. Interkingdom biofilms formed by these microorganisms have been shown to exacerbate the course of diseases compared to infections caused by either species alone. Methods: To address the limited number of studies examining fungal–bacterial interactions in wound environments, leucocyte-rich human plasma biofilm models (lhBIOMs) inoculated with S. aureus and C. albicans were prepared. The efficacy of the commonly used clinical antiseptics octenidine dihydrochloride/phenoxyethanol (OCT/PE) and polyhexamethylene biguanide (PHMB) was examined using the quantitative suspension method (QSM). In addition, spatial distribution and morphology of the microorganisms within this biofilm model were analyzed by confocal laser scanning microscopy (CLSM). Results: In this study, the presence of S. aureus triggered an increase in the formation of filamentation of C. albicans in contrast to the single-species biofilm. In addition, treatment with the tested antimicrobial agents was effective against C. albicans after repetitive applications and showed a clear reduction against S. aureus. Furthermore, the quantitative analysis of the co-culture revealed increased growth of S. aureus in the control culture compared to the single-species model. Conclusions: These findings highlight the pathogenic relevance of interkingdom biofilms in chronic wounds and emphasize the importance of effective species-independent antimicrobial treatment strategies. Full article

This study investigated the interactive pathogenic characteristics of co-infection with Streptococcus equi (S. equi) and Streptococcus dysgalactiae (S. dysgalactiae) isolated from diseased foals. Integrating genomic analysis, in vitro phenotypic assays, and a murine intranasal infection model, we comparatively analyzed the biological traits and pathogenic differences between single and dual infections. Comparative genomic analysis revealed divergent virulence profiles between the two isolates, and both species harbored open pan-genomes. In vitro, the biofilm biomass of strain F1 was higher than that of all other groups. No synergistic hemolytic activity was observed in co-cultures of the two isolates. In vivo infection results indicated that streptococcal infection significantly altered the structure and abundance of pulmonary microbiota in mice. Furthermore, single and dual infections displayed strain-specific and time-dependent microbial disparities at early and late infection stages, which were associated with differential enrichment of multiple immune- and metabolism-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including the Tumor Necrosis Factor (TNF) and Mitogen-Activated Protein Kinase (MAPK) signaling pathways. Notably, co-infection triggered the most severe pulmonary microbiota dysbiosis and aberrant functional pathway regulation. This study preliminarily characterized the distinct phenotypic and pathological alterations induced by dual streptococcal infection, providing experimental evidence and novel insights for further exploring the interactive features of equine streptococcal mixed infections. Full article

Periodontitis, a chronic inflammatory disease driven by plaque biofilm, is a leading cause of tooth loss in adults worldwide. Effective treatment requires not only infection and inflammation control but, more critically, functional regeneration of the periodontal ligament, cementum, and alveolar bone. Periodontal regeneration, however, is a highly ordered, multi-stage biological cascade involving temporally coordinated phases of blood clot formation, inflammatory regulation, tissue formation, and remodeling. Conventional single-drug or mixed-delivery strategies cannot distinguish the distinct demands of each healing phase and fail to replicate this natural rhythm. Sequential controlled-release systems address this gap by delivering multiple bioactive agents (antimicrobials, immunomodulators, and growth factors) in a programmed order tailored to the healing cascade, enabling precise modulation of the periodontal microenvironment and orderly tissue regeneration. This review systematically summarizes advances in these systems, classifying material platforms into four categories: (1) diffusion-barrier and degradation-kinetics systems, including multilayer films, core–shell fibers, porous microspheres, and microneedle arrays; (2) stimuli-responsive systems triggered by pH, matrix metalloproteinases, reactive oxygen species, or exogenous physical stimuli; (3) cell and extracellular vesicle-based systems exploiting the inflammatory tropism of M2 macrophage-derived exosomes for targeted immune reprogramming; and (4) asymmetric structural designs achieving spatiotemporal coordination of physical and biochemical signals through hierarchical architectures. These systems follow an anti-infection/anti-inflammation first, osteogenesis later therapeutic logic, circumventing temporal antagonism among bioactive factors. However, significant challenges hinder clinical translation, including individualized prediction of release kinetics, long-term biocompatibility of carrier materials, material retention under dynamic oral conditions, translational limitations of animal models, and precise regulation of complex factor networks. Future progress will likely depend on multi-responsive and logic-gated systems, deeper integration of biotechnology and immunomodulation, personalized precision medicine, AI-driven material design, and robust clinical translational research. Full article

