Biofilm

SynopsisO_ST_ABSBackgroundC_ST_ABSThe complexity of diagnosing and treating biofilm-associated infections necessitates a comprehensive strategy to mitigate the rising rates of antimicrobial resistance (AMR). Microtiter plate methods are used globally for determination of biofilm eradication concentrations (MBEC) but few have been adapted to observe pharmacodynamic observations. Here, we describe a method which allows for both static and pharmacodynamic assays of biofilm evaluation. MethodsA total of 150 clinical isolates from Southmead Hospital were assessed, representing five bacterial species (N=30 per bacterial species): Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. MBECs were determined using a developed method using 96 well plates and glass beads. MBECs of seven different antibiotics were compared to those determined using the established Calgary biofilm device (CBD). Dynamic pharmacodynamic evaluations to produce Biofilm Time Kill curve (BTKC) based on published planktonic time kill curve (TKC) data and ISO recommendations were carried out using the glass bead model for K. pneumoniae and ciprofloxacin, S. aureus and levofloxacin and S. pneumoniae and vancomycin. Quantification of biofilm biomass was assessed at 0, 2, 4, 8 and 24 hours and compared to planktonic culture survival under comparable challenge conditions. ResultsComparing MBEC results for all bacterial strains and antibiotic challenges showed no statistical difference between the glass bead and CBD methods (P <0.05). Biofilm BTKC AUBKC were inferior to planktonic equivalents but demonstrated specific pharmacodynamic patterns of biofilm reduction efficacy. MBEC correlated with biofilm BTKC penetration in line with clinical observations for S. aureus vs vancomycin and S. pneumoniae vs levofloxacin. ConclusionsThe glass bead biofilm models provide robust, reproducible alternatives to the traditional methods of determining MBEC and bridge the gap with biofilm pharmacodynamic evaluations. These methods also provide a low-cost option to current methods as only standard laboratory equipment is required, allowing for the generation of comprehensive data sets. This ensures greater translatability to complex in vitro models and clinical scenarios.

Clinically relevant pathogens are often tested for antimicrobial susceptibility using standard laboratory media that poorly reflect the in vivo environments in which they cause infections, leading to poor clinical outcomes. In this study, we aim to understand the impact of media on the global transcriptome, biofilm formation, and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus USA300 when cultivated in a physiologically relevant wound medium, such as simulated wound fluid (SWF), compared to cation-adjusted Mueller-Hinton broth (caMHB), a general-purpose medium. The transcriptomics analysis showed upregulation of 865 genes and downregulation of 792 in SWF compared to caMHB. Upregulated genes in SWF are associated with virulence, such as genes coding for fibronectin-binding proteins (fnaAB), serine proteases (splABCDE), as well as genes involved in antimicrobial resistance, such as multidrug efflux pump genes (norB, norC). Conversely, genes associated with transmembrane ion transport, including phosphate transport (pstSCAB, phoU) and potassium intake (kdpABCF), were significantly downregulated in SWF, as further confirmed by increased membrane disruption upon exposure to a membrane-potential-sensitive dye (DiSC3). Biofilm assay showed reduced surface attached biofilm but increased cell-to-cell attachement in SWF compared to caMHB. Antimicrobial susceptibility testing revealed a 2- to 4-fold increase in tolerance to clinically relevant antibiotics in SWF compared to caMHB. Overall, our findings revealed that media affects gene expression, membrane physiology, virulence, and antibiotic tolerance in MRSA, underscoring the need to use physiologically relevant media in routine antimicrobial susceptibility testing and the drug development pipelines.

