Microbiology

Phage therapeutics are re-emerging as adjuncts or alternatives to antibiotics and their clinical translation will be enhanced with production methods that minimise downstream processing. We evaluated whether an endotoxin-reduced E. coli strain developed for production of recombinant proteins, ClearColi(R), can serve as a useful, safe phage production host without compromising yield and whether targeted receptor complementation can expand its utility. The parent strain BL21(DE3), and its lipid A modified derivative, ClearColi(R), were compared with respect to infection and generation of phage. Across a panel of 31 phage, a similar host range was observed between BL21(DE3) and ClearColi(R). To expand host range ompC was genetically engineered into the chromosome of ClearColi(R), thereby adding OmpC-dependent phage to its production capacity. Production metrics were broadly comparable between the hosts; efficiency of plating and final titres for representative phage were not significantly different; burst size varied by phage but without consistent host bias. Endotoxin activity in ClearColi(R)-propagated lysates was reduced by over 1000-fold relative to BL21(DE3), reaching the low hundreds of endotoxin units (EU) versus hundreds of thousands for BL21(DE3). Intravesical administration of ClearColi(R)-derived phage (LUC4) into pigs elicited no clinical abnormalities and no significant increases in circulating cytokines up to 48 hours after administration. ClearColi(R) allows efficient production of diverse phage with low endotoxin, reducing the requirement for downstream processing. Although its minimal LPS reduces its capacity for producing some LPS-dependent phage and its growth is slower than BL21(DE3), requiring optimisation for maximal phage titre, the safety and simplified manufacturing process support further development of endotoxin modified strains for phage production. Impact statementAntibiotic resistance is a current global problem and treatments based on phage and phage products already have a proven track record with particular bacterial infections, especially in the urinary tract. While progress is being made on in vitro phage synthesis, large scale bacteriophage preparations require a bacterial host for production, consequently toxic components in the initial lysate need to be removed or significantly diluted for safe clinical use. This is a study of the potential to utilise an endotoxin-reduced E. coli strain, ClearColi(R), to produce safer phage therapeutics. Such endotoxin modified strains should minimise the processing steps required and reduce overall production costs of a phage preparation. The research demonstrates that the endotoxin-reduced strain was able to produce a wide range of phage and for studied examples at phage titres equivalent to the more toxic parent strain. We also show that the strain can be modified to increase its host range and confirm the very low endotoxicity of basic phage lysates produced by the strain. Replicating this process to engineer additional low-toxicity bacterial production strains will accelerate the development of safer, more cost-effective phage therapeutics.

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.

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.

Colistin is used to treat antibiotic resistant gram-negative infections, including those caused by Pseudomonas aeruginosa (Pa). Using a diverse collection of clinical isolates, we identified BWH047, a colistin-resistant isolate with an extremely high minimum inhibitory concentration (MIC, 1280 {micro}g/mL). To characterize the genes conditionally essential for colistin resistance in BWH047, we employed transposon insertion sequencing and identified 20 gene candidates. In-frame deletion validated 75% of the candidates and identified genes in several novel pathways that contribute to colistin resistance, including algU and wapH. We also identified several candidate genes from previously reported colistin resistance pathways (e.g. arn, pmrAB). We further investigated the impact of a colistin resistance-associated inner membrane DedA-family undecaprenyl phosphate flippase, which we named DpcA (DedA of Pseudomonas necessary for colistin resistance A). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 {micro}g/mL) and resulted in several unique changes to the structure of lipopolysaccharide (LPS), including production of decreased amounts of the colistin resistance-conferring 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification on lipid A. To date, this work represents the most complete analysis of colistin resistance in Pa and identifies novel intersecting pathways that contribute to extreme phenotypic resistance. Author summaryPseudomonas aeruginosa is a bacterium that causes a wide variety of infections. It is especially problematic given its propensity to become resistant to antibiotics. One antibiotic used to treat multidrug-resistant P. aeruginosa infections is colistin. In this study, we investigated colistin resistance mechanisms in a patient-derived, extremely phenotypically resistant P. aeruginosa isolate, BWH047, using transposon insertion sequencing and mass spectrometry. We identified 13 genes conditionally essential for colistin resistance and investigated the role of one of these genes, dpcA, on the composition of the bacterial outer membrane, the target of colistin. Additionally, our study identified novel colistin resistance genes residing in several intersecting pathways that could be targeted to prevent the development of antimicrobial resistance.

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.

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

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.

