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.

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.

Total laryngectomy is performed for advanced laryngeal and hypopharyngeal cancers. Following the surgery, the insertion of indwelling silicone-based voice prosthesis (VP) implants is considered the gold standard for speech rehabilitation. However, early VP failure due to biofilm formation and invasion of the VP surface can lead to routine use of nystatin and the need for frequent valve changes. Despite these burdens, the composition and impact of colonising pathogens on VP integrity are poorly understood. We have developed a rapid pipeline which permitted the transfer, fixation, staining, and imaging of explanted VPs from patients within 90 minutes of sample collection. Using this pipeline, we demonstrate a multimodal reflection contrast and fluorescence confocal laser scanning microscopy technique to simultaneously map the VP surface and visualise the biofilms in situ, providing new insights into the role of pathogens on VP failure. Our results reveal that complex fungal and bacterial biofilms form polymicrobial biofilms on the VP surface. We demonstrate that biofilms not only reside on the VP surface but can invade the device, releasing silicone particles that are visible within the volume of the biofilms and are separate from the VP surface. Our findings highlight the complexity of the biofilms that form on speech valves; complex polymicrobial biofilms require more advanced treatment than what is conventionally used. Work should focus both on the patient factors that may influence this and the material properties of the valve.

Background: Indwelling urinary catheters can cause pain, urethral irritation and trauma and provide surfaces for microbial colonisation and biofilm formation. The LubriShield(R) Foley catheter incorporates a covalently bonded, superhydrophilic antifouling hydrogel designed to reduce surface friction and limit biofilm accumulation without releasing an antimicrobial agent. This study evaluated its safety and clinical performance compared with an uncoated silicone Foley catheter, including usability and patient comfort, and explored catheter-associated microbial colonisation and biofilm formation. Methods: This prospective, randomised, single-blind, single-centre controlled clinical investigation enrolled 33 adults requiring indwelling urinary catheterisation for 72 hours to 14 days. Participants received either the LubriShield(R) Foley catheter (n = 16) or an uncoated Sterimed all-silicone Foley catheter (n = 17). The primary endpoint was safety based on adverse-event reporting; the prespecified safety criterion was the absence of a serious adverse device effect in the LubriShield(R) group. Secondary endpoints included investigator-rated ease and functionality of catheter insertion and removal and participant-reported pain and irritation/discomfort assessed using 0-10 numerical rating scales. Urine microbiology, catheter colonisation and catheter-level biofilm measurements were exploratory endpoints. Analyses were performed in the as-treated population. Numerical rating scale outcomes were compared using two-sided Mann-Whitney U tests; exploratory biofilm analyses used permutation tests with bootstrap confidence intervals. Results: No serious adverse events or serious adverse device effects occurred. Nine non-serious adverse events were reported and resolved completely. At least one adverse event occurred in 4/16 (25.0%) participants in the LubriShield(R) group and 4/17 (23.5%) participants in the control group. The prespecified safety criterion, defined as the absence of a SADE in the LubriShield(R) group, was met. LubriShield(R) was rated more favourably for ease of insertion (p = 0.0096), functionality during insertion (p = 0.0051), ease of removal (p = 0.0037) and functionality during removal (p = 0.0085). Participants reported significantly less pain and irritation/discomfort during insertion, while the catheter was indwelling and during removal (all p < 0.05). Outcomes 7 {+/-} 1 days after catheter removal were similar between groups. Absolute biofilm biomass and bacterial and fungal loads did not differ significantly. In a post hoc exploratory normalised analysis, the biofilm-to-bacterial-load ratio was lower with LubriShield(R) (median 0.052 vs 0.080; permutation p = 0.043), although the bootstrap 95% confidence interval for the between-group difference included zero. Conclusions: The LubriShield(R) Foley catheter demonstrated a favourable short-term safety profile and was associated with improved investigator-rated usability and participant-reported comfort compared with an uncoated silicone Foley catheter. A post hoc exploratory analysis was compatible with reduced biofilm accumulation relative to bacterial load, but this finding is hypothesis-generating only and requires confirmation in larger, adequately powered studies with prespecified biofilm endpoints.

