Microbiology

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.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 g/ml for CAZ, 8 g/ml for LVX, 16 g/ml for MEM, and 8 g/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 g/ml, 8 g/ml, and 16 g/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCEThe Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

Pairwise analysis of genomic and spatial data offers opportunities to identify and estimate the associations between covariates and the transmission of pathogens between individuals. However, such pairwise analyses are computationally intensive, and may not be feasible to conduct given the high dyad count in even moderately sized datasets. Here we compare two approaches to increase the efficiency of pairwise analysis for large datasets. We quantify and compare the performance of divide-and-conquer Bayesian model fitting and pairwise case-control approaches for estimating associations between individual- and pair-level covariates and shared membership in a transmission cluster. We utilize a large dataset (n=4,154) of spatially-referenced, genomically-sequenced Mycobacterium tuberculosis isolates collected from a single city for this analysis. We find that the case-control approach produces unbiased estimates of effect sizes with expected credible interval coverage and is more robust than the divide-and-conquer method when effect sizes are large. Thus, we recommend using the case-control approach with at least three controls per case to downscale datasets for pairwise analysis when analysis of the entire dataset is not possible. This approach mitigates the computational challenges of pairwise Bayesian modeling on datasets that require significant computational resources while maintaining desired inferential properties. Author SummaryPairwise analyses of large datasets to study pathogen transmission are computationally demanding because they typically require simultaneous analysis of each possible pair of individuals in a dataset; as datasets become larger these analyses often are not feasible to conduct even with access to high-performance computing resources. In this work, we compare a case-control approach and divide-and-conquer approaches for more efficient pairwise analysis of large datasets. Using a large dataset of Mycobacterium tuberculosis isolates including genetic and spatial data, we investigate the performance of each method for estimating the associations between host covariates and genetic clustering of isolates. We find that the case-control approach is generally preferred over methods which first divide the data into subsets and then combine results. While additional extensions of these analyses are needed to test the generality of these findings to other data settings, this work provides a practical way forward for the pairwise analysis of large datasets to study pathogen transmission.

During bloodstream infection, most bacterial pathogens maintain homeostatic levels of heme, which serves as an essential biochemical cofactor and iron source, but becomes toxic at high intracellular concentrations. Well-characterized, surface exposed heme binding and acquisition systems exist in several blood-borne bacterial species. However, some gram-positive bacteria that invade the bloodstream do not encode surface displayed heme acquisition systems, despite showing clear evidence of heme utilization in blood. An example is Streptococcus agalactiae (group B Streptococcus; GBS), which is a major cause of infection in neonatal and immunocompromised populations. Here we show that GBS uses its cell membrane as a dynamic heme reservoir, which functions as the primary site of environmental heme capture, sensing, and transmembrane flux. Using positive and negative genetic selection screens, targeted mutagenesis, membrane fractionation, and spectroscopic heme detection and binding assays, we demonstrate that heme is partitioned into the GBS cell membrane, where it is sensed by the histidine kinase HssS and extracted for intracellular use by the CydDC transporter. Genetically disrupting the function of either HssS heme sensing or CydDC membrane heme extraction attenuates bacterial survival in human whole blood and in a mouse model of bacteremia. These results suggest that cell membrane-localized heme homeostasis is a determinant of fitness during blood survival. This work expands the current models of bacterial heme physiology and provides evidence that membrane localized, homeostatic heme reservoirs may represent an underrecognized strategy for blood-borne pathogens that lack canonical heme acquisition systems.

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

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Background The COVID-19 pandemic has caused significant global mortality. Despite declining infection rates, new variants of SARS-CoV-2 continue to emerge, necessitating new prevention strategies. Objective This study aimed to evaluate the effect of four over-the-counter (OTC) antiseptic mouthwash/gargling solutions in the U.S., compared with a distilled water control, on SARS-CoV-2 viral load across multiple oral and oropharyngeal sample types. Methods This pilot single-center randomized controlled clinical trial enrolled adults in the San Francisco Bay Area, California, who tested positive for COVID-19. Participants were randomized to distilled water, chlorine dioxide, hydrogen peroxide, cetylpyridinium chloride, and essential oils. Participants were instructed to rinse and gargle four times daily for four weeks using standardized instructions to ensure protocol adherence. Samples were collected on Days 1, 7, and 28 and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The primary outcome was the change in SARS-CoV-2 viral load from baseline to Day 28, assessed using cycle threshold (Ct) values. Secondary outcomes included self-reported clinical symptoms and hospitalization. Results Forty-nine participants completed the study. No mouthwash demonstrated a statistically significant reduction in SARS-CoV-2 viral load over time. Cetylpyridinium chloride showed a transient increase in Ct values on Day 7 that was not sustained on Day 28. At baseline, throat swab samples had the lowest Ct values across all sample types. Due to limited subgroup sample sizes for secondary outcome measures, no statistical or moderator analyses were conducted. Conclusion Further large-scale randomized trials are needed before recommending antiseptic mouthwashes for SARS-CoV-2 prevention or management.