Rhodotorula mucilaginosa is an emerging opportunistic fungal pathogen increasingly associated with catheter-related bloodstream infections. Although biofilm formation is considered a major virulence trait for R. mucilaginosa, factors contributing to biofilm persistence on medical devices remain poorly understood. Here, we characterized the thermotolerance, biofilm formation, UV resistance, and cell surface hydrophobicity profiles of eight R. mucilaginosa strains representing clinical and non-clinical (laboratory, environmental, and marine mammal) isolates. All strains grew optimally at 30C and exhibited restricted growth at 35C and 37C, although one environmental isolate maintained robust growth at 37C. All strains exhibited moderate to high cell surface hydrophobicity. We then assessed biofilm formation for each strain, including adherence to two different plastic substrates, development of biofilm biomass, comparison of biofilm metabolic activity, and the effects of temperature on biofilm formation. Under static conditions, biofilm biomass of most isolates on 96-well polystyrene plates was greatest at 24C. Clinical isolates generally maintained higher biofilm metabolic activity at 37C than nonclinical isolates, while at lower temperatures, clinical and non-clinical isolates did not differ significantly in metabolic activity. All strains readily formed biofilms on polyurethane intravenous catheters under dynamic conditions, as confirmed by scanning electron microscopy and metabolic activity. While planktonic cells already displayed substantial UV-C tolerance, biofilm-associated cells remained viable following exposure to UV-C doses up to eightfold higher than those that impaired planktonic growth. These findings document differences in thermotolerance and biofilm formation by isolate origin and identify biofilm formation as a major factor promoting persistence of R. mucilaginosa on clinically relevant materials and reduced susceptibility to UV-C sterilization.

Bacterial biofilms on indwelling medical devices is a major driver of healthcare-associated infection despite significant advances in antifouling surface engineering, suggesting that laboratory antibacterial performance does not fully capture the determinants of clinical colonization. Here we show that adherent immune cells constitute a previously underappreciated conditioning layer that promotes biofilm formation on otherwise antifouling biomaterials. Pre-exposure of clinically used substrates to macrophages, monocytes, neutrophils or human peripheral blood cells markedly increased Staphylococcus aureus and Escherichia coli adhesion and aggregation. These studies also reveal that immune cells promote biofilms even after cell death with cellular debris acting as a conditioning agent. We demonstrate that reactive-oxygen-species amplification by incorporating bismuth telluride into a silicone composite converts adherent immune cells from passive conditioning agents into active bactericidal effectors. We note that this antimicrobial composite confers durable antibacterial protection across early, delayed and late infection time points in a murine implantation model. Together, these findings introduce a class of immune-coupled antibacterial materials as an alternative to the current antifouling paradigm.

Chronic Rhinosinusitis (CRS) is a common chronic inflammation of the paranasal sinus mucosa. Staphylococcus aureus contributes to its severity through biofilm formation. In this study, we isolated eight sequential methicillin-resistant S. aureus (MRSA) isolates from a patient with severe CRS over a period of 672 days (T1-T8). The isolates were phenotypically and genomically characterised, and the extracellular biofilm proteome analysed. We identified an accumulation of mutations that included the acquisition of an IS21 family insertion sequence inactivating the icaR gene and nucleotide variants in various genes including the transcription repair coupling factor (mfd). The genomic changes were associated with a switch to a mucoid phenotype from T3 onwards (Day 178), with a significant increase in biofilm-forming capacity and the secretion of multiple enterotoxins. Targeted mutagenesis confirmed mfd is a regulator of strain mucoidy with enhanced biofilm and enterotoxin production. These findings support mfd as a target for novel anti-virulence therapies.

Biofilm represents a complex aggregation of bacteria embedded within a self-produced extracellular polymeric substance (EPS). We investigated the characteristics of mycobacterial biofilm using Mycobacterium smegmatis (Msm) as model organism. By combining transcriptomic (RNA-seq) and proteomic (LC-MS) analyses, the research captures dynamic changes during the establishment and maturation of the biofilm. Transcriptomics analysis showed a distinct gene expression profile as compared to its planktonic form. Interestingly, clear differences were seen between initial (~2-day old) and mature (~5-day old) biofilm stages, highlighting phasic gene expression throughout biofilm development. Marked alteration in oxidative stress-related genes and energy metabolism from ATP to NADH was observed. Furthermore, quantitative mass spectrometry-based proteome examination of EPS showed an abundance of cytoplasmic proteins present differentially between initial and mature biofilm stages. Pathway enrichment revealed enhanced oxidative stress responses and metabolic shifts in mature biofilms, including upregulation of NADH dehydrogenase and downregulation of ATP synthase, indicating altered energy metabolism. Our findings thus provide insights into the molecular adaptations, including production of mycofactocin, occurring during mycobacterial biofilm establishment and maturation, and advance our understanding of mycobacterial biofilm physiology.