Catalytic nanozymes have emerged as a versatile class of engineered nanomaterials that combine enzyme-like catalytic activity with exceptional physicochemical stability, tunable composition, and scalable fabrication, offering significant advantages over natural enzymes for antimicrobial applications in food systems. Recent advances in materials engineering have enabled the development of nanozymes with enhanced catalytic efficiency, broad-spectrum antimicrobial activity, and improved resistance to harsh food-processing environments. Nevertheless, current research remains fragmented across diverse material platforms and application scenarios, while a comprehensive understanding of how engineering strategies govern catalytic performance, antimicrobial efficacy, and translational potential is still lacking. This review provides a critical and systematic analysis of catalytic nanozymes for antimicrobial food systems from a structure–activity relationship perspective. Emphasis is placed on the engineering principles that regulate enzyme-mimicking activities, including compositional tuning, crystal phase and facet engineering, defect creation, heterostructure construction, pore architecture, surface functionalization, and single-atom engineering. The relationships between these structural features and catalytic mechanisms, including peroxidase-, oxidase-, catalase-, and multi-enzyme-like activities, are discussed in relation to the generation of reactive oxygen species, membrane disruption, extracellular polymeric substance degradation, biofilm eradication, and pathogen inactivation. Representative applications in food-contact surface decontamination, antimicrobial packaging, fresh produce preservation, and intelligent food processing are critically evaluated using recent experimental evidence. Beyond antimicrobial performance, this review introduces a safe-by-design framework that integrates material engineering with toxicological assessment, nanoparticle migration, environmental fate, regulatory considerations, and scalable manufacturing. Emerging opportunities for artificial intelligence-assisted nanozyme design, high-throughput materials discovery, and data-driven optimization are also discussed as transformative approaches for accelerating industrial translation. By integrating materials science, catalytic mechanisms, food microbiology, and safety assessment, this review establishes a comprehensive framework for the rational development of next-generation catalytic nanozymes toward sustainable, effective, and industrially applicable antimicrobial food systems. Full article

Microbially influenced corrosion (MIC) poses a significant threat to buried metallic infrastructure, yet assessing MIC risks in complex, vertically stratified soil environments remains challenging. Unlike traditional models that focus on single canonical corrosion-related species, localized MIC is increasingly recognized as a community-driven process mediated by biofilm formation and stress adaptation. This study investigated the spatial and vertical distribution (0–2.5 m) of microbiomes and corrosion-associated functional genes along a transmission line in Guangxi, China, using shotgun metagenomic sequencing. Taxonomic profiling revealed pronounced site-specific divergence. Site C was enriched in Sphingomonas and nitrifying taxa that promote biofilm-mediated corrosion, whereas Site E was dominated by the iron-reducing Anaeromyxobacter, suggesting anaerobic corrosion susceptibility. Along all investigated sites, surface horizons were dominated by aerobic biofilm formers. Intermediate depths were enriched in dissimilatory iron reducers and nitrite oxidizers, while the deepest layers were dominated by acid-producing Streptomyces. Similarly, the corrosion-related functional genes exhibited a shared vertical stratification across all sites. Functional annotation identified a persistent baseline of corrosion-relevant functions—including oxidative-stress sigma factors (e.g., K03088), nickel-transport systems for hydrogenase assembly (e.g., K02035/K02033/K02034), biofilm-regulatory kinases (e.g., K12132), and ATP-binding cassette (e.g., K06147). These functional markers exhibited clear vertical stratification, with nickel-transport and stress-response genes peaking at intermediate depths. Correlation analyses suggested that K03088 was associated with high resistivity and oxidation-reduction potential, whereas the nickel-transport cluster K02035/K02033/K02034 was correlated with lower pH and reduced salinity. These findings suggest that MIC risk in soils is potentially driven by complex ecological networks, and provide candidate genetic biomarkers for the early warning and risk assessment of buried infrastructure corrosion. Full article