The crystal violet (CV) microtiter plate assay is widely used to compare microbial biofilm formation because it is inexpensive, simple, and readily adapted to 96-well formats. Its high-throughput design enables multiple strains, growth conditions, and treatments to be tested in parallel, but increasing experimental complexity can make downstream data handling difficult. Plate layouts, absorbance measurements, blank correction, replicate analysis, and visualization are often managed using separate spreadsheets or software, requiring repeated transfer and organization of experimental information. This can compromise consistency in linking measurements with their corresponding conditions and controls. We developed BiofilmQ-HT, a standalone, browser-based workflow that integrates plate annotation with direct import of plate-reader data, condition-specific blank correction, replicate summarization, growth-normalized specific biofilm formation (SBF) analysis, percentage-inhibition analysis, visualization, and data export. The workflow was applied to three representative datasets: a comparison of biofilm formation in 25% and 100% media, an analysis of bacteriophage- and antibiotic-associated antibiofilm activity, and a screening of biofilm formation among natural microbial isolates. These applications demonstrate a common analytical framework for comparative biofilm analysis, antibiofilm screening, and high-throughput isolate screening while retaining experimental conditions and controls. By linking plate organization to quantitative analysis and reporting, BiofilmQ-HT provides a reproducible and traceable approach for processing condition-rich CV assay datasets and facilitates consistent comparisons across experimental groups. The utility therefore improves consistency in data handling without altering the underlying experimental assay or requiring changes to the established assay.

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.

Broad-spectrum {beta}-lactam exposure can select for Enterococcus-dominated urinary communities in catheterized intensive-care patients, even when co-colonizing Escherichia coli remains susceptible. We investigated paired E. faecalis and E. coli isolates recovered before and after piperacillin-tazobactam (TZP) treatment using a catheter biofilm model and showed that their survival depends on mutualism and biofilm-dependent persistence. Without antibiotics, E. faecalis reduced E. coli biofilm formation yet promoted pre-attachment co-aggregation and reorganized mixed-biofilm architecture on the catheter. Despite TZP susceptibility and the absence of resistance determinants, catheter-associated biofilms and biofilm-dispersed cells survived concentrations 250- to 1000-fold above their MICs, whereas planktonic cells were eliminated. Survivors retained susceptibility but showed delayed regrowth, consistent with a transient persister-like state. In the post-treatment pair, each species sustained the other during recovery, coinciding with a nonsynonymous substitution in the enterococcal surface adhesin Esp. These findings show that antagonistic and cooperative interactions can coexist within catheter biofilms and enable susceptible polymicrobial communities to withstand {beta}-lactam treatment without {beta}-lactam resistance.

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.

Oral multispecies biofilms developing on dental implants drive peri-implant mucositis and peri-implantitis. They are shaped by the physicochemical conditions of their surrounding environment, among which oxygen availability is an important influential factor. However, while oxygen tension fluctuates significantly within the peri-implant pocket, whether these environmental shifts are sufficient to influence the structure and composition of an established microbial community remains unclear. For this purpose, we evaluated the structure and composition of a four-species oral biofilm model comprising the species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, which was cultivated under normoxic (21% O2), hypoxic (1% O2) and anoxic (0% O2;) conditions for a period of 21 days. Biofilm 3D architecture, volume and membrane integrity was analyzed by LIVE/DEAD staining combined with confocal laser scanning microscopy. Community composition was assessed by quantitative real-time PCR and complemented by Fluorescence In Situ Hybridization (FISH) to provide structural context. While normoxia transiently promoted higher early biofilm volume and membrane integrity, the community exhibited remarkable long-term resilience, consistently maintaining a V. dispar-dominated structure across all oxygen gradients. Crucially, the obligate anaerobic pathogen P. gingivalis persisted as a viable minority (~1%) even under 21-day normoxic cultivation, sheltered within the deeper layers of the biofilm. These results suggest that clinical oxygen levels alone are not able to drastically modulate established oral biofilm communities and that oxygen-sensitive pathogens persist in a subclinical sanctuary protected by the surrounding non-pathogenic species.

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.

