Biofilm

Urinary tract infections are common healthcare associated infections, a large subset of which are caused by indwelling catheters. Long term catheterization causes persistent, asymptomatic, polymicrobial colonization despite catheters changes and antibiotic usage. In these polymicrobial populations, P. mirabilis, E. faecalis, and E. coli were found as the most common co-colonizing species. We investigated how interactions between P. mirabilis, E. coli, and E. faecalis contribute to biofilm formation and colonization of urinary catheters. Our results show that the interaction between these three species leads to enhanced biofilm biomass driven by an increase in total protein content of the biofilm. Biofilm enhancement required all three species and was also media-dependent, especially for dual-species combinations. Importantly, triple species biofilms also demonstrate biofilm enhancement when established under flow conditions in a biofilm reactor model using silicone urinary catheters. Additionally, triple species biofilm enhancement occurred in co-colonizing isolates from catheterized patients and was found to be specific to interactions between these three species. Triple species biofilms also demonstrated a species-dependent resistance to two commonly used antibiotics, ciprofloxacin and nitrofurantoin. By examining priority effects, E. coli was found to be the main facilitator of biofilm enhancement in a flow model. Finally, proteomics revealed that an L-fucose utilization pathway in E. coli was a key contributor to triple species biofilm enhancement. Overall, our results demonstrate the significant impact of polymicrobial interactions on biofilm formation in the catheterized environment and highlight ways in which complex microbial interplay and priority effects can shape the establishment of persistent colonization.

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.

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.

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.

Chronic wounds are persistent and difficult to treat. Often this is because they are colonized by polymicrobial communities which contribute to changes in antimicrobial susceptibilities, making these infections harder to effectively clear. We explored the role a community can play in individual members survival when challenged by antibiotics, specifically looking at a community consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Acinetobacter baumannii. Our data shows that communities can contribute to both increases and decreases in susceptibilities depending on the species and the antibiotic. The changes in susceptibilities can be due to interspecies cooperation or competition, with identifiable mechanisms. We also demonstrated that current antimicrobial susceptibility testing (AST) methods used in hospitals, which focus on determining the minimum inhibitory concentration (MIC) via determination of visible turbidity breakpoints, are not able to truly indicate the clearance of bacteria, as species can persist in higher antibiotic concentrations after visible turbidity is gone. To combat decreases in antibiotic susceptibilities contributed to by the community, we used our data from individual antibiotics to determine a potentially effective antibiotic combination, similar to combinatorial therapy used in hospitals to treat recalcitrant infections. Our data proved useful, as the combination of gentamicin and cephalexin was able to overcome polymicrobial synergism and clear the desired bacteria. This demonstrates that it is possible to determine effective antibiotic treatments for polymicrobial infections, whether they be combinatorial in nature or not. One simply must account for the role of the community in order to prescribe the most effective treatment.

Biofilms pose a significant challenge to antimicrobial therapy. Bacteria in biofilms differ from planktonic counterpart in their altered metabolism, collective behavior, protective role of extracellular matrix and diversified microbial subpopulations. These attributions significantly influence bioavailability and activity of antibiotics. The presence of bacterial aggregates during acute infections expands the problem to many other conditions previously not discussed in the biofilm context. Klebsiella pneumoniae is a leading cause of life-threatening hospital-acquired infections and is included in the WHO Bacterial Priority Pathogens List due to increasing antimicrobial resistance. The combination of antimicrobial resistance and the ability to form biofilms severely limits the efficacy of antibiotic treatments. In this study, we investigated the in vitro susceptibility of mature biofilms to 13 antimicrobials of K. pneumoniae clinical isolates from a single hospital. The resistance profiles of the local clinical isolates were consistent with the global epidemiology of K. pneumoniae. Minimal biofilm eradication concentrations (MBEC) for mature biofilms were defined with two assays (biomass and metabolic activity measurements) and brought into relation with susceptibility breakpoints and plasma (Cmax). Colistin sulfate, tigecycline, cephalosporins and combination of imipenem with cilastatin were the most potent biomass eradicators, while suppression of metabolic activity was barely reachable. Moreover, we observed a notable increase in metabolic activity upon exposure to sub-MBEC concentrations of antibiotics. Finally, our data broach a subject of antibiotic prioritization with respect to biofilm tolerance. IMPORTANCEThis study addresses the critical gap between standard antibiotic susceptibility testing and the tolerance of biofilm and microbial aggregates during infections caused by K. pneumoniae. By systematically evaluating mature biofilms from a significant number of clinical isolates, we demonstrate that colistin and tigecycline show potent activity against both biofilm biomass and metabolic activity, whereas cephalosporins primarily reduce biomass without effectively suppressing bacterial metabolism, and other drugs have only weak effects on biofilms at clinically achievable concentrations. Furthermore, the alarming observation that sub-inhibitory biofilm eradication concentration (sub-MBEC) of antibiotic can paradoxically increase the metabolic activity of biofilms highlights a potential risk factor for therapy failure and resistance development. Our findings contribute to the necessary evidence base for prioritizing existing antibiotics in the limited armamentarium against biofilm-forming K. pneumoniae.

