Microbiology

Pseudomonas aeruginosa, an opportunistic pathogen, uses a trio of quorum sensing (QS) systems to regulate the production of some virulence factors. Two of these, the las and rhl systems, involve acyl-homoserine lactone signals; the third, called pqs, primarily uses the signal 2-heptyl-3-hydroxy-4(1H)-quinolone ("PQS"). We aimed to identify how interbacterial interactions are regulated between P. aeruginosa and Stenotrophomonas maltophilia, which co-occur in the airways of people with cystic fibrosis. We explored P. aeruginosa and S. maltophilia interactions using a co-culture model. S. maltophilia in co-culture with P. aeruginosa grows for 12 hours and thereafter exhibits a large decline in CFU, demonstrating that P. aeruginosa is killing S. maltophilia. Co-culture of S. maltophilia with P. aeruginosa deficient in las, rhl, or pqs QS resulted in greater S. maltophilia viability than co-culture with the wildtype. This inhibition was not attributable to las and rhl-regulated toxins. Therefore, we interrogated the role of PQS and found that co-culture of S. maltophilia with P. aeruginosa deficient in PQS biosynthesis showed similar CFUs to monoculture. Exogenous PQS did not complement this phenotype, suggesting that another quinolone is the effector. We found that S. maltophilia killing is reduced in competition with a mutant that cannot make the quinolone HQNO. We show that full killing of S. maltophilia by P. aeruginosa requires three components: HQNO, the chaperone PqsE, and intact PQS biosynthesis. Our work identifies quinolone biosynthesis as a driver for interactions between P. aeruginosa and S. maltophilia and, more generally, in modulating interbacterial interactions.

Considerable effort has focused on identifying alternatives to mouse models in research studies. In the field of bacterial pathogenesis, Galleria mellonella and epithelial cell lines have been widely used for this purpose, but the concordance of these models with mice remains unclear. To begin to address this knowledge gap, we used 105 clinical isolates of Pseudomonas aeruginosa for which the virulence had been previously determined in a mouse bacteremia model. A semistrong correlation was observed between G. mellonella median time to 50% mortality and mouse 50% pre-lethal dose (LD50) values (Spearmans rank correlation coefficient [{rho}] = 0.75), whereas percent A549 epithelial-like cell lysis during co-culture showed a weak correlation to mouse LD50 values ({rho} = -0.47). Given the stronger correlation between G. mellonella and mouse virulence, we next examined whether G. mellonella could substitute for mice when asking questions about the virulence of large numbers of P. aeruginosa isolates. Results from mice indicated that isolates with resistance to more antibiotics were significantly less virulent, and the use of G. mellonella identified the same inverse correlation. Furthermore, both models found no evidence for the existence of hypervirulent clonal lineages. In particular, isolates belonging to sequence types defined as high-risk clones were not consistently more virulent than other isolates, despite the known association of high-risk clones with poor clinical outcomes. These findings suggest that G. mellonella can serve as an adequate substitute for mice when addressing specific population-based virulence questions, although conclusions should be confirmed in mice. Author SummaryWe found that virulence measurements in a G. mellonella infection model showed a semistrong correlation with those from a mouse bacteremia model and that this insect larval model adequately detected population-level trends similarly to mice. In contrast, A549 epithelial-like cell lysis during bacterial co-culture correlated less well with mouse virulence. Together, these results support the use of G. mellonella as a scalable, low-cost, and humane first-line model for assessing P. aeruginosa virulence but also indicate that conclusions should be validated in mice.

