Microbiology

Colistin has emerged as an important last line of defence for the treatment of infections caused by antibiotic resistant Gram-negative pathogens. Here we investigate the responses of {approx}1,000 populations of an MDR strain of P. aeruginosa to a high dose of colistin. Colistin exposure resulted in rapid cell death, but a sub-set of populations eventually recovered due to the outgrowth of heteroresistant cells. Genome sequencing revealed that heteroresistance was primarily driven by mutations in the PmrAB two-component system that occurred at a rate ({approx}2x10-5 per cell division) that was 103-104 fold higher than typical resistance mutation rates. Crucially, this elevated mutation rate was only found in pmrB, demonstrating that hypermutability is localized to this gene. PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a high mutation rate as an adaption to generate mutants that are resistant to host antimicrobial peptides that are secreted during infection. Interestingly, we found no mutations in 1/3 of populations that recovered from colistin treatment, suggesting that phenotypic plasticity and/or persister cells contribute to the ability of Pseudomonas to adapt to colistin.

Adaptive radiation of a single lineage into novel ecological niches underpins the evolution of biodiversity. To study adaptation in the industrially and ecologically important bacterium Streptomyces, a long-term evolution experiment (LTEE) was undertaken in a strain lacking several antibiotic biosynthetic gene clusters, facilitating the study of epistasis in antibiotic biosynthesis. Streptomyces coelicolor is a filamentous soil organism that does not undergo sporulation under the LTEE conditions, affording the opportunity to study adaptation under relaxed selection on sporulation. Over 3000 generations, replicate populations showed parallel loss of sporulation, morphological transitions to fragmenting hyphae and diversification in carbon source utilisation. Genomic analyses revealed mutations in developmental and metabolic genes, while reintroduction of the actinorhodin cluster demonstrated pervasive negative epistasis affecting antibiotic production. These findings reveal adaptive diversification and antagonistic interactions in regulatory pathways under relaxed selection, providing insights into the evolution of complex multicellular bacteria and informing industrial antibiotic production strategies.

The rise of antibiotic-resistant bacteria has necessitated the development of alternative therapeutic strategies such as bacteriophage therapy, where viruses infect bacteria, reducing bacterial burden. However, rapid bacterial resistance to phage treatment remains a critical challenge, potentially leading to failure. Phage Steering, which leverages the evolutionary dynamics between phage and bacteria, offers a novel solution by driving bacteria to evolve away from virulence factors or resistance mechanisms. In this study, we examined whether Phage Steering using bacteriophage Luz19 could function in the presence of a competing pathogen, Staphylococcus aureus (USA300), while targeting Pseudomonas aeruginosa (PAO1). Through in vitro co-evolution experiments with and without the competitor, we observed that Luz19 consistently steered P. aeruginosa away from the type IV pilus (T4P), a key virulence factor, without interference from S. aureus. Genomic analyses revealed mutations in T4P-associated genes, including pilR and pilZ, which conferred phage resistance. Our findings suggest that Phage Steering remains effective even in polymicrobial environments, providing a promising avenue for enhancing bacteriophage therapy efficacy in complex infections. ImportancePhage Steering--using phages that bind essential virulence or resistance-associated structures-- offers a promising solution by selecting for resistance mutations that attenuate pathogenic traits. However, it remains unclear whether this strategy remains effective in polymicrobial contexts, where interspecies interactions may alter selective pressures. Here, we demonstrate that Pseudomonas aeruginosa evolves phage resistance via loss-of-function mutations in type IV pilus (T4P) when challenged with the T4P-binding phage Luz19, and that this evolutionary trajectory is preserved even in the presence of a competing pathogen, Staphylococcus aureus. Phage resistance was phenotypically confirmed via twitching motility assays and genotypically via whole genome sequencing. These findings support the robustness of Phage Steering under interspecies competition, underscoring its translational potential for managing complex infections--such as those seen in cystic fibrosis--where microbial diversity is the norm.

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together. ImportanceStaphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial pathogens that infect people with the genetic disease, cystic fibrosis (CF). They are often found together in CF-associated polymicrobial infections that are associated with worse patient prognosis. Understanding how these very different opportunistic pathogens influence each other in a shared environment is pertinent to improving the treatment of polymicrobial infections. While much attention has been brought to the interspecific interactions between S. aureus and P. aeruginosa, few studies have used experimental evolution methods to identify determinants of their competition and coexistence. Here, we use a serial transfer experimental evolution approach and identified a single genetic change associated with improved survival of S. aureus in the presence of P. aeruginosa. Our findings implicate metabolism of shared resources as an important factor in S. aureuss ability to survive in the presence of P. aeruginosa.

