Microbiology

Pseudolysogeny is a latent state in which phage development is delayed after infection and has been proposed to promote phage persistence under unfavorable conditions. Virulent phage T3 has been reported to establish pseudolysogeny after infecting starved E. coli, then resume lytic replication following transfer to nutrient-rich media, a phenotype linked to the T3 SAMase gene. Here, we revisited the findings of Krueger et al. (1975) to test pseudolysogeny in T3 and examine phage propagation under nutrient-limited conditions. Both T3 and T7 showed impaired propagation under nutrient limitation, with the most stringent conditions causing substantial losses in recoverable infective centers. T3 was modestly more resilient than T7 under these conditions, but we were unable to reproduce the reported phenotype in which T3 remained latent while T7 replicated normally. Supplementation of minimal medium with small amounts of LB supported propagation of both phages, and a repeat experiment designed to more closely match the historical protocol, including post-adsorption reduction of extracellular phage carryover, likewise failed to reveal a T3-specific pseudolysogenic state. Together, our results indicate that, in this experimental system, phage propagation dynamics are more consistently explained by nutrient conditions and media switching than by starvation prior to infection. These findings suggest that the previously reported T3 pseudolysogeny phenotype may depend on additional environmental or methodological factors and underscore the importance of revisiting historically reported phage behaviors using modern controls.

The acquisition of antibiotic resistance is typically associated with a fitness cost, reducing the competitive ability of cells in the absence of antibiotics. Our ability to predict these costs informs our ability to predict how likely it is that resistance will subside in the absence of treatment. Fitness costs are typically measured in laboratory cultures using a nutritionally rich medium for growth. Here, we tested the impact of infection-like environments on the prevalence and magnitude of fitness costs associated with antibiotic resistance mutations in the opportunistic pathogen Pseudomonas aeruginosa. We assessed two different clinically relevant infection niches, the lung and urinary tract, through the use of infection-niche mimicking media that models the chemical compositions of these niches. The fitness costs of resistance mutations in rich media did not reliably predict those in infection-like environments, on average costs were increased in the lung-mimicking environment and costly mutations were less prevalent in the urinary tract-mimicking environment. Synthetic human urine significantly enhanced the fitness of P. aeruginosa with resistance mutations in nfxB, encoding a multidrug efflux pump transcriptional repressor, and we identified oxalate as the likely component driving this effect. Our findings highlight the importance of environmental chemical composition for shaping the fitness costs of resistance in P. aeruginosa and suggest that nfxB mutants may have a selective advantage in the urinary tract, mediated by the presence of oxalate.

Intracellularly acting antibacterials must cross the bacterial inner membrane to reach their targets in the cytoplasm. Using a panel of isogenic Escherichia coli strains with single, double and triple deletions of sbmA, ygdD and mdtM the susceptibility to bleomycin, Oncocin, Api88 and Bac7(1-17), was assessed together with bacterial fitness in vitro and in vivo during mouse intestinal colonization. SbmA was the dominant susceptibility determinant, while YgdD contributed strongly to bleomycin and Api88 susceptibility and more modestly to Oncocin and Bac7(1-17) susceptibility. Simultaneous loss of YgdD and SbmA produced both a 128-fold increase in the bleomycin MIC and a 37% increase in doubling time, indicating that the two proteins share partly overlapping functions. Single mutants showed no detectable growth defect, but the {Delta}ygdD {Delta}sbmA, {Delta}ygdD {Delta}mdtM and {Delta}ygdD {Delta}sbmA{Delta}mdtM mutants showed significant growth defects. In mice, {Delta}sbmA and {Delta}mdtM showed reduced late competitive persistence. These findings support a compound-specific network of inner-membrane susceptibility determinants with context-dependent fitness costs.