Streptococcus mutans is a key contributor to dental caries, with its capacity to form structured biofilm microcolonies being a principal component of its cariogenic potential. Yet, most mechanistic studies rely on a limited number of laboratory strains and may not capture the functional diversity present across the species. Here, we characterized a panel of phenotypically and genomically diverse S. mutans isolates to determine how strain background influences biofilm architecture, extracellular matrix accumulation, acid-associated physiology, environmental responsiveness, and antimicrobial susceptibility. Quantitative high-resolution imaging revealed extensive heterogeneity in produced biofilm microcolony size, structure, and matrix composition, demonstrating that biofilm architecture is not a uniform species-level trait. Interestingly, the commonly used reference strain UA159 displayed an intermediate phenotype related to microcolony size and biofilm organization. Human saliva further altered biofilm structure and matrix accumulation in a strain-dependent manner rather than producing a standard species-wide response. Isolates also differed in growth and retained biofilm biomass under acidic conditions, while acid accumulation within mature biofilms varied independently of average microcolony volume, demonstrating that strains that produce larger microcolonies on average were not necessarily associated with greater acid accumulation. Susceptibility to the antiseptics chlorhexidine and cetylpyridinium chloride likewise differed among isolates and could not be predicted from formed biofilm architecture alone. Together, these findings demonstrate that disease-relevant traits commonly attributed to S. mutans are distributed unevenly and only partially coupled across strain backgrounds, with biofilm spatial organization failing to serve as a dominant phenotype linking acid accumulation, acid tolerance, and antimicrobial susceptibility.

S. mutans and V. parvula cooperate in dental plaque to assemble a healthy biofilm and are associated with increased caries risk. S. mutans produces lactic acid from carbohydrates resulting in a final biofilm pH[~]4, while V. parvula metabolizes lactate to acetic and propionic acids resulting in pH[~]5. This process results in healthier biofilms that still generate a pH capable of demineralizing tooth surfaces (pH<5.5). The purpose of this study was to identify V. parvula genes required for deacidification of S. mutans biofilms and determine whether the ability of V. parvula to deacidify S. mutans biofilms correlates with enhanced biofilm health. Using transposon mutagenesis in V. parvula we identified several genes required for deacidification of S. mutans biofilms. These included numerous V. parvula transposon mutations in the previously unstudied lutABC lactate utilization operon. To assess biofilm health, S. mutans in the presence of various V. parvula mutants were stained with a LIVE/DEAD stain and imaged by fluorescence microscopy. An intact lutABC operon was required to enhance biofilm health, as demonstrated by plasmid-based complementation of a lutB transposon mutant. Transposon insertions in other loci unrelated to deacidification had no impact on biofilm health. Addition of HEPES buffer at the time of S. mutans biofilm assembly prevented full acidification of the biofilm and resulted in improved biofilm health, even without the addition of V. parvula. Finally, we found V. parvula can use either nitrate or fumarate as a final ETC electron acceptor during lactate utilization. In all, we found the lutABC lactate utilization operon of V. parvula is critical for the ability of V. parvula to deacidify S. mutans biofilms and promote biofilm health. Interfering with this pathway would interrupt the mutually beneficial relationship between S. mutans and V. parvula that leads to their co-association in caries, root caries, and early childhood caries.