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. (i) This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. (ii) Because of the mode of action of PYOSa S. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. (iii) PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear the populations of S. aureus. Due to the ascent of a phenotypically diverse array of small colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone. Significance StatementThe increasing frequency of antibiotic-resistant pathogens has fostered a quest for alternative means to treat bacterial infections. Prominent in this quest is a therapy that predates antibiotics: bacteriophage. This study explores the potential of a phage, PYOSa, for treating Staphylococcus aureus infections in combination with antibiotics. On first consideration, this phage, isolated from a commercial therapeutic cocktail, seems ideal for this purpose. The results of this population dynamic and genomic analysis study identify a potential liability of using PYOSa for therapy. Due to the production of potentially pathogenic atypical small colony variants, PYOSa alone cannot eliminate S. aureus populations. However, we demonstrate that by following the administration of PYOSa with bactericidal antibiotics, this limitation and potential liability can be addressed.

Quorum sensing (QS) is a mechanism of cell-cell communication that connects gene expression to environmental conditions (e.g. density) in many bacterial species, mediated by diffusible signal molecules. Current functional studies focus on a dichotomy of QS on/off (or, quorate / sub-quorate) states, overlooking the potential for intermediate, graded responses to shifts in the environment. Here, we track QS regulated protease (lasB) expression and show that Pseudomonas aeruginosa can deliver a graded behavioral response to fine-scale variation in population density, on both the population and single-cell scales. On the population scale, we see a graded response to variation in environmental population density. On the single-cell scale, we see significant bimodality at higher densities, with separate OFF and ON sub-populations that respond differentially to changes in density; static OFF cells and increasing intensity of expression among ON cells. Together these results indicate that QS can tune gene expression to graded environmental change, with no critical cell mass or quorum at which behavioral responses are activated on either the individual cell or population scale. In an infection context, our results indicate there is not a hard threshold separating sub-quorate stealth mode and a quorate attack mode.

Antimicrobial resistance is a massive threat, but it can take decades to develop a new antibiotic. That time could be drastically reduced if we were able to anticipate desirable properties of a chemical, such as its potential to target specific bacterial compartments. This would provide the opportunity to prioritise the development of molecules that target, for instance, the cell membrane, as this does not rely on transporters and usually results in a fast-acting bactericidal effect. We used flow cytometry and a set of fluorophores together with a group of antibiotics to discriminate between antimicrobials acting on cell membrane versus intracellularly against two Gram-negative bacteria E. coli and A. baylyi. We then chose Rhodamine 123 to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects towards E. coli. This was confirmed with growth inhibitory assays in both E. coli and A. baylyi for Pantoprazole, Theophylline and Zoledronic acid. This represents an approach to the large-scale screening of small molecules with the potential to deliver fast-acting molecules that target cell membranes in Gram-negative bacteria. 3. Impact statementThe discovery of novel antimicrobials is essential to build resilience against infectious diseases. Understanding the mechanism of action of known as well as novel compounds is equally crucial. Achieving this understanding is a long and complex process that is usually addressed on an individual basis for an antibiotic, or a class of antibiotics. Using high-throughput pipelines such as those possible with flow cytometry can help to shorten this process. We present a flow cytometry-based approach capable of determining if a given molecule has rapid antimicrobial effects that involve increasing the permeability of the cell membrane. The latter is a property that usually makes a chemical a very effective antimicrobial. 4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.