Persistent high-risk human papillomavirus (HPV) infection drives cervical cancer, a leading cause of cancer-related mortality among women in low- and middle-income countries; its clinical course is shaped by the cervicovaginal microbiome, in which Lactobacillus-dominated communities are associated with enhanced viral clearance. Despite this, vaginal probiotic interventions often demonstrate limited colonization efficiency, and autochthonous strain libraries from Central Asia remain absent. We applied a tiered functional screening workflow to a collection of 235 vaginal lactic acid bacterial isolates recovered from 400 women undergoing routine gynecological examination in Astana, Kazakhstan. The workflow sequentially filtered isolates on (i) antimicrobial activity against seven urogenital indicator pathogens using the deferred antagonism assay, (ii) surface adhesion by the Brilis erythrocyte assay, and (iii) biofilm-forming capacity by crystal violet retention and laser-capture-microdissection (LCM) microscopy. Species-level identification of the selected candidate was performed by whole-genome shotgun sequencing followed by Kraken2 taxonomic classification. From 235 isolates, three rounds of phenotypic filtering identified four broad-spectrum antimicrobial candidates (127-3, 127-4, 107-2, 107-4) with non-overlapping inhibitory profiles against seven urogenital indicator strains. Adhesion phenotyping segregated candidates into low- and moderate-adhesion groups, with none reaching the high-adhesion threshold. Among all four candidates, only strain 127-4 produced a reproducible biofilm-associated signal (crystal violet retention OD490 = 0.09 ± 0.07 at 24 h; 0.08 ± 0.03 at 48 h), consistent with early surface attachment under static conditions. Whole-genome shotgun sequencing assigned 97.81% of classified reads to Lactiplantibacillus plantarum, supporting preliminary identification of the selected isolate as L. plantarum strain 127-4. Composite ranking confirmed 127-4 as the only isolate combining broad antimicrobial activity (5/7 indicators), moderate adhesion (specific adhesion index, SPA = 2.95), and a detectable biofilm-associated phenotype. We report the first systematic functional screening of autochthonous cervicovaginal lactic acid bacteria from a Central Asian population and identify L. plantarum 127-4 as a probiotic candidate with an integrated trait profile rarely identified through single-criterion screening approaches. Beyond candidate identification, this work establishes a transferable workflow for assembling functionally annotated vaginal Lactobacillus collections from underrepresented populations, providing a foundation for future population-specific probiotic interventions targeting cervicovaginal health. Full article

The rise in antimicrobial resistance and the resilience of microbial biofilms demand innovative strategies that combine, for instance, membrane-active agents with marketed drugs. Arginine-based surfactants are promising alternatives to conventional quaternary ammonium compounds, but comparative data on their antimicrobial, antibiofilm and modulatory activities remain limited. Five arginine-derived surfactants, the single-chain Nα-lauroyl-L-arginine methyl ester (LAM) and ethyl ester (LAE), together with their double-chain homologues LANHC₃, LANHC₅ and LANHC₈ were evaluated against Gram-positive and Gram-negative bacteria and four Candida spp. Minimum inhibitory (MIC) and lethal (MLC) concentrations were determined by broth microdilution method. Antibiofilm activity was assessed through minimum biofilm inhibitory (MBIC) and eradication (MBEC) concentrations. Checkerboard assays were used to evaluate the synergism between the surfactants and conventional therapeutic antibacterial and antifungal agents. LANHC₃ and LANHC₈ exhibited uniform antibacterial MICs of 19.53 µg/mL, while LAM and LANHC₅ showed MICs of 19.53 µg/mL for most strains, with Enterococcus faecalis requiring 39.06 µg/mL. LANHC₃ was the most potent surfactant over Candida spp. With MICs of 9.76 µg/mL for all species, and similarly to LAM, both were fungicidal at 39.06 µg/mL. LAM and LANHC₃ also showed the lowest MBIC and MBEC values, inhibiting the Candida biofilm formation at 39.06 µg/mL and eradicating mature biofilms at 78.12 µg/mL, while the other surfactants required higher concentrations to disrupt the microbial biofilms. Synergic or additive interactions were found between the surfactants and selected β-lactam and macrolide antibiotics, as well as azole antifungals, with no antagonism observed. LAM and particularly LANHC₃ combined broad-spectrum antimicrobial activity, relevant antibiofilm effects and the ability to potentiate the activity of conventional agents, supporting their choice as alternative or complementary antimicrobial adjuvants over resistant microorganisms and their biofilms. Full article