The role of spatial structure in microbial ecology and evolution is increasingly recognised and investigated, often with agar plates as a template for a spatially structured environment. While convenient, agar plates do not allow for the spatial and temporal control microbiologists have become accustomed to in the field of microfluidics with its tight environmental control for single cells and small populations, holding back research on surfaces and at larger length scales and population sizes. To close this gap, we developed a novel device with an agar sheet sealing indented channels through which media perfuses. As proof of principle, we grew populations of non-motile Escherichia coli and motile Pseudomonas aeruginosa for 60 hours with continuous propagation of the colonys front, in contrast to agar plates where growth declined much earlier and stopped after about 40 hours. To demonstrate the capabilities of spatial control, we grew P. aeruginosa along different temporally-stable gradients of the cephalosporin antibiotic ceftazidime and characterised the emerging bacterial growth patterns. The device is a step towards highly controlled studies of microbial populations in continuous, non-uniform spatially structured environments. Designed with cost and accessibility in mind, we believe that this novel device will enable new insights into microbial ecology and evolution.

Catheter-associated urinary tract infections (CAUTIs) are the most prevalent healthcare-associated infections globally, yet the ecological dynamics governing polymicrobial biofilm communities on indwelling catheters remain poorly understood under physiologically relevant conditions. Most prior work uses static in vitro models that fail to capture continuous urine flow and sub-inhibitory (sub-MIC) antibiotic gradients. We investigated how continuous flow and sub-MIC concentrations of ciprofloxacin and gentamicin reshape colony-forming unit (CFU) dynamics across attached biofilm and dispersed effluent fractions, and species dominance in mono- and polymicrobial biofilms of Pseudomonas aeruginosa (Pa), Klebsiella pneumoniae (Kp), and Enterococcus faecium (Ef) using silicone-coated latex catheter segments, volumetric infusion pumps, and ibidi {micro}-slide VI 0.4 microfluidic chambers. Under antibiotic-free conditions, Pa dominated both dual co-cultures (Pa+Kp, Pa+Ef) in static condition, but this dominance was not sustained under flow in the Pa+Ef pairing, where Ef rose to 62.5% relative abundance. Sub-MIC ciprofloxacin under flow promoted Kp dispersal (+15.87 log? fold change in dispersed-cell fraction(filter), cooperative Pa recovery via Ef co-occupancy, and pronounced Ef dominance in the triple-species community (64.71% relative abundance). Ef exhibited enhanced growth under sub-MIC gentamicin in static conditions that was abolished under flow. CLSM imaging revealed ciprofloxacin-induced Kp filamentation under flow, with Ef microcolonies localising at filament termini--a novel architectural interaction providing spatial scaffolding for the gram-positive partner. These findings establish that continuous flow and antibiotic class jointly determine polymicrobial dominance outcomes in ways invisible to static assays, underpinning Ef persistence in mature CAUTI biofilms and highlighting flow as a central ecological variable in infection pathogenesis.