Achromobacter species are emerging multidrug-resistant (MDR) pathogens in people with cystic fibrosis. Their increasing resistance has grown an interest in phage therapy as an alternative treatment strategy. However, the factors governing phage susceptibility remain poorly understood, thereby limiting the rational selection of phage candidates. Using 15 strictly lytic Achromobacter phages and 7 clinical cystic fibrosis isolates representing Achromobacter insolitus and Achromobacter xylosoxidans, we demonstrate substantial variation in infection efficiency across all 105 phage-host combinations, variation that could not be discerned from qualitative plaque assays alone. We integrated complete bacterial and phage genomes with quantitative efficiency-of-plating (EOP) assays and lineage-aware Bayesian mixed-effects modelling to show that phage infectivity in Achromobacter is governed predominantly by bacterial lineage and strain identity, accounting for 90% of total variance in log-normalised EOP, with individual strains varying substantially in permissiveness irrespective of species membership. After accounting for this lineage structure, no individual defence system, antimicrobial resistance gene class, or phage tail cluster retained a statistically significant independent or interaction association with infectivity. Together, these findings demonstrate that bacterial strain identity is the primary driver of infection outcome. Host defence systems and phage tail-associated genes remain biologically plausible contributors; their independent effect could not be resolved after accounting for lineage structure, indicating that infection outcomes are largely strain-dependent. This work shifts the question from which individual traits predict infection to how strain lineage and specific host-phage combinations jointly determine infectivity, and argues that quantitative phenotyping of individual phage-host pairs is essential for guiding phage candidate selection and supporting rational cocktail design against multidrug-resistant Achromobacter infections in cystic fibrosis. Impact statementChronic Achromobacter infections in cystic fibrosis are increasingly difficult to treat due to multidrug resistance and biofilm formation. Although phage therapy is a promising alternative, its development is limited by poorly understood and highly variable infectivity. Here, we show that infectivity within a phage host range spans a broad quantitative continuum spanning several orders of magnitude that cannot be captured by qualitative plaque assays. These infection efficiencies are primarily structured by bacterial lineage and strain identity, while the contributions of individual genomic features remain unresolved, given the current sample size. This work provides a framework for predicting phage-host compatibility and supports a shift from empirical screening toward rational, evidence-based phage selection for MDR Achromobacter infections.

Daptomycin is a membrane-targeting lipopeptide antibiotic used in the treatment of infective endocarditis caused by multidrug-resistant Gram-positive bacteria such as Staphylococcus aureus, enterococci and viridans group streptococci. Despite demonstrating excellent in vitro activity and a low prevalence of resistant isolates, treatment failure is a significant concern, particularly for enterococcal infection. We have shown recently that human serum triggers daptomycin tolerance in S. aureus, but it was not clear if a similar phenotype occurred in other major infective endocarditis pathogens. We found that Enterococcus faecalis, Streptococcus gordonii or Streptococcus mutans grown under standard laboratory conditions were efficiently killed by daptomycin, whereas bacteria pre-incubated in human serum survived exposure to the antibiotic, with >99% cells remaining viable. Incubation of enterococci or streptococci in serum led to peptidoglycan accumulation, as shown by increased incorporation of the fluorescent D-amino analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a >10-fold reduction in serum-induced daptomycin tolerance, demonstrating the important contribution of the cell wall to the phenotype. We also identified a small contribution to daptomycin tolerance in E. faecalis from cardiolipin synthases, although this may reflect the inherent susceptibility of cardiolipin-deficient mutants. In summary, serum-induced daptomycin tolerance is a consistent phenomenon between Gram-positive infective endocarditis pathogens, but it may be mitigated using currently available antibiotic combination therapy.

Streptococcus sanguinis is a prevalent member of human microbiome capable of acting as a causative agent of oral and respiratory infections. S. sanguinis competitive success within the infection niche is dependent on acquisition of metal ions and vitamins. Among the systems that bacteria use for micronutrient uptake is the energy coupling factor (ECF) transporter system EcfAAT. Here we describe physiological changes arising from EcfAAT transporter disruption. We found that EcfAAT contributes to S. sanguinis antibiotic sensitivity as well as metal and membrane homeostasis. Specifically, our work found that disruption of EcfAAT results in increased polymyxin susceptibility. We performed assessment of cell-associated metal content and found depletion of iron, magnesium, and manganese. Furthermore, membrane composition analysis revealed significant enrichment in unsaturated fatty acid species resulting in increased membrane fluidity. Our results demonstrate how disruption of a single EcfAAT transporter can have broad consequences on bacterial cell homeostasis. ECF transporters are of interest within the context of infection biology in bacterial species other than streptococci, hence work described here will further the understanding of how micronutrient uptake systems contribute to bacterial pathogenesis. ImportanceProficiency in micronutrient uptake is key for pathogen success in bacteria-bacteria and bacteria-host interactions within the infection context. Micronutrient uptake mechanisms are of interest in furthering the understanding of bacterial physiology within infection niche and as targets for design of antimicrobials. Here we describe how a deletion of a nutrient uptake transporter in S. sanguinis alters bacterial sensitivity to antibiotics. We also show that a defect in this candidate nutrient uptake system has consequences on the intracellular metal content, and also results in changes in membrane fatty acid composition and fluidity. This study demonstrates how disruption of a single nutrient uptake system disrupts bacterial physiology resulting in increased antibiotic sensitivity.