The continuing emergence of antimicrobial resistance (AMR) poses one of the most urgent threats to public health in the 21st century, causing [~]1.2 million deaths per year. A promising alternative to traditional antimicrobials is phage therapy, which has proved effective in the treatment of antimicrobial-resistant infections, including methicillin-resistant Staphylococcus aureus (MRSA). However, considerable variation in the outcome of phage therapy remains, with phage that show high efficacy in vitro often showing little to no effect when applied in vivo. To allow for the efficient clinical use of phage, it is vital to understand how both bacterial virulence and phage efficacy vary in vivo and determine how well in vitro and in vivo measures of phage efficacy correlate. Here, we infected 4,968 Galleria mellonella larvae with 64 phylogenetically diverse Staphylococcaceae isolates, both with and without co-inoculation of the bacteriophage ISP, and recorded mortality and melanisation over 24 hours. We found that bacterial virulence varied among Staphylococcaceae strains, and that a large proportion of this variation could be explained by the evolutionary relationships between bacteria. These results indicate that pathogen phylogeny may be a useful tool for explaining variation in both the severity of clinical infections and the virulence of novel emerging pathogens. The addition of phage significantly improved the survival of G. mellonella larvae, with an average 15.2% increase in endpoint survival and 10.1% reduction in endpoint melanisation across Staphylococcaceae strains. Phage efficacy in vivo showed phylogenetic repeatability, but no detectable phylogenetic heritability across bacteria species, in contrast to previous in vitro studies of phage infections. Concurrently, we found no evidence of a correlation between in vivo and in vitro measures of phage efficacy across bacterial isolates, demonstrating that environment plays a major role in determining the outcomes of bacteria-phage interactions. This highlights that caution should be used when extrapolating from in vitro measures of phage efficacy to select phages for therapeutic uses.

Escherichia coli ST131 is a globally disseminated multidrug-resistant lineage frequently associated with recalcitrant urinary tract infections (UTIs) and bacteraemia. While bacteriophages offer a promising alternative treatment to antibiotics, their efficacy is often limited in physiologically relevant conditions in comparison to laboratory media. In this study, we have investigated the mechanisms by which the representative ST131 strain, EC958, evades elimination by a model phage, LUC4. We observed that in the urine environment, EC958 can transiently resist phage infection by a density dependent mechanism and by the production of protective polysaccharides. Based on this understanding, we developed a phage treatment strategy that can sterilise an EC958 culture in urine-based medium, even at high bacterial densities. The rational design of the successful phage therapy strategy utilises a tailored phage cocktail containing phage that encode depolymerase enzymes to degrade bacterial surface carbohydrates and the targeting of multiple receptors to prevent the emergence of fixed genetic resistant mutants. We found the addition of specific carbon sources renders the bacteria more susceptible to phage infection. By combining these findings with a simulated bladder wash to model voiding, we successfully achieved elimination of EC958 cultures in a urine environment. This study provides a framework for overcoming both fixed and reversible phage resistance, offering a translatable strategy for effectively treating urinary tract infections with phage. Author SummaryIn this study, we investigated how bacterial populations can overcome a phage infection. Phage are viruses that naturally kill bacteria and provide an alternative treatment to antibiotics. We focussed on a particularly aggressive and antibiotic resistant strain of E. coli, EC958, which belongs to a group of E. coli strains that are a leading cause of urinary tract infections and life-threatening bloodstream infections worldwide. We found that in a simulated bladder environment, these bacteria do not rely on genetic mutations to survive but they employ a range of hide and seek strategies. We showed that bacteria can coat themselves in a protective layer to block the phage and use social signalling to enter a dormant state when cell density is high. When they are in this sleep-like state the phage cannot successfully infect. To overcome these bacterial defences, we developed a treatment strategy combining effective phage with specific naturally occurring additives, that trick the bacteria into waking up and becoming vulnerable again to phage infection. By also simulating a clinical bladder wash to reduce bacterial numbers and therefore reduce social-signalling, we were able to eliminate the bacterial population. Our findings suggest that by understanding bacterial strategies we can design more effective and personalised phage therapies to treat bacterial infections.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