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, contributing to membrane integrity and environmental interactions. Genome-wide studies defining bacterial gene functions have been extensively performed in the model strain Escherichia coli K-12 which lacks O-antigen (OAg), and therefore does not produce smooth LPS (S-LPS). Consequently, the genetic requirements for S-LPS production in this model system remain incompletely defined. Here, a functional wbbL gene was introduced into the E. coli K-12 KEIO single-gene deletion mutant library to restore OAg synthesis, enabling genome-wide analysis of S-LPS production by screening with colicin E2 (ColE2) and validated with LPS silver staining. This identified 319 mutants with increased sensitivity to ColE2 in the presence of OAg, suggesting broader envelope-associated effects during screening. In addition, 27 mutants showed defects in S-LPS production, corresponding to genes involved in OAg biosynthesis, LPS core and sugar precursor synthesis, OAg ligation and regulation, and enterobacterial common antigen biosynthesis. A further 18 mutants initially appeared defective in S-LPS production but could not be validated upon reconstruction, and whole-genome sequencing revealed secondary mutations responsible for the observed phenotypes. This study provides a validated genetic framework for S-LPS production in E. coli K-12 and highlights the importance of rigorous validation in genome-wide screening approaches.

The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300{Delta}femT to assess its function. Although growth of USA300{Delta}femT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300{Delta}femT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 {micro}M palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300{Delta}femT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport. IMPORTANCEThe FemT efflux pump of S. aureus is co-expressed in an operon with femX encoding an essential enzyme needed to complete the synthesis of peptidoglycan precursor Lipid II. Although this alluded to a specific role for FemT in supporting peptidoglycan synthesis, our data are instead consistent with a general role in efflux of cellular isoprenoids and carotenoid lipids that are susceptible to oxidation during routine cellular functions. Consequently, S. aureus became strongly dependent on FemT function when exogenous host-derived fatty acids were being actively metabolized. This represents a significant advance in our understanding of the role of an RND efflux pump in supporting routine growth-related functions of S. aureus and exposes a function that could be targeted to impair S. aureus growth on exposure to host-derived fatty acids.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

People with intellectual disabilities and their communities are rarely involved in setting priorities for research. Our study addressed this gap through consultations with the UK communities of three genetic syndromes in which intellectual disabilities are common: Down syndrome (DS), Fragile X syndrome (FXS) and Williams syndrome (WS). The study aimed to provide an understanding of (1) the views of the DS, FXS and WS communities on current UK research; (2) their priorities for future research; and (3) participants views of engaging with UK research. We conducted focus group discussions with 39 community members including: children and adults with DS, FXS and WS; parent/carers of people with DS, FXS and WS; practitioners and researchers who work with these communities. Our study was carried out in collaboration with a Steering Group and two Advisory Groups of DS, FXS and WS community members. We identified three themes. First, participants shared their dissatisfaction with the current research landscape and wanted a more balanced landscape, with more research with direct application to the daily lives of people with DS, FXS and WS. Second, community members emphasised the importance of translating research into practice, advocating for better access to research and more meaningful participation to research of individuals with lived experience. Third, our study not only identified what should be the focus of future research on DS, FXS and WS, but also how researchers should conduct their research. Whilst including children in our sample was a strength, there were some limitations to the diversity of our sample; children with FXS were not represented and gender, ethnic and geographic diversity could have been broader. Nevertheless, we hope that our findings will change the future of research in this field so that research carried out in the name of individuals with intellectual disabilities such as DS, FXS and WS, is of direct use to these communities.

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

Background: Pseudomonas aeruginosa is a ubiquitous opportunistic bacterial pathogen associated with nosocomial infections and is a leading cause of infection in persons with cystic fibrosis (pwCF). The front-line treatment for multidrug-resistant P. aeruginosa infections is ceftolozane-tazobactam (C/T). While previous research has characterized clinical P. aeruginosa isolates that evolved resistance to C/T, the collateral effect of evolved resistance on susceptibility to bacteriophages has not been explored. Methods: We collected paired P. aeruginosa clinical isolates from 10 pwCF and 18 non-pwCF who developed treatment-emergent C/T resistance. We compared genetic relatedness, acute and chronic virulence phenotypes, and antibiotic and phage susceptibilities between each pair of susceptible baseline and treatment-emergent C/T-resistant isolates. Results: Treatment-emergent C/T-resistant isolates were genetically closely related to baseline isolates in all patients. Virulence phenotypes did not differ between pre- and post-C/T exposure isolates, but isolates from pwCF demonstrated differences in protease production, twitching motility, and amino acid auxotrophy compared to isolates from non-pwCF. Treatment-emergent C/T resistance was associated with increased resistance to ceftazidime and ceftazidime/avibactam, but no other trends in antibiotic or phage susceptibility were detected. Conclusions: Treatment-emergent resistance to C/T does not cause predictable alterations in phage susceptibility across genotypically and phenotypically diverse multidrug-resistant P. aeruginosa clinical isolates.