Pseudomonas aeruginosa is an opportunistic pathogen that often adopts persistent phenotypes -- such as biofilm formation-- that are associated with chronic infections including those observed in the cystic fibrosis (CF) lung Recently, highly effective modulator therapy (HEMT) such as elexacaftor/tezacaftor/ivacaftor (ETI) has significantly improved the quality of life of people with CF (pwCF). Yet a potential direct impact of ETI on the physiology of P. aeruginosa during growth to a remodeled CF lung environment has remained unexplored. To address this, we conducted an experimental evolution using P. aeruginosa PA14 grown in CF-like conditions in the presence or absence of ETI. We observed a marked reduction in biofilm formation and in the number of small colony variants (SCVs) for P. aeruginosa populations evolved under ETI treatment. Also, sequencing of specific evolved clones exhibiting distinct morphotypes revealed two major observations: (i) P. aeruginosa-evolved communities exposed to ETI retained a wild type-like morphotype and, (ii) P. aeruginosa populations evolved in the absence of ETI adopted a SCV-like phenotype with mutations acquired in the Wsp chemosensory pathway. Furthermore, analysis of evolved populations revealed that ETI treatment likely modulates c-di-GMP pools by driving mutations in an enzyme catalyzing the degradation of this second messenger. Overall, our work suggests that ETI has the potential to hinder the acute to chronic biofilm transition of P. aeruginosa thereby limiting the emergence of variants typically associated with long-term CF lung colonization.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) remains a common cause of skin and soft tissue infections, and topical agents that combine antimicrobial activity with wound compatibility are needed. C12-alkyl(ethylbenzyl)dimethyl ammonium chloride is a novel C12 quaternary ammonium molecule (hereafter, EQ12) related to benzalkonium chloride but designed to avoid the compositional heterogeneity and varied potency of conventional C8-C18 benzalkonium mixtures. We evaluated whether topical EQ12 is tolerated in healing wounds and whether it reduces MRSA burden in a splinted murine excisional wound infection model. MethodsFemale CD1 mice underwent 5-mm full-thickness dorsal excisional wounding, silicone splinting, and transparent dressing placement. For tolerability studies, uninfected wounds received EQ12 at 0.1 or 1 mg/mL or phosphate-buffered saline (PBS) and were followed for 14 days. For efficacy studies, wounds were inoculated with 1 x 104 CFU MRSA and treated topically every 8 hours for 3 days beginning 4 hours after infection with EQ12 (1 mg/mL), bacitracin (500 U/mL), or PBS. Wound tissues were harvested on days 2, 4, and 6 for quantitative culture. Dressing-associated biofilm was evaluated by scanning electron microscopy. ResultsEQ12 did not alter body weight, wound inflammation scores, wound area, or percent wound closure compared with PBS in uninfected animals. In infected wounds, day 2 tissue burdens were not significantly different among treatment groups. By day 4, EQ12 significantly reduced MRSA burden compared with PBS, whereas bacitracin did not. By day 6, EQ12-treated animals had significantly lower MRSA burdens than both bacitracin- and PBS-treated controls. Mean tissue burden in the EQ12 group declined from 6.27 log10 CFU/g on day 4 to 5.53 log10 CFU/g on day 6, while bacitracin- and PBS-treated wounds remained persistently colonized. SEM revealed minimal adherent cocci or matrix-like bridging on dressings from EQ12-treated wounds, in contrast to dense microcolonies and biofilm-like structures on bacitracin and PBS dressings. ConclusionsTopical EQ12 was compatible with gross wound healing and reduced MRSA burden in a dressed, splinted murine wound model. These data support continued development of EQ12 as a topical anti-staphylococcal wound-directed antimicrobial.

Blood banks represent highly controlled healthcare environments where microbiological surveillance has traditionally focused on blood products rather than environmental microbial reservoirs. Despite their critical role in transfusion safety, the ecology of surface-associated microorganisms and the persistence traits that enable their long-term survival remain poorly understood. Here, we combined scanning electron microscopy, culture-based microbiology, phenotypic characterization, MALDI-TOF mass spectrometry, and whole-genome sequencing to investigate whether surfaces within a public blood bank facility constitute reservoirs of environmentally derived bacteria with enhanced persistence potential. Samples collected from a public blood bank in Tucuman, Argentina yielded 37 culturable bacterial isolates, predominantly Gram-positive environmental taxa together with a limited number of opportunistic Gram-negative species. More than 30% of the isolates exhibited multidrug resistance, while several strains displayed strong biofilm formation, amyloid-like fiber production, motility, and hemolytic activity, indicating multiple phenotypic strategies associated with long-term surface persistence. Whole-genome sequencing of six representative isolates confirmed species identity, identified genes related to antimicrobial resistance, adhesion, biofilm formation, stress adaptation, and cytotoxicity, and revealed frequent genotype-phenotype discordance, highlighting the importance of integrating genomic and phenotypic analyses. Notably, one isolate exhibited less than 92% average nucleotide identity with publicly available genomes, suggesting the presence of a previously undescribed environmental species. Thus, blood bank surfaces function as selective ecological niches favoring bacteria with persistence-associated traits rather than simply reflecting contamination from blood products. These microorganisms may constitute latent biosafety hazards if environmental barriers fail, particularly in facilities handling biological materials intended for vulnerable patients. Our results support the incorporation of integrated bioimaging, phenotypic characterization, and genome-resolved environmental surveillance into infection prevention strategies and transfusion biosafety programs within a One Health framework.