It is well established that Staphylococcus aureus can incorporate straight-chain unsaturated fatty acids (SCUFAs) into its lipids in addition to the normally biosynthesized branched-chain and straight-chain saturated fatty acids. Incorporation of oleic acid into S. aureus lipids has recently been shown to significantly enhance S. aureus growth at low temperatures due to the greater fluidity imparted to the membrane. Here, we show that low-temperature growth of S. aureus is not limited to oleic acid but enhanced also by various antimicrobial SCUFAs when present at low concentrations. A fakA-deficient strain did not show SCUFA-induced growth stimulation, which indicates that the fatty acid kinase is necessary for SCUFA incorporation into membrane lipids to promote low-temperature growth. Determination of total lipid fatty acid composition showed that incorporated SCUFAs make up [~]12% or less of the total fatty acids. Lipidomic investigations revealed elevated synthesis of diglucosyldiglyceride in the absence or presence of SCUFAs. SCUFAs were incorporated into diglucosyldiglyceride to a greater extent than phosphatidyglycerol at both 12 {degrees}C and 37 {degrees}C. The presence of SCUFAs at low temperatures also enhanced production of the carotenoid staphyloxanthin. The results suggest that multiple strategies are at play in the membrane adaptation of S. aureus to low temperatures. Inclusion of oleic acid in media decreased the minimum growth temperature of S. aureus, suggesting that the presence of SCUFAs in food may facilitate the growth of S. aureus at low temperature. Also, incorporation of SCUFAs into lipids may promote the disruption of the membrane by SCUFAs.

Microbes have evolved a variety of strategies to survive exposure to naturally occurring and synthetic antimicrobials. These strategies have been investigated extensively in model bacterial organisms, whereas less is known about under explored pathogenic bacteria such as bacteria within the Yersinia genus. In this study we investigated the inhibitory effect and bactericidal activity of antibiotics from five different classes and of the disinfectant hydrogen peroxide against Yersinia pseudotuberculosis, the ancestral species from which Yersinia pestis and Yersinia enterocolitica have emerged. We found that Y. pseudotuberculosis is able to survive exposure to clinical antibiotics and disinfectants by employing a variety of strategies, with persisters and the Eagle effect playing a role in survival to quinolones, tolerance playing a role in survival to ceftriaxone and overexpression of catalases and peroxidases playing a role in survival to hydrogen peroxide. Our findings suggest that future research should focus on informing new, effective ways to treat infections caused by Yersinia species. IMPORTANCEAntimicrobial resistance is routinely investigated by measuring the minimum inhibitory concentration of antimicrobials needed to stop microbial growth. Here we show that the bacterial pathogen Yersinia pseudotuberculosis is not killed when antibiotics and disinfectants are used at these concentrations and that, in some cases, increasing antibiotic concentrations decreases their activity against this bacterium, therefore posing a potential risk to human and animal health.

AbstractBeneficial associations between bobtail squids (Cephalopoda: Sepiolidae) and Vibrio bacteria encompass a unique association where symbionts are obtained environmentally from the surrounding environment. Vibrio symbionts are susceptible to a number of ecological pressures such as protozoan grazing whilst in their free-living state. Impacts of grazing have several consequences for symbiosis characteristics such as biofilm formation, a trait crucial for survival both in and outside the squid. Therefore, in order to ascertain how biotic factors such as grazing in the environment effect symbiotic success, two V. fischeri strains, ES114 and ETBB1-C were experimentally evolved in separate biofilm grazing experiments with the amoeba, Acanthamoeba castellanii and ciliate Tetrahymena pyriformis. Both ES114 and ETBB1-C biofilms were evolved up to 50 generations through serial passaging. At 50 generations, ES114 biofilms displayed variability in response to predation by both predators, whereas ETBB1-C biofilms were more stable across generations of grazing. A. castellanii decreased in population numbers when co-inoculated with ETBB1-C, whereas T. pyriformis increased in numbers with biofilm growth. Growth of V. fischeri biofilms in the presence of grazers such as T. pyriformis has an important role in inducing biofilm growth by acting as a chaperone for recycling nutrients back into the environment. Additionally, V. fischeri colonization fitness in the host was dependent on which grazer was used to evolve the biofilms. Such variation in response by V. fischeri to different types of predation demonstrates the versatility of this symbiont in its free living state and has subsequent impacts on the eventual association with squids. ImportanceThis manuscript demonstrates the importance of biotic factors (such as protozoan grazing) in the environment that effect host colonization in a beneficial symbiosis. Using an experimental evolution approach, this work demonstrates how symbiotic biofilms can adapt to pressures such as grazing that subsequently influences the ability to colonize its invertebrate host.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