The Gram-positive obligate anaerobe Finegoldia magna is a member of the healthy human microbiota, but also acts as an opportunistic pathogen to cause persistent, biofilm-associated infections. Despite its prevalence on the human host, little is known about F. magna biology or the mechanisms underlying its clinically relevant phenotypes, partly due to the lack of genetic tools. We address this gap by establishing genetic approaches for investigating gene function in F. magna, which we apply to identify genetic determinants of aggregate biofilm formation. We found that F. magna isolates are naturally competent, allowing for targeted chromosomal integration of linear DNA constructs via homologous recombination. Transformation frequency varied substantially among strains and was also affected by factors such as homologous flank length, DNA concentration, and incubation method. To identify genes that mediate aggregate biofilm formation, we used experimental evolution to select for F. magna mutants that had lost the ability to aggregate. This approach identified a conserved locus encoding a putative adhesin that we named FafA (Finegoldia adhesion factor A). Next, we applied targeted mutagenesis tools to show that deletion of fafA markedly reduced autoaggregation but does not impair other modes of biofilm formation, including surface attachment or aggregation during agitation. Finally, we demonstrate that FafA-mediated aggregation protects F. magna from antibiotic and oxidative stress. Together, these findings establish a genetic framework for mechanistic studies in F. magna and identify FafA as a conserved adhesin that promotes aggregation and stress tolerance in this anaerobic pathobiont.

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.

The use of antimicrobial compounds in humans, animals and in agriculture leads to environmental antimicrobial contamination through domestic, industrial and agricultural wastewater. Efforts have been made to perform environmental risk assessments based on the potential of these compounds to select for antimicrobial resistance (AMR) at certain concentrations in bacteria. This has resulted in predicted no effect concentrations (PNEC) which determine the minimum thresholds required to select for resistance. However, the effects of these compounds on intracellular transposition within bacterial isolates, a major driver of AMR, have not been previously assessed. Here, we assess the effect of differing sub-inhibitory concentrations of the third-generation cephalosporin, ceftriaxone, on the rate of intracellular transposition in combination with colistin and kanamycin. Two triple replicons systems (RS1 and RS2) were developed to assess this, each containing a chromosome, plasmid and entrapment vector. We show that sub-inhibitory concentrations of ceftriaxone exert hormetic effects on the intracellular transposition rate in RS1 and a steady linear increase in RS2. This defines a predicted no effect concentration for transposition (PNECT) for ceftriaxone as 320 ng/L in RS1 and 3200 ng/L in RS2. This provides a minimum threshold for the environmental impact of ceftriaxone on biological systems at the sub-cellular scale, which is applicable to industrial standards of waste management, where consideration of ecological impact is central.

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.

Chronic Pseudomonas aeruginosa lung infections are becoming harder to treat due to global escalation of antimicrobial resistance (AMR). Bacteriophage (phage) therapy has emerged as a promising adjunct to conventional antibiotics, especially in chronic lung infections such as those seen in cystic fibrosis (CF). However, phage monotherapy may be limited by the emergence of phage-resistant bacterial populations and there remains limited preclinical evidence evaluating both antimicrobial efficacy and host safety in physiologically relevant human airway models. Here, we evaluated the safety and antimicrobial activity of Kara-mokiny 3, a myovirus bacteriophage, alone and in combination with subinhibitory concentrations of tobramycin using fully differentiated paediatric primary airway epithelial cells (pAECs) cultured at the air-liquid interface (ALI). Kara-mokiny 3 rapidly reduced P. aeruginosa viability and exhibited synergistic activity with tobramycin, resulting in significantly greater bacterial killing than either treatment alone. Importantly, phage treatment replicated efficiently in the presence of its bacterial host while preserving epithelial morphology, mucin production and epithelial barrier architecture., without inducing cytotoxicity or excessive IL-6 and IL-8 inflammatory responses. These findings demonstrate that phage-antibiotic combination therapy can enhance antimicrobial activity while maintaining epithelial safety in a physiologically relevant human airway model. This study represents one of the first comprehensive evaluations of phage-antibiotic combination therapy in differentiated primary airway epithelial cultures, providing important preclinical evidence supporting the development of personalised phage-based therapies for the treatment of MDR pulmonary infections. ImportanceThe rise of MDR P. aeruginosa has created an urgent need for alternative treatment strategies for chronic lung infections. Although phage therapy is receiving increasing clinical attention, there is limited evidence evaluating its safety and efficacy in physiologically relevant human airway models. Using differentiated primary airway epithelial cultures, we demonstrate that a phage-antibiotic combination reduces bacterial burden without compromising epithelial integrity and toxicity or excessive inflammatory responses. These findings provide translational evidence supporting phage-antibiotic combination therapy and highlight the value of primary airway epithelial models for the preclinical assessment of emerging antimicrobial interventions, supporting the translation of personalised phage therapies.