ObjectivesFosfomycin (FOS) remains an important therapeutic option for urinary tract infections caused by uropathogenic Escherichia coli (UPEC), but specific virulence traits as biofilm formation, metabolic adaptation, and antimicrobial resistance may limit its efficacy. This study investigated whether the natural compound, trans-cinnamaldehyde (t-CA) potentiates FOS activity against UPEC and explored the underlying mechanisms of its effect MethodsThe interaction between t-CA and FOS was assessed using checkerboard assays, time-kill analysis. We evaluated biofilm viability and structure using confocal and scanning microscopy as well as catheter-associated biofilm models. Next, effects on membrane integrity, cell-surface properties, membrane potential, intracellular pyruvate levels, and resistance evolution during serial passage were evaluated. Molecular docking was used to explore potential interactions of t-CA with enzymes involved in pyruvate metabolism. Galleria mellonella infection model was employed to evaluate in vivo therapeutical efficiency. Resultst-CA potentiated FOS activity against laboratory, reference, and clinical UPEC strains, with synergistic or additive interactions observed across the tested collection. The combination enhanced bacterial killing, reduced biofilm viability and biomass, and disrupted biofilm architecture. In catheter-associated biofilms, combined treatment markedly impaired surface-associated UPEC communities. t-CA reduced extracellular matrix abundance and altered cell-surface hydrophobicity and membrane potential without inducing detectable oxidative stress. Mechanistically, t-CA affected pyruvate homeostasis, reduced intracellular pyruvate levels, and phenotypically intersected with the BtsSR pyruvate-sensing pathway. Serial exposure to FOS alone rapidly increased MIC, whereas t-CA limited this phenomenon and did not itself promote reduced susceptibility. The compounds combination also improved survival of UTI89-infected G. mellonella larvae. Conclusionst-CA enhances FOS activity against UPEC through complementing the antibiofilm and metabolic effects. By weakening biofilm matrix integrity, perturbing pyruvate homeostasis, and limiting FOS-associated MIC elevation, t-CA represents a promising adjuvant candidate for improving FOS efficacy against biofilm-associated UPEC infections.

Lung transplantation (LT) is the ultimate treatment option for patients suffering from end stage cystic fibrosis (CF). Most LT-patients, colonized pre-LT by Pseudomonas aeruginosa witness colonization of their non-CF allograft within a few days or weeks post-LT, thereby compromising graft and life expectancy. How P. aeruginosa isolates adapted for years to the specific CF lung environment efficiently colonize and survive in the non-CF allograft environment remains unclear. To address this question, we collected sequential isolates from CF LT-recipients and non-CF LT-recipients and performed phenotypic and genetic analyses of pairs of early and late isolates from LT-patients. We found evidence for mutations compatible with a switch from biofilm to planktonic lifestyle as well as loss of mucoid phenotypes. Hypermutators, characteristic of chronic CF-adapted isolates, were also found in four LT-patients. Their persistence in the non-CF allograft environment suggests a continuous seeding from the sinuses. Our results suggest that in CF LT-recipients efficient colonisation by P. aeruginosa of the allograft implies both adaptation and continuous seeding from the sinuses to the lower respiratory tract.