AimsBacteriophage (phage) propagation has traditionally relied on bacterial culture media containing animal-derived ingredients; however, safety concerns with animal-derived materials for production of phages for therapeutic use limit their acceptability. We compared animal-free and traditional media formulations, and evaluated their effects on phage yield, bactericidal activity, and genomic characteristics, hypothesizing no significant differences would be observed. Methods and ResultsPhages targeting Pseudomonas aeruginosa (n=8) and Staphylococcus aureus (n=1) were propagated in solid and liquid media containing animal-free (AF) or animal-derived (LB) peptones. Kinetic assays were used to assess phage suppression of host bacterial growth. In a mock therapeutic phage screen, spot tests, Efficiency of Plating (EOP) and kinetic assays were performed against novel bacterial targets. Whole genome sequencing of phages and their bacterial hosts propagated in AF or LB broth was used to observe genomic differences between formulations. Animal-free peptone did not impact phage yield, with both AF and LB phage stocks growing to high titers ([≥]108 PFU/mL). Kinetic assay results showed similar suppression indices for AF and LB-grown phages. Likewise, phage screen spot test, EOP, and kinetic assay results were similar between AF and LB phages. Comparisons of phage and bacterial genome annotations showed no major differences arising from media formulation. ConclusionsFindings suggest animal-free peptones do not significantly alter phage yield, bactericidal activity, or genomic characteristics, supporting use of animal-free medium for medicinal phage manufacture. This is one of the first studies to systematically combine phenotypic and genomic assessment of phages and hosts across animal-free and traditional media. Impact StatementPhage therapy is increasingly used to treat antimicrobial resistance infections. Emerging guidelines and regulations for the manufacture of phage therapeutics will impact laboratory processes and materials used for phage production. Here, we explored the use of an animal-free medium for medicinal phage propagation, providing data on phage yield and metrics of phage activity.

The human gut microbiome is a complex community that plays an important role in health, where perturbations can result in dysbiosis and disease. Bacteriophages (phages) can provide treatment for bacterial gastrointestinal disease, and commercial preparations such as the Intesti bacteriophage cocktail can be taken orally to target bacterial pathogens. However, interactions between these phages and the native gut microbiota are understudied. To investigate the impact of phage treatment, we used simulated gut models seeded with healthy donor microbiota from three individuals, sequenced the DNA, and analysed the bacterial and viral portion from samples obtained over time. Each donor had a unique bacterial composition which diverged with time. When comparing phage treated to control samples, we observed that Escherichia coli abundance accounted for the largest portion of bacterial community variance and was more associated with the controls. The lower abundance in phage treated samples may have resulted from the lytic action of phages from the cocktail. Additionally, our analyses of the viral portion revealed a phage bloom exclusive to phage treated samples. A highly abundant phage in this bloom was matched with the Intesti bacteriophage cocktail, showed similarity to Enterobacteria phage phi92, and provided evidence of productive infection within the model. While we did observe fluctuations in relative abundance of additional viral sequences in the presence of the phage cocktail, these changes were often transient. Furthermore, we detected only slight differences to typical members of the virome, and low numbers of active prophages. Our experiments suggest that the phage cocktail had minimal interruption to the native gut microbiota within the model. Impact statementBacteriophages are increasingly investigated and tested for their efficacy in treating infections and are a key component in fight against antimicrobial resistant bacterial infections. Because of their specificity, it has become almost a dogma to state that they do not alter the gut microbiome. We have now tested this in an in vitro study using a commercially available cocktail and real human faecal microbiota. We show minimal effects on the composition of the healthy microbiota with an individual-specific effect on Escherichia coli caused by productive infection of one phage in the cocktail.

BackgroundAcinetobacter baumannii is a common bacterial pathogen in nosocomial infections. It has become one of the greatest threats to human health for its growing resistance to last resort antibiotics, which has led to a revival of phage therapy as a potential treatment. However, conventional methods for isolating A. baumannii-infecting phages are labor-intensive and often unsuccessful. MethodsOur approach involves a computational pipeline to identify temperate phages (prophages) integrated into A. baumannii genomes, followed by mitomycin C (MMC) induction of those strains to screen for active prophages. ResultsHere we show a prophage analysis for nearly 900 A. baumannii genomes. We observed MMC-triggered excision of nine prophages from eight A. baumannii strains by PCR and sequencing. Further we show four prophage form virions detectable by transmission electron microscopy, and two which can plaque on other A. baumannii isolates. ConclusionThis work demonstrates the utility and diversity of prophages for further development as therapeutics for antibiotic resistant A. baumannii.