Host-associated bacteria navigate complex nutrient landscapes where metabolites act as both growth substrates and cues that shape behavior. Yet, for most commensal and pathogenic bacteria, the nutrients and metabolisms that support persistence in the host remain unknown. Finegoldia magna is an obligate anaerobe that normally colonizes human skin and mucosal surfaces but also causes persistent biofilm-associated infections on implanted medical devices and in chronic wounds. Here, we developed a defined medium to investigate how nutrients influence F. magna physiology. We found that F. magna has a remarkably restricted metabolism that is specialized to use a limited set of host-relevant nutrients, including glycine, fructose, nucleosides, and betaine. Carbon-source screens showed glycine was the only substrate that supported growth as a sole carbon source. Instead, growth typically required two carbon substrates: a compatible electron donor-acceptor pair, suggesting redox balance imposes major constraints on its metabolism. To determine whether these constraints extend to host environments, we cultured F. magna in media derived from human chronic wound tissue. Despite its chemical complexity, F. magna displayed a similarly restricted metabolic profile, primarily consuming peptides, nucleosides, and betaine. These nutrients also directed biofilm behavior, with different metabolites promoting surface attachment or aggregation. Our findings show that although F. magna lacks metabolic flexibility, this opportunistic pathogen appears specialized to exploit host-derived products of skin physiology, tissue damage, and inflammation. This work suggests that host-associated bacteria with highly specialized metabolisms may be especially responsive to nutrient availability, linking local metabolite composition to key persistence behaviors like biofilm formation.

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.

Onion tissues produce antimicrobial thiosulfinates after tissue damage and cellular decompartmentalization. Burkholderia gladioli pv. alliicola (Bga), a common onion pathogen, encodes a thiosulfinate tolerance gene (TTG) cluster that protects the bacterium during thiosulfinate exposure. Previous work showed that the TTG cluster contributes to foliar infection but has little effect on infection of onion bulb tissue. To further examine Bga-thiosulfinate interactions in foliar and bulb tissues, we used a thiosulfinate-responsive PaltR-Lux reporter strain to determine when and where Bga encounters thiosulfinates. In leaves, Bga-induced necrosis was associated with de-repression of the PaltR-Lux reporter and coincided with a contribution of the TTG cluster to bacterial population size, indicating thiosulfinate exposure during foliar infection. In contrast, TTG mutants and wild-type (WT) strains showed similar growth in scales, and PaltR-Lux signal declined as scale necrosis progressed, suggesting limited thiosulfinate exposure during bulb colonization. However, when necrosis was induced by the non-native toxin pantaphos, PaltR-Lux was de-repressed and recovery of the TTG mutant was reduced. These results indicate that Bga encounters thiosulfinates during foliar infection but largely avoids exposure during bulb infection. Preconditioning the TTG mutant in onion scale tissue did not alter its thiosulfinate sensitivity in vitro, arguing against an infection-associated thiosulfinate exclusion mechanism. In contrast, partial rescue of the TTG mutant by the WT strain in zone-of-inhibition co-plating assays suggests extracellular thiosulfinate detoxification. Together, these findings indicate that Bga detoxifies thiosulfinates released during bulb necrosis, limiting thiosulfinate exposure during onion bulb infection. The molecular basis for detoxification and tissue specificity remain unresolved.