Pseudomonas aeruginosa is a major opportunistic pathogen, responsible for healthcare-associated urinary tract infections. Its metabolic flexibility and genomic plasticity promote its survival and adaptation in complex environments. Here, we conducted an in-depth analysis of three pairs of sequential P. aeruginosa urinary isolates, named "early" and "late", from three patients to investigate metabolic and phenotypic changes during urinary tract adaptation. An integrated multi-omics approach combining RNA sequencing and metabolomics was performed on isolates grown in human urine (HU) and trypticase soy (TS) medium, and compared with previously published proteomics data. Late isolates showed down-regulation of genes encoding type VI secretion system in both media, while oxidative phosphorylation associated-genes were up-regulated in HU. These late isolates also displayed significant down-regulation of amino-acid metabolism suggesting an increased use of carbon sources available in urine. As previously observed in proteomics, siderophore- and iron-related genes were significantly down-regulated for all late isolates in HU but not in TS, supporting convergent adaptation to the low-iron urinary environment. Metabolomic profiles of HU supernatants from late isolates clustered together, showing common metabolite production in HU. The differential metabolic profile between early and late isolates included phosphatidylcholines, acylcarnitines, biogenic amines and amino-acids, highlighting their importance in urinary adaptation. While long-term survival in HU and TS was similar between isolates, biofilm formation was reduced or lost in late isolates in line with the down-regulation of biofilm-related genes. These findings highlight the transcriptomic and metabolic reprogramming as well as the phenotypic changes occurring during P. aeruginosa adaptation to the urinary tract. ImportancePseudomonas aeruginosa is a major opportunistic bacterial pathogen responsible for healthcare difficult-to-treat urinary tract infections. Understanding how this bacterium adapts and persists in the human urinary tract is essential for improving the management of persistent infections. Through an integrated analysis of six sequential clinical isolates from three patients, this study shows that P. aeruginosa undergoes coordinated metabolic and phenotypic changes that promote survival in the urinary environment. During adaptation, the bacterium reduces traits involved in bacterial competition and biofilm formation while reprogramming its metabolism to the nutrients available in urine. These changes emerged independently in different patients, revealing convergent adaptation to this challenging environment. By identifying biological processes consistently associated with urinary tract persistence, this work provides new insight into the mechanisms that support long-term colonization and highlights adaptive metabolic pathways that may represent future targets for therapeutic intervention.

Summary/Abstract (this is copy paste of abstract)Human supragingival plaque (SUPP) is a polymicrobial biofilm whose contents undergo dysbiotic transitions during multiple oral diseases. The study of healthy SUPP may lead to future pro or prebiotic therapies, to help prevent or revert dysbiosis during disease. However, many oral plaque models focus on the cultivation of oral pathogens and do not well cultivate commensal SUPP populations. Here, we use a 16S microbiome guided iterative approach to develop a low-cost high sample number SUPP model. Our model demonstrates several findings including a surprisingly minimal impact on salivary preparation methods on model microbiota and the ability to test microbial interactions with added oral strains to assess their fitness. This model provides a reductionist system for the study of healthy oral commensals in a complex polymicrobial framework in the absence of host immune responses.

Antibiotic resistance is a growing global health crisis, yet resistance is almost exclusively quantified under aerobic laboratory conditions that fail to reflect the complex microenvironments bacteria encounter during infection. Many clinically important infection sites, such as airways of individuals with cystic fibrosis or chronic wounds, are microaerobic to anoxic. To address this, we investigated how anoxia alters antibiotic resistance determinants, hypothesizing that anaerobic metabolism might change the fitness effects and selection of resistance mutations. We used experimental evolution to propagate Pseudomonas aeruginosa populations for approximately 200 generations under conditions differing in oxygen availability (oxic vs. anoxic), growth mode (biofilm vs. planktonic), and tobramycin (TOB) exposure (subinhibitory increasing to inhibitory concentrations). Subinhibitory exposure was sufficient to achieve resistance 2-4x greater than ancestral levels, with anoxic populations consistently showing higher minimum inhibitory concentrations than oxic comparisons. Resistance developed through condition-dependent genomic targets: mutations in amgS were selected in oxic populations, while fusA1 and ptsP mutations arose across all conditions. Notably, mexT mutations were nearly universally selected, particularly under anoxic or tobramycin-exposed conditions. mexT inactivation may also enhance virulence through altered quorum sensing and increased rhamnolipid production. Anoxic populations additionally exhibited significantly increased biofilm formation, some exceeding 1000% of ancestral levels, reduced twitching motility driven by type IV pilus gene mutations, and greater competitive fitness. Together, these findings demonstrate that oxygen availability shapes resistance evolution in P. aeruginosa, with the anoxic environment selecting for a more virulent, sessile, and antibiotic-resistant phenotype.