Chinese Cordyceps (Ophiocordyceps sinensis) and Xinjiang Cordyceps (Paraisaria gracilis) are related entomopathogenic fungi that occupy different elevations and habitats. Whether their holobiont architectures have diverged accordingly is unknown. In this hypothesis-generating study based on samples from single locations (Altai Mountains for Xinjiang Cordyceps and Nagqu, Tibet for Chinese Cordyceps), we compared the two species using amplicon sequencing, untargeted metabolomics, and comparative genomics. Chinese Cordyceps from the sampled site comprises a specialized parasitic fungus and host-adapted bacteria for nutrient acquisition. Xinjiang Cordyceps from the Altai site contains diverse saprotrophic fungi and a rhizosphere-like bacterial consortium enriched in oxidative defense and biofilm genes, a finding that may explain why its sclerotia remain intact for 3–5 years in this population. Metabolomic profiles distinguish the two species at these sites. Xinjiang Cordyceps shows upregulation of tyrosine and porphyrin pathways, and its bacterial community shows functional enrichment in the same pathways, suggesting cross-kingdom coordination. P. gracilis has lost many gene families, and the retained species-specific genes are linked to cell adhesion and acyltransferase activity. Xinjiang Cordyceps is not a simple substitute for Chinese Cordyceps but appears to represent a different ecological strategy shaped by genome streamlining and host–microbe coadaptation. Our findings generate testable hypotheses for future large-scale, multi-population investigations. Full article

Carbon quantum dots (CQDs) exhibit multiple antibacterial mechanisms, making them more effective than conventional antibiotics, which typically act through a single mode of action. These mechanisms include membrane disruption, biofilm inhibition, reactive oxygen species (ROS) generation, and photodynamic (PDT) or photothermal (PTT) effects under light irradiation. Extensive research has been conducted to reinforce these mechanisms and improve the antibacterial performance of CQDs, aiming to reduce required CQD dosages and combat bacterial resistance. This review systematically summarizes structural and functional design strategies reported since 2020. We categorized these strategies into selecting antibacterial molecules as precursors, controlling particle size, surface modification, doping with non-metal and metal elements, and forming functional composites to enable light activation, synergetic effects, and multifunctionality. For each category, we provide representative CQD examples, in terms of their preparation, physicochemical properties contributing to antibacterial performance, and possible structure–activity relationships. Finally, the review highlights limitations and proposes future research directions for developing antibacterial CQDs for clinical translation. Full article

Background/Objectives: Biofilms are a form of defense that enables bacteria to withstand antibiotic pressure and demonstrate antibiotic resistance. It is crucial to develop anti-biofilm strategies in order to combat chronic and persistent multidrug-resistant (MDR) infections. Methods: In this study, we developed 3D biofilms of single-, dual-, and poly-species MDR ESKAPE components, including the pathogens P. aeruginosa S. aureus and K. pneumoniae, in CF Mu3Gel. We evaluated the efficacy of using a phage, a di-hetero phage cocktail or a poly-hetero phage cocktail in combination with ciprofloxacin to eliminate mature biofilm biomass after 72 h or one week in a single treatment. Results: The phage components mostly exhibited synergistic behavior when combined with ciprofloxacin and with each other in di- and poly-hetero-cocktails. The reduction in 72-h dual- and poly-species biofilms was one log higher than that of one-week biofilms treated with the phage–antibiotic combination. The greatest reductions were observed in the 72-h single-species biofilm with combination therapy, at 1.4–3.0 log. Reductions of 2.16 and 1.6 log were observed in the dual-species P. aeruginosa and S. aureus biofilm and the poly-species biofilm, respectively. Conclusions: This study examined how a single application of phages or phage cocktails, either alone or in combination with ciprofloxacin, impacted established biofilm models, and how this affected the proportion of microcolonies of different species within each model. These insights will facilitate the development of strategies for multiple follow-up treatments, as well as the reordering of phages, phage cocktails, and combinations with antibiotics, to improve outcomes. The 3D biofilm models developed here could be used to screen phages or phage cocktails either on their own or alongside other therapies. This would facilitate the application of in vitro findings to real physiological settings. Full article