Antimicrobial resistance (AMR) is a major global public health threat, and Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) represent some of the most common and concerning pathogens in clinical settings. Importantly, the dissemination of these resistance mechanisms is largely driven by mobile genetic elements (MGEs), particularly plasmids. Advancing our understanding of AMR evolution through experimentation requires moving beyond domesticated laboratory strains and towards clinically relevant isolates. However, despite the abundance of genomic data in public repositories, there is a lack of well-characterised clinical collections available for experimental work. Here, we characterise the RyC collection, which includes 50 multidrug-resistant, ESBL-producing Escherichia coli and Klebsiella spp. strains isolated from the gut microbiota of hospitalised patients at Hospital Universitario Ramon y Cajal (Madrid, Spain). We generated high-quality genome assemblies for all strains using a combination of short- and long-read sequencing technologies. From these data, we performed a comprehensive characterisation of the pangenome, mobilome, resistome and defensome of the collection. We present the RyC collection as a robust and experimentally tractable resource to study AMR evolution and MGEs dynamics in clinically relevant bacterial backgrounds. Impact statementAntimicrobial resistance (AMR) is a growing global health threat driven by the rapid dissemination of resistance genes among clinically relevant bacteria. A major challenge in studying AMR evolution is the reliance on domesticated laboratory strains, which poorly represent the complexity of pathogens circulating in hospitals. Here, we introduce the RyC collection, a set of well-characterised, multidrug-resistant Enterobacterales isolates obtained from hospitalised patients. By combining high-quality genome sequencing with detailed analyses of their gene content and mobile genetic elements (MGEs), this collection provides a realistic and experimentally tractable system to study how resistance evolves and spreads. The RyC collection will facilitate research on AMR dynamics, plasmid biology and host-MGEs interactions, ultimately contributing to the development of more effective strategies to combat antibiotic-resistant infections.

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

Necrotizing soft tissue infections (NSTIs) are fulminant bacterial diseases characterized by rapid tissue destruction, systemic deterioration, and high mortality. Aeromonas hydrophila is an important causative agent of NSTIs, but the system-level bacterial mechanisms that coordinate tissue destruction, in vivo expansion, dissemination, and host lethality remain incompletely understood. Here, we investigated the contribution of the GspCD-dependent type II secretion system (T2SS) to A. hydrophila pathogenesis using transposon mutants, extracellular protein analyses, and a mouse NSTI model. Mutants carrying transposon insertions in gspD and gspC showed defective secretion of a FLAG-tagged truncated AerA construct and markedly reduced hemolytic activity in culture supernatants. Comparative analysis of extracellular proteins further showed that disruption of gspC altered the extracellular protein landscape, with reduced abundance of multiple known or predicted virulence-associated factors, including AerA, Ahh, lipase, and metalloprotease. In the mouse NSTI model, both mutants exhibited attenuated virulence, including reduced serum markers of tissue injury, less severe histopathological damage, impaired in vivo expansion and dissemination, and decreased lethality. These defects were more pronounced in the gspC mutant than in the gspD mutant. Together, these findings show that the GspCD-dependent T2SS functions as a coordinated extracellular secretion system that drives tissue destruction, in vivo expansion, dissemination, and lethal outcome during A. hydrophila NSTI. IMPORTANCENecrotizing soft tissue infections (NSTIs) are rapidly progressive, life-threatening bacterial infections, and Aeromonas hydrophila is an important causative agent. Here, we show that the GspCD-dependent type II secretion system (T2SS) drives A. hydrophila virulence in a murine NSTI model. Transposon mutants in gspC or gspD exhibited impaired extracellular protein secretion, reduced hemolytic activity, attenuated tissue damage, decreased bacterial proliferation and dissemination, and markedly reduced lethality. Comparative analysis further indicated that T2SS disruption alters the extracellular virulence landscape rather than affecting a single toxin alone. These findings provide in vivo evidence that coordinated T2SS-dependent secretion is a central determinant of severe A. hydrophila soft tissue infection.

Members of the Mammalian Cell Entry (MCE) superfamily interact with other proteins to form diverse architectures for the transport of hydrophobic molecules across the cell envelope in Gram-negative bacteria. Some of these trans-envelope MCE protein complexes include a PqiC-like outer membrane (OM) lipoprotein component. The best-studied member of this group of OM lipoproteins is E. coli PqiC, from the PqiABC system, which can form an octameric ring. How PqiC-like lipoproteins interact with their MCE protein binding partners to facilitate transport is not well understood. Here we report the cryo-electron microscopy structures of Pseudomonas aeruginosa PA3214, a homolog of PqiC, in the context of the full MCE transport PA3211-PA3214 system. Our structure provides insight into the biological assembly of the lipoprotein and interactions with its binding partner, MCE protein PA3213. We utilize deep mutational scanning to identify functionally important sites in E. coli PqiC in an unbiased manner. Through phenotypic and biochemical experiments, we characterize the interactions of the lipoproteins PqiC and PA3214 with their associated MCE proteins PqiB and PA3213, thus providing a model for how some MCE proteins employ a C-terminal peptide to mediate key interactions with their cognate lipoproteins at the OM.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While several studies have characterised the composition and roles of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used 16S rRNA gene sequencing to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Analysis of 239 samples revealed a set of dominant bacterial genera commonly associated with other fish species, including Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter, that constituted a core skin microbiota across lochs. Microbiome composition was primarily shaped by environmental variables, particularly habitat, water pH, conductivity, and metal concentrations, with pH emerging as a key driver of community structure. Host sex also influenced microbiome variation, with several taxa differing in relative abundance between males and females. Alpha-diversity was higher among stickleback fish from lochs with a neutral pH compared with those from alkaline and acidic environments. Differential abundance analyses identified 27 and 24 amplicon sequence variants (ASVs), respectfully, associated with variations in pH and host sex, including members of Psychrobacter, Sphingobacterium, Carnobacterium, Chryseobacterium, and Arthrobacter, highlighting the combined influence of environmental and host factors on microbiome composition in wild fish populations in freshwater environments.