AimsPseudomonas aeruginosa biofilm-associated infections pose a significant clinical challenge due to their inherent antibiotic tolerance. This study aimed to evaluate the antibacterial and antibiofilm activity of Placentrex, a standardised aqueous placental extract, against P. aeruginosa and to elucidate its molecular mechanism of action using RNA sequencing (RNA-seq). Methods and ResultsPlacentrex exhibited potent bactericidal activity against P. aeruginosa at 50 mg/mL. Biofilm formation was significantly inhibited by [~]87% at 50mg/mL after 72 hours. Preformed biofilms were eradicated by [~]93% and [~]89% at 50 and 25 mg/mL, respectively. Interestingly, biofilm viability was reduced by [~]93% and [~]87% upon treatment with 50 mg/mL and 25 mg/mL of Placentrex, respectively. EPS characterisation revealed that the EPS contain a single large polysaccharide, and chromatography data suggested that it is made up of glucose as a monomer. RNA-seq identified coordinated downregulation of seven key genes, namely, flp major pilin (surface attachment), extracellular solute binding protein (ABC transporter-mediated nutrient sensing and biofilm maintenance), gntP permease (carbon metabolism), AraC family transcriptional regulator (quorum sensing and polysaccharide biosynthesis), ureE (urease nickel metallochaperone), aromatic amino acid permease (pyoverdine and PQS biosynthesis), and MFS transporter (efflux and autoinducer export). ConclusionsPlacentrex exerts comprehensive antibiofilm and antibacterial activity through simultaneous disruption of surface attachment, nutrient-sensing-driven biofilm maintenance, quorum sensing, carbon metabolism, urease virulence maturation, and efflux-mediated persistence. This polypharmacological mechanism supports Placentrex as a promising multi-target antibacterial agent against P. aeruginosa biofilm-associated infections. Impact statementPlacentrex is a potential anti-biofilm agent against Pseudomonas aeruginosa.

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.

Staphylococcus epidermidis biofilm oxygen gradients are spatially mapped during biofilm development and after vancomycin treatment using a solution-based ratiometric imaging platform. By integrating the oxygen-sensitive, tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate with oxygen-insensitive, water-soluble CdSe/ZnS quantum dots, we achieve in situ microscale resolution of dissolved oxygen (DO) concentrations within biofilms. The oxygen-sensing platform is calibrated within alginate hydrogels to mimic probe confinement within biofilms and subsequently validated in biofilms using chemical oxygen depletion (sodium sulfite) and thermal inactivation (60{degrees}C). We demonstrate that the probes do not significantly alter planktonic bacterial growth or biofilm development. Using confocal laser scanning microscopy and quantitative image analysis, 3D microscale oxygen maps of biofilms are visualized and evaluated. During S. epidermidis biofilm development from 12 to 24 hours, average biofilm DO concentrations decrease from 2.62{+/-}0.22 mg/L to 2.02{+/-}0.47 mg/L, corresponding with increased S. epidermidis biofilm biomass and bacterial metabolic activity. While treatment of S. epidermidis biofilms with vancomycin at the minimum inhibitory concentration (MIC) (2 {micro}g/mL) results in negligible DO decreases and biofilm biomass and metabolic activity comparable to untreated biofilms, higher vancomycin concentrations (20, 200 {micro}g/mL) lead to increases in biofilm DO, higher dead-cell biovolumes, and decreased metabolic activity. This work establishes a microscale, solution-based ratiometric platform for quantifying the interplay between biofilm oxygen gradients, structure, and metabolic activity, providing a framework for understanding biofilm resilience during antimicrobial treatment.

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.

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.