It has been known for decades that bacteriophages encode tRNA genes, but their function and the factors contributing to their acquisition and retention are unclear. Although tRNAs are found in a variety of phages infecting a variety of bacteria, many large-scale computational studies investigating tRNA acquisition and retention in phages are specific to Mycobacterium phages; however, these findings may not be representative of other phages or bacteria. This work uses a broader sampling of phages and hosts to investigate the relationships between codon usage bias, infection cycle, and tRNA gene numbers in phage genomes. We analyzed 154 phages infecting 7 host genera, including Gram-negative (Escherichia, Shigella, Salmonella) and Gram-positive (Bacillus, Lactobacillus, Staphylococcus, Mycobacterium) bacteria. Phages included temperate and virulent representatives, plus a range of tRNA numbers and morphologies. All phages and hosts were analyzed using four metrics: GC content, Effective Number of Codons, Relative Synonymous Codon Usage, and tRNA Adaptation Index. On a global scale, virulent phages with many tRNA genes show greater differences in codon usage and codon adaptation compared to their respective hosts. Gram-negative bacteria and their phages generally exhibit greater differences in codon usage compared to Gram-positive bacteria and their phages. Phages infecting Gram-negative hosts also tend to encode more tRNA genes. In nearly all genus-level comparisons, Mycobacterium phages were different from any other host and from global patterns. This suggests previous computational studies performed in Mycobacterium phages are likely not applicable on a global scale or to phages infecting other host genera. AUTHOR SUMMARYBacteriophages, or phages, are viruses infecting bacteria. They are abundant in all environments, yet how they interact with their bacterial hosts is still not well-understood. Like other viruses, phages must rely on the host translational components to replicate and form new phage particles; and similarly to other parasites, phages have genomes that differ significantly from their hosts in terms of composition. In this work, we explore the relationship between phage lifestyle, number of tRNA genes encoded, and genome differences from the host using a variety of phages and their associated hosts. Phages can be either virulent (do not integrate into the host genome) or temperate (capable of integrating into the host genome), with differences from the host genome more pronounced in virulent phages. There are many phages that also carry tRNA genes, and having higher numbers of tRNAs is associated with larger differences from the host genome. The findings here indicate that virulent phages carrying large numbers of tRNAs diverge the most from host genome composition.

Mycobacteria have a hydrophobic cell envelope that makes uniform growth in liquid culture challenging. Non-ionic detergents including Tween-80 and tyloxapol are commonly added to media when culturing mycobacterial species in the laboratory. Tyloxapol was reported to exhibit anti-tuberculous activity during animal infection with M. tuberculosis in the 1950s. In the 1980s, microscopy studies suggested that tyloxapol impacted the inter-action between M. tuberculosis and the phagosomal membrane, preventing mycobacterial access to the cytoplasm. It is now known that the ESX-1 Type VII secretion system mediates the interaction between pathogenic mycobacteria and the phagosomal membrane. Mycobacterium marinum is a pathogenic mycobacterial species that has been widely used to understand the molecular mechanisms and host responses to the ESX-1 system. The hemolytic activity of M. marinum allows the study of ESX-1 lytic activity outside of the context of a host cell. We found that tyloxapol inhibits the hemolytic activity of M. marinum in a concentration dependent manner. The addition of 100-fold less tyloxapol than commonly used for mycobacterial growth differentially inhibits the production and secretion of ESX-1 substrates required for lytic activity. Our findings directly impact how the field interprets data from studies where M. marinum, and potentially other mycobacterial species were grown in tyloxapol. Our findings may explain the original ob-servations linking tyloxapol to anti-tuberculosis activity. Author SummaryTuberculosis, which is caused by Mycobacterium tuberculosis, is one of the worlds deadliest diseases. We lack a clear understanding of how M. tuberculosis and related mycobacterial species cause disease. In the 1950s, it was reported that treating M. tuberculosis infected animals with tyloxapol improved the survival and in some cases protected the animals from death. Tyloxapol is a detergent that is commonly added to mycobacterial cultures to promote dispersed growth in the laboratory. Later studies suggested that tyloxapol altered the interaction between M. tuberculosis and the phagosomal membrane during macrophage infection. The ability to escape the phagosome is essential for mycobacteria to cause disease, and is mediated by a Type VII protein secretion system, ESX-1. Using M. marinum, a well-established model for understanding the molecular mechanisms of ESX-1 secretion, we show that tyloxapol used at more than 100-fold less than what is commonly used to grow mycobacteria in the lab, inhibits ESX-1 secretion. Our findings have widespread implications on how we interpret our findings as a field, and may explain why tyloxapol impacted M. tuberculosis infection of both animals and macrophages. Our study also indicates that tyloxapol can be used as a tool to understand the molecular mechanisms of ESX-1 protein secretion.