Several mechanisms have been described to explain how bacterial species colonize and persist in the mammalian gut. However, biofilm formation remains underexplored as a mechanism for gut microbiota symbiont persistence. While evidence of biofilm formation by individual gut symbionts is beginning to emerge, its occurrence and relevance in multispecies gut microbiota communities remain poorly studied. Here, we established an in vitro biofilm model for the Oligo-Mouse Microbiota 12 (OMM12) consortium, a defined community of murine gut isolates, and used it to investigate community biofilm formation and responses to bile acids, host-derived detergent-like molecules released into the gut that can perturb bacterial growth and community structure. We identified distinct contributions of two OMM12 members: removal of Enterococcus faecalis strongly reduced community biofilm biomass, whereas removal of Bacteroides caecimuris had limited effect on biomass but strongly altered species associations. These results, together with monoculture assays, show that individual biofilm capacity does not directly predict community-level contribution. Although planktonic and biofilm communities had broadly similar compositions, their response to bile acid stress were markedly distinct. Planktonic cultures, while more susceptible to bile, impaired in biomass and species associations, showed resilience by recovering biomass within 24 hours upon bile stress removal. Community biofilms, in contrast, showed greater tolerance to bile acid stress and preserved or recovered more species associations. Overall, our findings support biofilms as a community-level lifestyle that can buffer defined gut microbiota communities against host-associated chemical perturbations. ImportanceDespite decades of research, how the gut microbiota maintains diversity and persistence remains to be completely understood. Gut bacterial species must withstand harsh host-derived stresses while navigating complex interspecies interactions, many of which being highly competitive. In host-associated contexts, biofilms have largely been viewed as a detrimental trait because of their role in pathogen persistence and protection from clearance, leaving the potential contribution of commensal gut biofilms to microbiota stability underexplored. Our work establishes a simple and adaptable experimental framework to study biofilm formation in a defined multispecies gut bacterial community. We show that biofilms alter how this community responds to bile acids, host-derived molecules that can disrupt bacterial growth and community structure. Our findings support biofilm formation as a protective lifestyle that can help gut symbionts withstand bile acid stress, raising the possibility that community biofilms contribute to microbiota persistence under chemical stress encountered in the host.

Scanning electron microscopy (SEM) is widely used to investigate bacterial surface architecture; however, sample preparation protocols may introduce structural artefacts that compromise the interpretation of morphometric and ultrastructural features. This issue becomes especially relevant for extremophilic microorganisms, whose specialized cell envelopes may respond differently to chemical fixation. In this study, we evaluated the effects of two aldehyde-based fixation protocols, 2.5% glutaraldehyde and Karnovskys solution, combined with different fixation times (1, 3, and 24 h) and the presence or absence of osmium tetroxide (OsO4) post-fixation, on three Gram-positive bacterial strains: the polyextremophiles Exiguobacterium sp. S17 and Nesterenkonia sp. Act20, and the mesophile Kocuria rosea CH-021. Morphological preservation was assessed using morphometric parameters, including cellular area and surface-to-volume ratio, together with texture analysis based on Haralick descriptors derived from grey-level co-occurrence matrices (GLCM). Results showed that fixation conditions significantly affected morphometric and textural features in a strain-dependent manner. Although overall morphology appeared preserved, quantitative analyses revealed marked differences in surface texture and structural integrity among treatments. Osmium tetroxide modified morphometric and textural parameters, although its effects varied among strains and fixation conditions. These findings highlight the importance of integrated quantitative approaches combining cellular geometry and surface texture analysis for the evaluation of SEM preparation protocols in structurally specialized microorganisms.