BackgroundPhage therapy is increasingly considered a promising alternative for treating multidrug-resistant (MDR) infections. However, its clinical application remains limited by challenges in isolating effective phages against resistant clinical strains and by the limited ability of in vitro assays to predict performance in real biological environments. While biological matrices are known to influence phage activity, these effects are not well characterised. MethodsA phage-resistant Pseudomonas aeruginosa isolate from a patient with recurrent MDR urinary tract infection was used as the model organism. Conventional isolation methods failed to recover effective phages, leading to the development of TEASER-i (Transient EDTA- and Ion-Assisted Sequential Enrichment & Recovery). Recovered phages were characterised using adsorption assays, one-step growth kinetics, and time-kill experiments. Their antibacterial activity was evaluated both in vitro and in ex vivo human matrices (whole blood, serum, plasma, and urine). Phage efficacy was quantified using maximum log reduction (Emax), area under the curve (AUC), and phage-to-bacteria ratio (PBR). ResultsA novel TEASER-i method optimised for difficult-to-treat Gram-negative infections, enabled recovery of a functionally effective Osewage-derived P. aeruginosa phage, which outperformed a Ourine-derived P. aeruginosa phage that showed slower replication and lower burst size. Phage activity varied significantly in blood, serum, and plasma. Urine supported the most sustained antibacterial effect. In many cases, early bacterial reduction was followed by regrowth. Sustained activity was associated with maintenance of favourable PBR values, while negative PBR corresponded to treatment failure. At 96 h, only two conditions maintained favourable phage load (log 10 PBR > 0): the S. aureus phage in urine (+1.66) and the sewage-derived P. aeruginosa phage in serum (+1.32). ConclusionsPhage efficacy depends not only on intrinsic lytic capacity but also on the ability to persist and amplify within specific biological environments. Conventional isolation and in vitro screening may therefore overestimate therapeutic potential. Combining optimised isolation strategies with ex vivo evaluation provides a more realistic framework for phage selection and clinical translation.

The architecture of Bacillus subtilis biofilms is influenced by the coordinated regulation of cellular specialization, matrix assembly, and metabolism. B. subtilis can form different types of biofilm in diverse physical and chemical environments. Understanding the molecular mechanisms that drive biofilm heterogeneity and adaptation to different environmental niches is crucial for developing more effective strategies to control their formation. In this study, we developed a tightly dual-regulated CRISPR interference (CRISPRi) system and employed multi-scale imaging to investigate the functions of individual genes in two distinct biofilm models: the floating pellicle and the intricate, three-dimensionally structured macrocolony, which develop at the liquid-air and solid-air interfaces, respectively. Our findings validated the CRISPRi approach as a powerful method for studying biofilm development over extended periods and revealed that numerous small non-coding RNAs are involved in regulating biofilm growth dynamics and architecture. The CRISPRi approach was also applied to a pool of 507 genes and transcription units, including protein-coding genes and non-coding RNAs, to screen for cell fitness in these two biofilm models. We discovered that, while both biofilm forms rely on fundamental processes such as cell wall synthesis and nucleotide metabolism, they exhibit different genetic dependencies with regard to matrix composition, motility, and signaling. Exopolysaccharide production, motility, and chemotaxis are crucial for pellicle formation. In contrast, macrocolony development is influenced by {gamma}-polyglutamate synthesis and nutrient acquisition functions. Genes of unknown function were also identified to play a differentially important role in the two biofilm forms. Additionally, the CRISPRi screens revealed further non-coding RNAs regulating biofilm architecture and growth dynamics, adding to the existing layers of post-transcriptional control. Collectively, these results demonstrate that biofilm formation at different physical interfaces is governed by a combination of shared and unique genetic pathways tailored to the specific biofilm environment, thereby opening research avenues into the molecular mechanisms specific to the solid-air and liquid-air interfaces.