Background: Given their intraoral service for clinically relevant periods, it is important to clarify whether additive manufacturing parameters influence the surface characteristics of 3D-printed provisional restorations and thereby affect microbial colonization. Methods: This study evaluated the effects of printing orientation and layer thickness on surface roughness, wettability, and Streptococcus mutans biofilm formation of LCD-printed provisional restorations. Disk-shaped specimens were fabricated from a methacrylate-based provisional resin at two orientations (0° and 90°) and two layer thicknesses (50 and 100 µm) (n = 7 per group). Surface roughness (Ra) was measured by contact profilometry, wettability by sessile-drop contact angle analysis, and biofilm formation by crystal violet staining after 72 h. Results: Data were analyzed by two-way ANOVA, aligned rank transform (ART) ANOVA, and correlation analysis (α = 0.05). Orientation alone did not affect Ra (p = 0.992), whereas layer thickness (p = 0.012) and the orientation × layer thickness interaction (p = 0.002, η² = 0.339) were significant. At 50 µm, 90° oriented specimens showed higher Ra than 0° (p = 0.021); this pattern reversed at 100 µm (p = 0.020). Neither parameter significantly affected contact angle or biofilm formation (p > 0.05). Conclusions: Both printing orientation and layer thickness altered the surface microtopography of the specimens; however, no significant differences were observed in short-term S. mutans biofilm formation among the tested groups. Within the limitations of the present single-species 72 h in vitro model, the findings suggest that material-related characteristics may have contributed more prominently to the observed biofilm behavior than the printing-induced surface differences evaluated in this study. Full article

Staphylococcus aureus biofilms formed on titanium surfaces are highly relevant to orthopedic implant-associated infection and remain difficult to control after maturation. This study aimed to evaluate whether ultraviolet A (UVA, 365 nm) combined with cinnamaldehyde (CA) could improve antibiofilm activity against titanium-associated S. aureus biofilms in a stage-resolved in vitro model and to examine whether the observed responses were associated with reactive oxygen species (ROS). Early stage (8 h) and 24 h biofilm models were established on total hip arthroplasty (THA)-derived titanium discs. After condition screening, 0.5 mM CA combined with 5 min UVA exposure was selected for subsequent experiments. Biofilm biomass was assessed by crystal violet staining, bacterial viability by live/dead staining and colony-forming unit (CFU) enumeration, ROS-associated fluorescence by dihydroethidium (DHE) imaging, and biofilm-associated gene expression by quantitative real-time PCR (qRT-PCR). Chondrocyte viability was also evaluated under the selected antibiofilm-effective conditions. The combined treatment showed stage-dependent antibiofilm effects, with greater biomass reduction in the 8 h biofilm model and marked impairment of bacterial viability and culturability in both models. ROS-associated fluorescence increased under combined exposure and was partially attenuated by N-acetyl-L-cysteine (NAC) in the 24 h biofilm model. In parallel, CA + UVA was associated with lower expression levels of clfA, icaA, and icaD in the 8 h biofilm model and of icaA, icaB, and icaD in the 24 h biofilm model, with partial NAC attenuation in the latter. Chondrocyte viability was lower in all treatment groups than in the untreated control, although the combined treatment did not show an obvious additional decrease compared with the single-treatment groups. These findings indicate that UVA combined with CA exerts stage-dependent antibiofilm effects in an in vitro titanium-associated S. aureus biofilm model. The observed ROS-associated responses were consistent with, but do not establish, mechanistic involvement. The current treatment setting also requires further optimization before translational applicability can be more confidently considered. Full article

Dental implants are widely used to replace missing teeth, but peri-implantitis remains a major biological complication associated with bacterial biofilm formation on implant surfaces. The increasing incidence of peri-implant infections underscores the need for alternative antimicrobial strategies that effectively decontaminate complex titanium implant surfaces. This study evaluated the inhibitory effect of low-temperature microwave argon plasma on bacteria in an experimental model simulating peri-implant conditions and compared the responses of microorganisms with different biological characteristics. A 3D-printed mandibular bone segment model with an inserted Straumann BLX Roxolid^® dental implant was used to reproduce the peri-implant environment. Bacterial suspensions of Streptococcus mutans NBIMCC 1786 and the extremophilic bacterium Chromohalobacter canadensis NBIMCC 9077 have been exposed to a microwave non-equilibrium argon plasma jet (2.45 GHz, atmospheric pressure) for 1–7 min. Optical density measurements and colony growth analysis were used to assess antimicrobial effects. Plasma treatment induced a pronounced reduction in bacterial growth during the early post-treatment period. In C. canadensis, growth inhibition reached a plateau (~47–55% at 24 h) regardless of exposure time. In contrast, S. mutans showed a nonlinear response, with stable inhibition after short exposures (1–3 min) and partial recovery after longer treatments (5–7 min). These findings indicate that microwave argon plasma exhibits significant antimicrobial activity under controlled in vitro conditions, although its effectiveness depends on microorganism-specific biological characteristics. Because the present model was based on simplified single-species systems, direct clinical extrapolation remains limited and should be addressed in future studies using polymicrobial peri-implant biofilm models. Full article