Escherichia coli, the Escherichia type species, is present in mammalian and avian intestinal microbiota, and includes both commensals and pathogens. Other Escherichia species are understudied because they are less commonly associated with human disease and because of paucity of tools that can correctly delineate them from E. coli. However, other species of this genus including Escherichia albertii and Escherichia fergusonii are repeatedly reported as diarrhoeagenic. We hypothesized that some bacteria fitting the definition of enteroaggregative E. coli (EAEC) belong to species other than E. coli. We used phylogeny to determine the species of 2,818 Escherichia genomes from diarrhoea epidemiology studies in Nigeria. Phylogeny speciation was confirmed using GTDB-tk and ClermonTyping. Virulence genes were detected using ARIBA/Virulencefinder database and multilocus sequence typing performed using the Achtman scheme. Fourteen non-coli Escherichia genomes were identified-- Escherichia clade I ST485 (11), Escherichia ruysiae ST5792 (2) and Escherichia fergusonii ST5636 (1). All the Escherichia clade I ST485 carry EAEC virulence genes aap, aar, astA and air, as well as hlyF, eatA, tsh, traT, and chuA virulence genes. Interestingly, 62% of enteroaggregative Escherichia clade I ST485 genomes listed on Enterobase are from Africa isolates, despite only 3% of genomes overall coming from the continent. Our results suggest that non-coli Escherichia species are infrequently isolated from human stool, but, when they are, they are misidentified as E. coli so that their significance is largely overlooked. Escherichia clade I ST485 is a globally disseminated enteroaggregative Escherichia clade I lineage that is common in Africa. Author SummaryEscherichia clade I are rarely associated with disease and because of the difficulty in differentiating them from Escherichia coli in routine laboratory, they are often misidentified as Escherichia coli leading to the underestimation of their impact on the burden of disease. Additionally, some clones of Escherichia clade I also carry genetic markers that have been used to define Enteroaggregative Escherichia coli (EAEC), a cause of persistent diarrhoea in developing countries and travellers diarrhoea in developed economies. EAEC has also been associated with malnutrition and poor growth among children in developing economies. We here describe clones of Escherichia clade I (ST485) that carries enteroaggregative genes and in some cases, recovered from diarrhoeal cases. We show from genomes deposited on Enterobase and our study, that this clone is globally disseminated, often associated with human infections and often misidentified as Escherichia coli. We also describe other non-coli Escherichia other than Escherichia clade I isolated from humans. We suggest that the Escherichia clade I clone carrying enteroaggregative genes may be described as Enteroaggregative Escherichia clade I.

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.

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:

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.

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

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.

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.

BackgroundEnteroaggregative Escherichia coli (EAEC) are a heterogenous pathotype, implicated in acute and persistent diarrhoea especially in developing countries. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) are Type V Secretory System trypsin-like proteases repeatedly reported from EAEC. This study aimed to determine SPATE encoding-gene prevalence among EAEC and their association with diarrhoea. We screened 881 EAEC genomes from four recent epidemiological studies in Nigeria for 23 SPATE-encoding genes, initially using ARIBA and the Virulencefinder database. ResultsInitial screening inflated SPATE gene content, particularly in genomes with multiple SPATEs, due to cross detection of highly similar sequences and other artefacts. We developed and validated refined methodology, which detected 478 of 1,156 original SPATE calls and also identified SPATE miscalls from previous datasets in the literature. The most prevalent SPATE-encoding gene in our EAEC collection was sepA 297(33.71%), closely followed by sat 360 (29.74%). pic, encoding a SPATE with mucinase activity, was found in 65 (7.4%) genomes and associated with diarrhoea (p=0.00004). EAEC strains belonging to E. coli phylogroups A, B1 or C carried, on average, one SPATE gene per genome while >1 was typically detected in phylogroup B2 EAEC. Other EAEC carried few or no SPATE genes. ConclusionsOur study shows that multifunctional genome analysis tools may have to be refined for certain gene families to avoid overestimation. SPATEs are not as prevalent as previously thought but they remain common among EAEC, particularly among phylogroup A, B1, B2 and C, pointing to the possibility that they make lineage-specific contributions to disease.

ObjectivesBiofilms are the dominant mode of bacterial life. The gut microbiota itself has characteristics of a biofilm that grows on the intestinal mucosa. C. difficile and VRE are commonly co-isolated from patients but biofilm formation has not been studied in a multi-species context. Here we study the interactions between C. difficile and VRE in surface adherent community. ResultsWe found that VRE inhibits C. difficile biofilm formation in dual-species culture in the presence of excess glucose. Robust dual-species biofilms were produced when the carbon source was changed to a non-fermentable sugar such as fucose and xylose. We observed a high level of vancomycin tolerance in C. difficile biofilms that was not affected by the presence of VRE. Finally we also found that a nutrient step-change is sufficient to induce dispersion of single and dual-species biofilms. ConclusionsVRE can inhibit the development of C. difficile biofilms in the presence of a fermentable carbon source. VRE does not appear to affect vancomycin tolerance or nutrient-induced dispersion of C. difficile biofilms. Highlights- VRE inhibits C. difficile biofilm formation in the presence of fermentable glucose. - Stable VRE - C. difficile biofilms are formed by managing the available carbon source. - VRE does not affect C. difficile vancomycin tolerance in this model. - A 10-fold increase in available nutrients is sufficient to induce biofilm dispersion in C. difficile and VRE.