Enterococcus faecalis is a Gram-positive intestinal commensal and opportunistic pathogen capable of causing serious infections, including urinary tract infections, endocarditis, and wound infections. A major contributor to its persistence during infection is the ability to form biofilms on host tissues and medical devices. Biofilm cells have higher phenotypic tolerance to antimicrobial treatment than planktonic bacteria. While mechanisms governing biofilm assembly in E. faecalis have been widely studied, the processes that regulate biofilm dispersion, the final stage of the biofilm life cycle, remain poorly understood. In this study, we found that dispersion is triggered by a tenfold step-change increase in nutrient availability and by cell free supernatant (CFS) of E. faecalis OG1RF cultures. Cells released from biofilms regain sensitivity to antibiotics similar to planktonic cells but maintain a high potential for adherence. We characterized the glycosyltransferase epaOX, which contributes to the structure of the enterococcal polysaccharide antigen as necessary for nutrient step-change induced dispersion, CFS induced dispersion, and adhesion of dispersed cells. Supplementation of epaOX mutant CFS with galactose and N-acetylgalactosamine was sufficient to restore CFS induced dispersion. Together these data suggest that dispersion in OG1RF occurs with fast kinetics, affects antibiotic sensitivity and is regulated in part by known virulence factors. ImportanceE. faecalis causes difficult to treat infections at numerous body sites in human patients. E. faecalis biofilms are adherent populations that require high levels of antibiotics for treatment. Biofilms undergo a disassembly process named dispersion that allows individual cells to leave the biofilm and colonize new locations. Dispersed cells in other species are killed by lower amounts of antibiotics than biofilm cells. Here we showed that dispersion occurs in E. faecalis and lowers the level of antibiotics needed to kill dispersed cells. Dispersion triggers could be used in the future to design treatments that increase the effectiveness of antibiotics.

Background. Scanning electron microscopy (SEM) is widely used to determine fibrin fiber structural properties such as fiber diameter and fiber length. However, conventional SEM preparation protocols are time-consuming and typically require conductive sputter coating. The coating process introduces an additional layer onto the sample surface and may influence measurements of nanoscale fiber structure. Furthermore, preparation of purified fibrinogen clots often follows protocols originally developed for plasma clots, resulting in unnecessary processing steps. Objective. To evaluate indium tin oxide (ITO) as a flat, conductive substrate for SEM imaging of fibrin fibers, investigate the effects of sputter coating on measured fiber diameter, and develop a simplified SEM preparation protocol for purified fibrinogen clots. Methods. Platelet-poor plasma clots and purified fibrinogen clots were formed on ITO substrates and imaged by SEM following 0 s, 45 s, or 90 s sputter coating. Fibrin fiber diameters were quantified and compared across coating conditions. For purified fibrinogen clots, an ITO-based simplified preparation protocol, in which clots were formed and imaged directly on the conductive ITO surface, was compared with a previously developed, standardized SEM protocol, in which clots were formed in microtube lids and subsequently transferred onto carbon tape for imaging. Results. Fiber diameter measurements were affected by sputter coating duration, with increasing coating time resulting in larger apparent fiber diameters. Plasma and purified fibrinogen clots exhibited distinct fiber diameter distributions and coating responses. For purified fibrinogen clots, the simplified ITO-based protocol produced fiber diameter measurements that were not significantly different from those obtained using the standardized lid-to-carbon-tape workflow when identical coating times were applied. Conclusions. ITO provides a practical conductive substrate for SEM imaging of fibrin fibers and enables substantial simplification of purified fibrinogen clot preparation. When coating conditions are matched, the simplified ITO-based protocol yields fiber diameter measurements comparable to those obtained using the previously standardized lid-to-carbon-tape workflow. These findings support the use of ITO as an alternative conductive imaging substrate and provide a simplified workflow for SEM analysis of purified fibrinogen clots. By reducing washing and transfer steps, this workflow may also provide a useful platform for future controlled studies of fibrin interactions with added proteins or other associated components.

The search for antimicrobials with a low propensity to select resistance has intensified in response to the global antimicrobial resistance crisis. Norway spruce resin (Picea abies) has long been used in Northern European wound care traditions and has shown broad antimicrobial activity in earlier microbiological studies. In the present study, we evaluated whether prolonged exposure to medical-grade spruce resin promotes reduced susceptibility in clinically relevant bacterial species. A 20-day serial-passage experiment was performed with Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis using sub-inhibitory resin concentrations and broth microdilution readouts at baseline, day 10, and day 20. Resistance development was predefined as a [≥]4-fold increase in inhibitory concentration. Baseline inhibitory concentrations were 1.25% for S. aureus, 5.0% for P. aeruginosa, and 2.5% for E. faecalis. After 20 days, inhibitory concentrations were 2.5%, 10.0%, and 2.5%, respectively, corresponding to at most 2-fold changes and remaining below the predefined threshold for resistance development. Validation and vehicle-control arms indicated that these shifts were not attributable to medium transfer or solvent-related bias. These findings suggest that medical-grade Norway spruce resin has a low short-term tendency to select for reduced susceptibility under serial-passage conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723837v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@160479forg.highwire.dtl.DTLVardef@1fe1e95org.highwire.dtl.DTLVardef@89dec3org.highwire.dtl.DTLVardef@17ff134_HPS_FORMAT_FIGEXP M_FIG C_FIG