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. The biofilm defects in the mutants were confirmed using SEM, and cryoSEM was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

Two different individuals can consume an identical diet but experience different physiological outcomes. While there are many potential mechanistic reasons for this, one increasingly recognized reason is that differences in an animals microbiome can lead to differences in the processing of dietary nutrients. Thus, though a diet might start out the same, how it is experienced by a host is dependent on their microbiome. While exciting, mechanistic studies of diet-microbiome-host effects are often limited by the lack of high throughput laboratory techniques to identify and define interactions between dietary metabolites, microbial metabolism, and host biology. We hypothesized that the model organism Caenorhabditis elegans is an advantageous animal model for rapidly identifying and genetically dissecting interactions between dietary nutrients, microbial metabolism, and host physiology. Here, we used an established model of the effects of dietary glucose on insulin resistant mutant animals (daf-2/IR mutants) to study how differences in bacterial metabolism influence the consequences of dietary sugars on animal physiology. We found that the effect of dietary sugars on daf-2 mutant physiology is dependent on how the microbiome metabolizes dietary sugars. We found dietary sugar suppresses multiple daf-2 mutant phenotypes in the presence of some bacteria but has no effect in the presence of others. To determine how bacteria mediate the effects of dietary sugars on host physiology we screened 5,000 transposon mutations in the canonical C. elegans dietary bacteria, E. coli OP50 for effects on animal insulin signaling. From this, we found that the effects of exogenous sugars on the phenotype of daf-2 mutant animals is dependent on the function of pyruvate dehydrogenase in bacteria and that the loss of bacterial pyruvate dehydrogenase genes (ex. aceE) is sufficient to mimic the effects of dietary sugars on dauer formation, longevity, and gene expression in insulin signaling deficient animals. Collectively, our findings further support the growing body of evidence that the effects of dietary nutrients on animal physiology can be influenced by the gut microbiome. In addition, these studies demonstrate the advantages of the C. elegans model system for studying 3-way diet-microbiome-host interactions that are difficult to dissect in other model systems.

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

Plasmids play a key role in the spread of virulence and antimicrobial resistance genes to new genetic backgrounds. Genetic variation in the transfer operon, the genes responsible for conjugation, can lead to substantial differences in transfer potential even between closely related plasmids. However, it is not clear how much genetic diversity there is in transfer operons of natural bacterial populations. Here, we analyze the prevalence and transfer potential of F-like plasmids, a clinically important family of plasmids in Enterobacteriaceae. Using 1200 Escherichia coli genomes isolated from three livestock-associated environments, we find that the fraction of F-like transfer operons that are functionally complete was significantly higher in poultry than in bovine and swine associated bacteria. This difference was not captured in methods that use the presence of replication genes to estimate plasmid prevalence. Confounders such as the phylogenetic relatedness of E. coli or the presence of antibiotic resistance could not explain these significant differences in transfer potential. Instead, it seems the poultry environment selects for plasmids with high transfer potential, as it also contained more conjugative plasmid types per isolate. While we find environment specific differences in overall plasmid frequency, patterns of transfer gene presence/absence were similar across the three environments. Regulatory and exclusion genes are the exception to this pattern, suggesting environment specific modulation of transfer rates. This highlights the use of genomic data to uncover environment specific differences in plasmid prevalence and transfer potential, revealing the selection pressures shaping horizontal gene transfer in these environments.