HIV remains among the worlds most serious healthcare challenges, with adolescent girls and young women in sub-Saharan Africa at particularly high risk of infection. Bacterial vaginosis (BV) is a key risk factor for HIV acquisition, however current treatment strategies are limited. Optimal vaginal Lactobacillus spp. protect against BV and HIV, largely through immunoregulatory and antimicrobial activities mediated in part by lactic acid. Towards the development of a Lactobacillus-containing live biotherapeutic for African women, we sampled 181 vaginal Lactobacillus isolates from 25 BV-negative South African women. Fifty isolates were selected for evaluation of inflammatory responses using vaginal epithelial cells, D- and L-lactate and lactic acid production and culture acidification. Aside from a single Lactobacillus salivarius strain, L. crispatus isolates acidified the culture media the most and produced the most D- and L-lactic acid. Inflammatory cytokine responses to Lactobacillus strains were variable, with L. crispatus eliciting the lowest levels of cytokine production. When all properties were evaluated collectively, L. crispatus strains exhibited the most desirable biotherapeutic characteristics. Whole genome sequence analysis of ten L. crispatus isolates showed that the majority were more closely related to one another than to isolates from other geographical regions. This supports the need for live biotherapeutics to be tailored for the population of intended use. No antimicrobial resistance elements were detected, while putative bacteriocins and intact prophage sequences were identified in all isolates. L. crispatus isolates displayed characteristics essential for optimal live biotherapeutic performance, however additional analysis is required to determine the functionality of identified putative prophages. ImportanceHIV is a leading cause of morbidity and mortality in sub-Saharan Africa, where adolescent girls and young women are three times more likely to acquire HIV than their male counterparts. A key risk factor for HIV is bacterial vaginosis (BV), a condition characterised by the loss of beneficial Lactobacillus species and increased abundance of non-optimal, inflammatory bacteria. Although BV affects approximately 25% of women in sub-Saharan Africa, effective therapeutics are lacking. Live biotherapeutics containing optimal Lactobacillus spp. represent a promising strategy to improve BV treatment outcomes and reduce HIV infection risk. We isolated 181 vaginal Lactobacillus spp. from 25 BV-negative South African women and characterized 50 selected isolates. This led to the identification of live biotherapeutic candidates for African women with distinct genomes compared to isolates from other geographical regions. This study contributes to current knowledge of the characteristics that should be considered when screening novel isolates for this purpose.

Background/ObjectivesGlucagon-Like Peptide-1 (GLP-1) is a key incretin hormone that regulates glucose homeostasis and energy metabolism. Impaired GLP-1 signaling contributes to the development of obesity, metabolic syndrome, and type 2 diabetes. Emerging evidence indicates that gut microbiota-derived components can influence GLP-1 secretion, highlighting the therapeutic potential of microbial modulators. Akkermansia muciniphila, a next-generation probiotic associated with improved metabolic health, remains underexplored for its capacity to stimulate GLP-1 release. This study aimed to investigate the GLP-1- stimulatory effects of live and pasteurized (dead) A. muciniphila strains in human enteroendocrine cells. MethodsHuman enteroendocrine L-cells (NCI-H716) were treated with varying doses of live and dead A. muciniphila from Vidya Herbss proprietary VHAKM strain and a commercially available marketed strain (dead form). Following incubation, GLP-1 levels were quantified from culture supernatants using enzyme-linked immunosorbent assay (ELISA). Comparative analyses assessed differences in GLP-1 secretion between strains and treatment forms. ResultsBoth live and pasteurized VHAKM strains significantly increased GLP-1 secretion compared to untreated controls. The live VHAKM strain exhibited higher GLP-1 stimulatory activity than its pasteurized counterpart and the marketed strain. The results suggest a strain-specific and viability-dependent modulation of GLP-1 secretion in human L-cells. ConclusionsThis study demonstrates that A. muciniphila VHAKM enhances GLP-1 secretion in a strain- and form-dependent manner, with live cells showing superior efficacy. These findings provide foundational insights for developing microbiome-targeted interventions to boost endogenous GLP-1 levels and improve metabolic health outcomes.