Necrotizing soft tissue infections (NSTIs) carry 10 to 30% mortality. Current empirical antimicrobial guidance derives almost entirely from Western cohorts dominated by Streptococcus pyogenes and aerobic-anaerobic consortia, yet whether this microbial paradigm applies to tropical, high-antimicrobial-pressure settings has not been tested with culture-independent methods. We did a prospective cohort study of 169 patients with intraoperatively confirmed NSTI at a North Indian tertiary center (2021 to 2024). Wound tissue underwent aerobic and anaerobic culture, QIIME2-based 16S rRNA gene amplicon sequencing (V3-V4), and targeted SYBR Green quantitative PCR (qPCR) for Acinetobacter baumannii and S. pyogenes. The wound microbiota was overwhelmingly Gram-negative and polymicrobial, anchored by A. baumannii (culture, 33.7%; metagenomics, 49.1%; qPCR, 37.9%), Escherichia coli (32.0%), and Klebsiella pneumoniae (20.7%); S. pyogenes contributed only 4.7% of culture-positive cases. Polymicrobial wounds had higher Shannon diversity (2.59 versus 2.33; P = 0.048) and discrete community composition (PERMANOVA R2 = 0.511; P = 0.010). Culture-metagenomics agreement ranged from almost perfect for Escherichia ({kappa} = 0.849) to slight for Streptococcus ({kappa} = 0.131). North Indian NSTIs present a microbial picture distinct from the Western paradigm, with implications for empirical therapy.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation or ciprofloxacin resistance have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotic challenge remains incompletely understood. In this study we employed a whole genome screen to assay the impact of gene disruptions or altered gene expression on survival of P. aeruginosa biofilms exposed to different concentrations of ciprofloxacin. Genes impacting fitness in the biofilm context were identified by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Genes associated with c-di-GMP regulation and Gac/Rsm signalling were identified as primary regulators for biofilm formation in the presence and absence of ciprofloxacin. In addition, a group of genes involved in respiration, metabolism (especially polyamine metabolism), and various transporter and efflux systems were identified as important for biofilm fitness. Ciprofloxacin specifically imposed a selective pressure on flagellar function and Psl production which were essential for survival in early biofilms. Moreover, transposon insertions within the CPA gene clusters (PA5448-PA5451 and PA5455-PA5456) and the salvage peptidoglycan recycling pathway showed reduced fitness in late biofilms at high concentration of ciprofloxacin, indicating that cell envelope integrity is beneficial for mature biofilms. This study identifies important determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin and implicates potential therapeutic targets for antibiofilm drug development.

Abstract Introduction Postal sputum sampling represents a potential strategy for patient-led, efficient, and regular sampling, yet little is known about the validity of posting samples for clinical and research purposes in bronchiectasis. This study aimed to validate postal sputum sampling for clinical and research applications in chronic P. aeruginosa infection. Methods Sputum was collected from 12 participants with bronchiectasis and known P. aeruginosa infection. Each sputum sample was divided into four aliquots: two were sent immediately for analysis with or without DNA-Shield (shield-fresh and non-shield-Fresh), while two were transported through the UK postal service, with or without DNA-Shield (shield-posted and non-shield-posted). All aliquots were sent at ambient temperature and subsequently processed for bacterial enumeration through selective culture, detailed antimicrobial susceptibility testing, quantitative PCR (qPCR), 16S microbiome sequencing, metabolomics, and proteomics. Results During postage, there was a median of four days (range, 2-7) between sample collection and processing. 7/12 patients were positive for P. aeruginosa by culture of fresh samples, with 100% agreement in posted samples. Postage did not affect cultured (p=0.81) or amplified load of P. aeruginosa (p=0.94), and no differences were observed in AST profiles across 140 isolates for P. aeruginosa cultured from fresh or posted samples. Metabolomics and proteomics revealed that variation between individuals was significantly greater than between fresh and posted samples, and no significant differences in microbial taxa were observed between samples. No differences were associated with the addition of DNA Shield by qPCR (p=0.19), however, freeze-thaw from -80{degrees}C increased amplified load (p=<0.01). Conclusions We found little evidence of an effect of postage on sputum positivity, recoverable load, AST profile, microbiome, proteome or metabolome in sputum samples. These data suggest postal sputum samples may be a valuable tool for clinical and research applications.

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.