Bacillus subtilis is a major model organism for studying population heterogeneity in clonal bacterial cultures due to its high genetic tractability and ability to differentiate into subpopulations with distinct biological functions. It is also a key industrial production host, responsible for synthesizing a range of commercially valuable enzymes and metabolites. However, cell differentiation processes can pose a challenge for the optimal biotechnological utilization of B. subtilis, particularly when emerging subpopulations do not contribute to product biosynthesis. Here, we present robust assays that facilitate the analysis of two previously difficult-to-study population properties of B. subtilis: (i) the energization levels of individual cells within dense cultures and (ii) the extent of cell lysis that can occur under such conditions. Our findings reveal an unappreciated level of heterogeneity in cell energization within dense B. subtilis cultures, and a surprisingly high degree of cell lysis in seemingly healthy, actively growing populations. These insights add to our understanding of the biological complexities and single-cell heterogeneities present in superficially simple bacterial clonal cultures, establish analytical tools to study the associated processes, and provide a foundation for further optimizing B. subtilis as an industrial production host. IMPORTANCEBacillus subtilis and its close relatives are important industrial microorganisms, responsible for the production of a range of commercially valuable enzymes, antibiotics and metabolites. In recent years, considerable research efforts have been aimed at increasing the productivity of these organisms. However, their ability to undergo physiological and morphological differentiation processes at high cell densities ultimately limits their productivity. Our research reveals how the resulting heterogeneity impacts the population-level energy status of individual cells in the culture and the surprisingly high extent of population-level cell lysis. It also provides tools for determining these important productivity criteria as well as guiding the development of the production host.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobials mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpress to identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpress identified that all three analogues targeted the lipid IVA biosynthetic pathway in E. coli and Salmonella Typhimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues in E. coli when lpxD was overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in both E. coli and S. Typhimurium. LPS composition was a likely reason for differences between E. coli and S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpress has proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations. Data SummaryNucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13250. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

BackgroundInvasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM. MethodsTwo Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett-Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes. ResultsBoth strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure ConclusionsThese findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.

The secreted phospholipase C/sphingomyelinase, PlcH, is the heat-labile hemolysin of Pseudomonas aeruginosa and one of its important secreted virulence factors. While there are known and suspected genes that impact PlcH production in P. aeruginosa, we sought to identify additional genes by screening the PA14 transposon mutant library to measure extracellular PlcH enzyme activity induced by choline. The library as a whole had a log2-normal distribution of NPPC activity with notable tails that included the genes of interest. These outlier genes included nearly all of those known to be important for PlcH production in response to choline, including those required for choline metabolism, glycine betaine sensing, and secretion through the outer membrane. Interestingly, higher PlcH production was also seen in mutants of the protease associated genes lon, mucD, and clpA, as well as other genes. Additionally, we identified genes impacting baseline levels of PlcH production, which include genes in the dimethylglycine metabolism locus involved in choline metabolism. The high hit rate of known and suspected genes supports the power of this screen and our verification of these genes by clean deletion in strain PA14 confirm the broad importance of these systems across P. aeruginosa, as previous work was confined to strain PAO1. There were many genes identified in this screen that were not individually examined and the complete screen results reported here should allow others to identify intersection of their genes of interest with PlcH production. ImportancePseudomonas aeruginosa is an important opportunistic pathogen that employs multiple independent virulence factors to cause infection, one of which is the hemolytic phospholipase C/sphingomyelinase PlcH. Using a whole genome screen, we identified both known and previously unknown genes contributing to P. aeruginosa PlcH production. Our findings provide insight into the integration of various cellular processes with PlcH production and identify potential genes that may impact the PlcH expression heterogeneity seen in P. aeruginosa clinical isolates.