Antimicrobial resistance represents one of the most formidable global health crises of the 21st century, driven by the diminishing efficacy of conventional antibiotics due to bacterial adaptation and biofilm formation. In response, antimicrobial nanoformulations have emerged as a transformative therapeutic paradigm, offering multifaceted and innovative mechanisms to combat resistant pathogens. This comprehensive review delineates the broad scope and distinct novelty of nano-enabled antimicrobial strategies, moving beyond the single-target limitations of traditional drugs. We systematically explore the diverse architectural classes of nanoformulations—including metallic, polymeric, and self-assembling nanostructures—and elucidate their unique mechanistic actions. These encompass (1) physical disruption of microbial membranes via electrostatic interactions; (2) catalytic generation of reactive oxygen and nitrogen species to induce an ‘oxidative storm’; (3) intracellular sabotage of essential metabolic pathways; (4) the ‘Trojan horse’ strategy for enhanced drug delivery and bioavailability; (5) efflux pump bypass to counteract a major resistance mechanism; (6) penetration and eradication of resilient biofilms; and (7) disarming pathogens through quorum sensing and virulence inhibition. Furthermore, this review highlights the immunomodulatory potential of nanoformulations; their activity beyond bacteria against fungi, viruses, and parasites; and the critical role of the nano-bio interface defined by surface physicochemistry. We also address the translational pathway, considering challenges in nanotoxicology, scalability, and regulatory approval, alongside the ecological impact and economic horizon of these technologies. This sector is projected to reach USD 5.4 to 8.96 billion by 2033 to 2034, with compound annual growth rates of 11 to 21% across antimicrobial nanomaterials, nanocoatings, and nanomedicine applications. By integrating insights from computational modeling and in silico design, this review underscores how nanoformulations leverage synergistic, multi-target approaches to overcome resistance, enhance therapeutic efficacy, and represent a significant leap forward in the future of infectious disease management. The novelty lies in the holistic and mechanistic synthesis of how nanotechnology is redefining antimicrobial warfare, offering a promising arsenal to avert a post-antibiotic era. Full article

Background/Objectives: Bacterial conjunctivitis is a common ocular infection requiring prompt treatment, particularly in vulnerable patients, and may influence perioperative outcomes. This study aimed to characterize conjunctival bacterial isolates phenotypically and genotypically, to evaluate their biofilm-forming capacity, and to investigate the relationship between resistance gene carriage, resistance phenotypes, and biofilm-associated antimicrobial resistance (AMR). Methods: A prospective, single-center, cross-sectional study was conducted on bacterial isolates from conjunctival samples of patients examined in an ophthalmology department. Antimicrobial susceptibility testing (AST) was performed to determine the minimum inhibitory concentrations (MICs). Resistance genes were detected by quantitative PCR. Biofilm-forming capacity was assessed using the microtiter plate assay, and biofilm susceptibility to amikacin (AK) and levofloxacin (LEV) was evaluated using a biofilm susceptibility assay. Results: A total of 78 isolates were analyzed; Gram-positive cocci prevailed (GPC, 84.6%), being significantly more frequent than Gram-negative bacilli (GNB, p < 0.001). Among GPC, 65.2% were multidrug-resistant, with Staphylococcus epidermidis emerging as the most frequent species (p < 0.001). Resistance gene carriage was detected in 33.3% of GNB. Strong biofilm formation was observed in 22.7% of GPC versus 58.3% of GNB. It should be noted that the relatively small number of GNB may limit the statistical robustness of comparisons between Gram-positive and Gram-negative groups. A statistically significant association between resistance genes and biofilm capacity was found only in Staphylococcus aureus (p = 0.027). Biofilm-embedded bacteria showed increased antimicrobial tolerance, particularly for AK in S. aureus and for both AK and LEV in S. epidermidis (p < 0.001). Conclusions: The prevalence of multidrug-resistant conjunctival isolates and their biofilm-forming capacity highlights the clinical importance of biofilm-related resistance and support integrating AMR profiling with biofilm assessment to optimize empirical therapy in bacterial conjunctivitis. Full article