Pseudomonas aeruginosa is an opportunistic pathogen that occupies diverse ecological niches, including soil, water, and the human host. Environmental cues encountered across these habitats trigger adaptive responses that promote survival and persistence through regulation of virulence-associated traits, including secreted factors, siderophores, and biofilm exopolysaccharides (EPS). One major change experienced during the transition from environmental reservoirs to the host is an increase in temperature. Although temperature is a key signal encountered during host transition, its global impact on P. aeruginosa physiology remains poorly understood. We therefore investigated the effects of temperature on planktonic and biofilm-populations, comparing both growth states at 23{degrees}C and 30{degrees}C, representing environmental temperatures, and at 37{degrees}C and 40{degrees}C, representing normal and febrile host temperatures. Transcriptomic and phenotypic analyses revealed extensive temperature-dependent regulation of virulence determinants in both growth states. Expression of the type VI secretion system was elevated at environmental temperatures, whereas pyoverdine biosynthesis and the type III secretion system were upregulated at host temperatures. Building on our previous finding that biofilms formed at environmental and host temperatures differ in architecture, biomass, and EPS composition, we next examined how these structural differences influence stress tolerance. Biofilms grown at 23{degrees}C and 30{degrees}C exhibited substantially greater tolerance to antibiotic stress than biofilms grown at 37{degrees}C and 40{degrees}C. Growth temperature therefore establishes biofilm properties that subsequently influence the stress-tolerance profile of the population. Collectively, our findings identify temperature as a major environmental cue that reprograms P. aeruginosa physiology in ways likely to support persistence across distinct ecological niches.

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.

Mycoplasma pneumoniae is a leading cause of community acquired pneumonia and bronchitis in people, particularly children and young adults. Like many bacterial pathogens, M. pneumoniae can grow in biofilm towers on both biotic and abiotic surfaces. Current models indicate that planktonic M. pneumoniae are simply individual cells that are dispersed from biofilm towers due to physical shear forces, and do not represent a bona fide planktonic growth state. However recent data indicate that these fractions may be physiologically distinct and may differentially influence disease outcome. We observed that experimental infection of mice with inoculum containing both adherent and non-adherent fractions of M. pneumoniae produced more severe lung pathology than inoculum prepared from M. pneumoniae adherent biofilms alone. Direct comparison of experimental infection with titer matched non-adherent (P-Mp) and adherent (B-Mp) fractions revealed that P-Mp produces more severe lung pathology compared to infection with B-Mp. Furthermore, experimental infection with a lower dose of P-Mp resulted in similar lung pathology relative to B-Mp infected mice. This increase in pathology was not associated with differences in bacterial burden, bacterial dispersal during inoculum preparation, nor due to differences in the phenotype of host immune responses. Comparative proteomics revealed that certain known and putative virulence factors associated with immune evasion are upregulated in P-Mp, potentially explaining the difference in virulence between the two fractions. Furthermore, comparative proteomics revealed distinct metabolic phenotypes between the two fractions, consistent with distinct planktonic and biofilm growth states, indicating that P-Mp likely represents a bona fide planktonic growth state. Collectively, these data implicate the growth state of M. pneumoniae as a determinant of in vivo virulence for this atypical human pathogen.

In the environment, bacteria mainly exist in multicellular, surface-adhered communities called biofilms. One key feature of the biofilms is a self-produced extracellular matrix that embeds cells within a structured community and contributes to their adhesion, organization, and retention. The matrix of most characterized biofilms contains three primary components: polysaccharides, protein, and extracellular DNA (eDNA). Upon biofilm induction, Bacillus subtilis produces and secretes a polysaccharide known as exopolysaccharide (EPS). The 15 genes of the epsA-O operon encode proteins that coordinate the assembly, export, and polymerization of EPS oligosaccharide (glycan) subunits. While much progress has been made to elucidate the chemical structure of EPS, further work is needed to determine the contributions of some eps genes to EPS biosynthesis experimentally. Here, we generated a collection of marker-less, in-frame deletions targeting each eps gene in the epsA-O operon in B. subtilis strain NCIB 3610 using the Cre system. Biofilm phenotypes of resulting mutants were used to assess each gene's importance to biofilm development. Impact The epsA-O single-gene deletion collection was designed to enable systematic assessment of the contribution of each eps gene to EPS production and biofilm-associated phenotypes. When combined with current knowledge of EPS structure, this collection will help clarify the roles of individual genes in EPS biosynthesis. This approach can also be easily adapted for the systematic analyses of other large, complex operons in diverse bacterial species.