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO dyes should be used with caution on live samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/560001v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b8bab4org.highwire.dtl.DTLVardef@e9a84forg.highwire.dtl.DTLVardef@291058org.highwire.dtl.DTLVardef@1f0245d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Differentially culturable (DC) Mycobacterium tuberculosis (Mtb) phenotypes reduce the sensitivity of sputum culture and may be associated with adverse treatment outcomes among tuberculosis patients. Accurately quantifying DC Mtb remains an important research objective, with current approaches tending to combine Most Probable Number (MPN) assays and culture filtrate (CF) supplementation. These assume that growth is equiprobable across all bacterial inoculum densities - an untested assumption for Mtb. We performed a half-logarithmic dilution series of Mtb from 70,000-0 CFU/mL, culturing each inoculum in either standard 7H9 or CF and monitoring growth by optical density. Inocula of [≥]2,000 CFU/mL were 33 times more likely to grow than inocula of [≤]700 CFU/mL. CF increased the odds of growth five-fold, and reduced the time-to-positivity by 294 hours ([~]12 days) compared to 7H9 alone. However, CFs growth-promoting effects diminished with increasing inoculum density, becoming negligible at 70,000 CFU/mL. Notably, CF broadly altered Mtb cell physiology, producing shorter bacilli that were less likely to incorporate the mycomembrane probe, DMN-trehalose. These data indicate that Mtb is poorly culturable from low inoculum densities - a limitation only partially overcome by CF. This non-uniform growth probability suggests that unsupplemented MPN assays may systematically underestimate Mtb CFU. Moreover, while CF promotes Mtb growth, its density-dependent activity and broader effects on cell physiology suggest that its influence extends beyond simply resuscitating DC Mtb. Improved methods are needed for detecting DC Mtb phenotypes, as these may support clinical care and shed light on factors that govern mycobacterial replication at different population densities. HighlightsO_LIMycobacterium tuberculosis (Mtb) culturability decreases at low inoculum densities C_LIO_LIMost Probable Number (MPN) assays may systematically underestimate Mtb CFUs C_LIO_LICulture filtrate (CF) selectively promotes Mtb growth from low densities C_LIO_LIMtb requires secreted factors in culture filtrate to initiate growth at low cell numbers C_LIO_LICF-supplemented MPN assays may be inadequate for detecting DC Mtb phenotypes C_LI

Soil is a competitive environment containing a variety of resources used for bacterial growth. Complex polysaccharides, like chitin or starch, require the secretion of enzymes that degrade these resources into smaller units before they can be consumed. However, exoenzymes and the products they create are public goods, meaning they can be used by competitors, called "cheaters", who benefit from public goods even if they do not produce enzymes themselves. Here, we test the hypothesis that antibiotics produced by Streptomyces are used to privatize public goods by restricting access to resource cheaters. Using experiments with Streptomyces coelicolor and Bacillus subtilis, we first show that B. subtilis cheating significantly reduces S. coelicolor fitness on complex medium (starch) but not on a simple carbon source (maltose) which does not require exoenzyme secretion. Next, we show that antibiotics produced by S. coelicolor markedly increase fitness against resource cheaters, despite evidence that antibiotic production is metabolically costly. Finally, we find that the benefits of antibiotic production and the costs of resource cheating are both higher during growth on lower resource concentrations. Our results provide novel insights into the context-dependent costs and benefits of antibiotic secretion in Streptomyces and highlight the role of resource complexity and concentration in mediating competitive strategies in bacteria.

The bacterial cell wall is a covalently linked meshwork of peptidoglycan (PG) that establishes cell shape and prevents osmotic lysis. This structure must be flexible enough to accommodate transenvelope protein complexes, but strong enough to withstand high intracellular pressure. In order to elongate and divide, cells must remodel the cell wall through the concerted action of PG synthesis and degradation. Endopeptidases, a class of PG degrading enzymes, facilitate cell growth by hydrolyzing PG crosslinks. Vibrio cholerae encodes several functionally redundant endopeptidases, two of which are nearly identical: ShyA and ShyC. To investigate differential roles of these enzymes, we assessed growth and morphology of ShyA and ShyC mutants. We found that ShyA, but not ShyC, is required for normal adaptation to low osmolarity medium. Cells lacking ShyA exhibited longer lag phase and aberrant morphology during adaptation, and reduced survival in the presence of a beta-lactam antibiotic. Lastly, our experiments revealed that cells lacking ShyAs LysM domain exhibited more severe defects than cells lacking ShyA altogether, implicating the LysM domain in proper regulation of ShyA activity.