Bone infections, which are predominantly caused by Staphylococcus (S.) aureus, can be difficult to treat and have high rates of chronicity and reoccurrence. We previously identified that osteoclasts, the cells that break down bone matrix, may contribute to disease progression by allowing S. aureus to replicate intracellularly. There we identified that this bacteriums ability to grow intracellularly is tied to the maturation of osteoclasts. In this study we addressed whether osteoclast differentiation supports intracellular growth by changing the host cells response to infection or by altering the host cell environment to better support S. aureus. Using dual species RNA-sequencing we analyzed host and bacterial transcripts of infected osteoclast and precursor bone marrow macrophage (BMM) cultures. Host transcript analysis suggests that infected osteoclasts are slow to upregulate bacterial response genes compared to BMMs. We also identify that the S. aureus transcriptional response is primarily determined by the host cell type, and that bacteria in osteoclasts upregulate carbon metabolism genes compared to those inside BMMs. By utilizing intracellular survival assays on S. aureus mutants deficient in carbon metabolism and related pathways we determine that S. aureus require glycolysis, acetyl-CoA synthesis, and aspartate biosynthesis for proliferation inside osteoclasts, although bacteria can survive without them. With differentiation, osteoclasts increase glutamine uptake, and this metabolite is required for S. aureus intracellular growth. Taken together, these findings suggest that osteoclasts support S. aureus intracellular growth by providing nutrients required to replicate in the context of a blunted antimicrobial response. IMPORTANCEInfectious osteomyelitis, bone infection, is frequently caused by the bacterium Staphylococcus aureus. Intracellular infection of cells that build bone, osteoblasts, and cells that resorb bone, osteoclasts, have been implicated in disease progression by providing a niche for immune evasion. While S. aureus in osteoblasts are largely quiescent, bacteria in osteoclasts proliferate and therefore may be a source of reemergent infection. Factors that promote this growth in osteoclasts are poorly characterized. In this study we find that osteoclasts have a diminished transcriptional response to infection and show that S. aureus acquire glucose and glutamine, which have high flux in osteoclasts, to support intracellular growth. We further observe that S. aureus in osteoclasts require aspartate synthesis to grow intracellularly. This work highlights the importance of host cellular metabolism for the intracellular fate of S. aureus as an added factor beyond the direct antimicrobial response.

Carbapenem-resistant Gram-negative bacteria pose a critical public health threat. The role of mobile genetic elements in driving their transmission and persistence remains poorly defined. In 2022, we investigated a suspected outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB) in a Nigerian adult intensive care unit (ICU), using short-read whole genome sequencing (WGS) of carbapenem-resistant clinical and environmental isolates during the cluster period. Mobile element dynamics were then inferred from hybrid assemblies of Illumina and Oxford Nanopore reads. The suspected CRAB outbreak was ruled out by WGS but a carbapenem-resistant Enterobacter hormaechei ST114 bloodstream isolate was found to be indistinguishable from two environmental isolates, all recovered during the Acinetobacter surge. Hybrid assemblies revealed a strikingly conserved [~]19 Kb resistance island shared across all ST114 genomes. The island contained a blaNDM-5 cassette alongside many other antimicrobial resistance genes, within class 1 integronns and flanked by insertions sequences, located on a 46,176 bp plasmid. Using the ST114 plasmids hybrid assembly as scaffold, the same plasmid was identified in the genome of a Klebsiella pneumoniae ST15 isolate from the ICU environment during the same period. Additionally, re-interrogation of genomic surveillance data uncovered four clonal 2020 ST109 Enterobacter bloodstream isolates from the same facility that carried the resistance genes in the same context on a large 267,242 bp plasmid. Carbapenem resistance in hospital Enterobacterales is driven by both clonal expansion and horizontal spread of mobile resistance elements. These findings underscore the need to track mobile elements alongside bacterial lineages to inform evidence-based infection control, especially in low-resource settings. Impact StatementCarbapenem resistance among Enterobacterales remains a major public health threat, yet how mobile genetic elements contribute to their persistence and spread in hospital settings is still poorly understood. In this study, we investigated a suspected outbreak of carbapenem-resistant Acinetobacter baumannii in an adult intensive care unit in Nigeria. Although the outbreak was eventually ruled out, genomic analysis has shown the importance of careful interpretation of suspected outbreak cases in hospital settings. Our findings highlight the importance of close monitoring of ICU environments, the implementation of blood culture-based diagnostics, and the value of genomic support in outbreak investigations. These findings demonstrate that carbapenem resistance in hospital Enterobacterales is driven not only by clonal expansion but also by the horizontal dissemination of a highly stable blaNDM-5-associated MDR island capable of integrating into diverse plasmid backbones. This study emphasizes the need for genomic surveillance that tracks both mobile elements and bacterial lineages to strengthen outbreak investigations, especially in low-resource settings. It further underscores the links between clinical and environmental AMR reservoirs and reinforces the value of a One Health approach to controlling carbapenem resistance. Data summaryFASTQ sequences were deposited in the NCBI BioSample database under accession numbers SAMN55915584 - SAMN55915597.