Bacillus subtilis is the best-studied Gram-positive microorganism, but little is known about how the presence and utilization of diverse carbon sources impacts its production of biofilm. Here, we have identified gluconate as a carbon source that induces biofilm wrinkling in B. subtilis colony biofilms. Targeted phenotypic analysis revealed that the genes encoding the canonical structural biofilm components (tasA, epsA-O, and bslA) are required for this response. Import and metabolism of gluconate, however, are not required to induce these phenotypic changes. We show that the effect of gluconate on B. subtilis is linked to iron availability: production of the siderophore bacillibactin is decreased by gluconate and B. subtilis mutants unable to import bacillibactin grow better and wrinkle more in the presence of gluconate. In addition, supplementation of single-carbon media with iron dramatically increased growth and wrinkling of a B. subtilis bacillibactin mutant grown on gluconate but did not impact B. subtilis grown on glucose. We conclude that gluconate acts as a signal to increase biofilm in B. subtilis and increases iron availability in a bacillibactin-independent manner.

The cell envelope of many bacteria possesses components that play central roles in bacterial pathogenesis. Unique to mycobacteria is the presence of a lipid-rich cell wall which is critical for the virulence of pathogenic mycobacteria such as Mycobacterium tuberculosis (Mtb). The biosynthesis of mycobacterial cell wall lipids has been well characterized, but the mechanisms of lipid transport still remain largely unknown. MmpL (mycobacterial membrane protein large) proteins have been implicated in the biosynthesis and/or transport of mycobacterial cell wall lipids, but due to their cellular location, it is still unclear how cell wall lipids are transported beyond the inner membrane. Here, we further investigate the role of two conserved lipid transport pathways LprG-Rv1410c and MmpL11 in cell envelope biogenesis during biofilm formation in Mycobacterium smegmatis. We found that deletion of the lprG-rv1410c operon homologues MSMEG_3070-3069 and mmpL11 (MSMEG_0241) simultaneously led to similar biofilm defects as observed in the MSMEG_3070-3069 and mmpL11 mutants. Analysis of pellicle biofilms, total lipid extracts, gene expression and cellular c-di-GMP levels revealed that the shared biofilm defect is directly correlated with significant decreases in cellular c-di-GMP levels, but may also be due to changes in the synthesis and/or localization of glycopeptidolipids (GPLs), and changes in the expression of GPL biosynthesis and transport genes. Our findings therefore suggest that while both LprG-Rv1410c and MmpL11 pathways are involved in modulating cellular c-di-GMP levels during biofilm formation, only LprG-Rv1410c are important for regulating GPL synthesis and/or surface localization, and MmpL11 may play a broad role in fine-tuning GPL levels

Infection following high-energy open fractures remains a major challenge. Open fractures are exposed to environmental and skin pathogens, which, along with traumatized tissue and implanted hardware, create a favorable environment for infection. 24-83% of open fractures yield positive culture results. Despite culture-positive wounds being at higher risk for infection, culture data are rarely used in clinical decision-making. Culture-positivity in open fractures is believed to indicate higher wound bioburden, but this hypothesis remains unconfirmed. This study aimed to correlate culture positivity and quantitative wound bioburden using next-generation sequencing (NGS) and to characterize patterns of bioburden and bacterial diversity amongst patients with open fracture and Fracture Related Infection. We hypothesized that higher wound bioburden would be associated with culture positivity. This secondary analysis of the METRC Bioburden Study included 78 patients with severe open tibia fractures who underwent standardized tissue sampling at definitive wound closure and at later fracture site revision surgery. DNA extracted from intraoperative tissue was analyzed via 16S rRNA gene amplicon sequencing. Quantitative bioburden metrics included bacterial read percentage and bacteria-to-human read ratio (BHR). Associations between sequencing-based bioburden, culture results, and clinical infection (CDC-defined) were evaluated using Mann-Whitney U tests, McNemars chi-squared tests, and unsupervised clustering analyses. Culture-positive samples exhibited significantly higher bacterial read percentages and higher log(BHR) values than culture-negative samples. Optimal thresholds yielded high sensitivity and specificity for both bacterial reads and BHR for predicting culture positivity. Neither baseline culture nor NGS positivity predicted later infection. However, follow-up samples from patients with established infection showed significantly higher bioburden and distinct microbiome structures. Clustering analyses identified two infection-associated microbial profiles: one with high bioburden and high diversity (polymicrobial infection) and one with low bioburden and low diversity (single-dominant pathogen). Culture positivity in open fractures correlates strongly with increased bacterial bioburden, indicating that positive cultures may primarily reflect microbial load rather than specific pathogens. Quantitative NGS-based measures, including bacterial read proportion and BHR, may offer a biologically grounded approach to assess wound contamination and may inform infection risk. Distinct bioburden-diversity patterns suggest heterogeneous infection phenotypes that may require tailored surgical and antimicrobial strategies.

Biofilms are widely distributed in both natural and engineered systems and play a fundamental role in microbial ecology and biotechnological applications. When grown on agar substrates, biofilms often exhibit macroscopic structural features due to mechanical instabilities driven by matrix production. These wrinkles encode relevant information about biofilm growth and structural development and have even been shown to possess some functional roles. Despite their relevance, currently available approaches for wrinkle detection from images rely on manual annotation, which is time-consuming, not scalable, and strongly dependent on the annotator. In the present study, we developed an end-to-end machine learning pipeline, named AutoWrinkleID, that starts from bright field images of biofilms and performs wrinkle identification, characterisation, and quantification across multiple bacterial species and strains. The name reflects the automated identification and subsequent characterisation and quantification of biofilm wrinkles. The annotations used to generate the masks, namely images containing only the wrinkle structures, were obtained both through manual labelling and using constitutive fluorescence and motility fluorescence. Interestingly, we found that models trained using only manual annotations perform worse than those trained using motility fluorescence markers. A comparison between motility and constitutive fluorescence masks further indicates that motility based annotations consistently outperform constitutive fluorescence across all strains, strongly suggesting that motile phenotypes are tightly associated with wrinkle structures. Additional computational analysis is conducted on the same dataset to assess the robustness and limitations of the results as a function of training dataset. We also observed that Sholl analysis, originally developed for the study of neuronal dendrites and later applied to wrinkle analysis, can effectively characterize wrinkle patterns, although its applicability is strongly limited by wrinkle shape and by the quality of the masks.

Delafloxacin (DLX) is a novel fluoroquinolone with enhanced antibacterial activity in acidic environments, a property that may be advantageous for treating Pseudomonas aeruginosa infections in cystic fibrosis (CF), where airway surface liquid pH is typically reduced (pH 5.5-6.7). However, the propensity for resistance development and the underlying mechanisms in P. aeruginosa remain incompletely defined. We conducted serial passage experiments on six clinical P. aeruginosa isolates (three CF-derived and three non-CF-derived) exposed to sub-inhibitory concentrations of DLX or ciprofloxacin (CIP) at pH 6.0 and 7.3 over nine days. Susceptibility was assessed by broth microdilution (BMD), and resistance mechanisms were characterized by whole-genome sequencing (WGS), efflux pump expression analysis (qRT-PCR), and functional validation using CRISPR/Cas9-mediated genome editing and complementation assays. DLX minimal inhibitory concentrations (MICs) rose only 10.1- to 28.5-fold over 9 days, compared with 77.6- to 97.8-fold for CIP, indicating a substantially higher genetic barrier to resistance. This barrier was most pronounced under acidic conditions: only 38.9% of DLX-passaged samples crossed the resistance breakpoint, compared with 94.4% at neutral pH, whereas CIP resistance reached 100% regardless of pH. Cross-resistance was asymmetric: exposure to DLX consistently selected for CIP cross-resistance (97.2% of samples), whereas exposure to CIP induced DLX cross-resistance efficiently at neutral pH but only partially under acidic conditions. A previously undescribed gyrA mutation (p.Ala51Val) conferred a 4-fold increase in DLX MIC when introduced by CRISPR/Cas9, and upregulation of the MexEF-OprN efflux pump, reversible by mexS complementation, emerged as a prominent resistance mechanism. Overall, DLX exhibited a markedly higher genetic barrier to resistance than CIP in P. aeruginosa, particularly under the acidic conditions characteristic of the CF airway. However, its use may co-select for CIP cross-resistance through efflux upregulation, underscoring the need for careful stewardship in CF.