Francisella tularensis is a highly infectious, Gram-negative intracellular bacterium and the causative agent of tularemia, a potentially fatal disease. Owing to its low infectious dose, ease of aerosolization, high virulence, lack of an effective vaccine, and potential use as a bioterrorism agent, F. tularensis is classified by the CDC as a Tier 1 Category A Select Agent. Despite its clinical importance, the mechanisms underlying F. tularensis virulence remain incompletely understood. In this study, we generated a partial Tn5 transposon insertion mutant library in the F. tularensis live vaccine strain (LVS) and identified a mutant disrupted in the FTL_0690 gene through screening under macrophage-like conditions. FTL_0690 encodes an acyl-CoA synthetase. Characterization of both a transposon-insertion mutant and a targeted deletion mutant ({Delta}FTL_0690) revealed critical roles for this enzyme in F. tularensis pathobiology. Loss of FTL_0690 increased sensitivity to oxidative stress and impaired intracellular growth within macrophages compared to wild-type F. tularensis LVS. Lipidomic profiling of the {Delta}FTL_0690 mutant revealed disruptions in fatty acid metabolism, membrane lipid remodeling, and redox homeostasis. Altered lipid-derived and membrane-associated metabolites indicated defective phospholipid incorporation and altered membrane composition, likely contributing to oxidative stress sensitivity and reduced intramacrophage survival. Collectively, these findings demonstrate that FTL_0690 which encodes long-chain acyl-CoA synthetase, contributes to lipid homeostasis, membrane integrity, and oxidative stress resistance of F. tularensis. ImportanceThis work addresses critical gaps in our understanding of Francisella tularensis virulence by identifying lipid metabolism as a central determinant of intracellular survival and stress resistance. By integrating transposon mutagenesis, targeted gene deletion, and lipidomic profiling, this study provides mechanistic insight into how metabolic remodeling supports pathogenesis. Our identification and characterization of FTL_0690 as a long-chain acyl-CoA synthetase essential for lipid homeostasis, membrane integrity, and oxidative stress resistance reveals a previously unappreciated link between fatty acid metabolism and intramacrophage survival of F. tularensis.

Background: Microbiota dysbiosis of the skin has been implicated in ulcer formation. Individuals with diabetes remain at high risk for diabetic foot ulcers (DFUs) even after ulcer healing. Topical chlorhexidine gluconate (CHG) is a broad-spectrum antiseptic commonly used to reduce microbial burden. In a prior randomized clinical trial comparing daily CHG foot treatment with soap-and-water treatment, no statistically significant reduction in new DFUs was observed, prompting evaluation of whether CHG produced durable changes in the skin microbiota. Objective: To compare changes in foot skin microbiota (including bacterial bioburden, diversity, and community composition) associated with daily CHG versus soap-and-water use over one year in people with diabetes and prior foot complications. Methods: In a single-center, double-blind, placebo-controlled randomized trial, 87 participants were randomized to daily CHG wipes or soap-and-water wipes for 12 months. Foot swabs were collected at baseline, 3 and 12 months, and 4 weeks post-treatment. Bacterial bioburden was quantified. Microbiota composition was assessed using 16S rRNA and ITS amplicon sequencing. Key Results: CHG treatment significantly reduced bacterial bioburden, increased microbial diversity, and altered community composition, including sustained reductions in Staphylococcus abundance. Several microbiota changes persisted more than 4 weeks after treatment cessation. Soap-and-water treatment showed similar but smaller and largely nonsignificant trends. Conclusions: Daily CHG use durably modifies foot skin microbiota in high-risk individuals with diabetes. However, this alone may be insufficient to prevent new foot complications, highlighting the need for additional interventions. These findings have implications for long-term CHG use in populations at risk for staphylococcal infections.

The gut microbiome plays a central role in host metabolism, immune function, and overall health, with disruptions in microbial composition (dysbiosis) being associated with a range of metabolic, inflammatory, and infectious conditions [1,2]. Consequently, strategies aiming to modulate the microbiome require selective activity that preserves beneficial commensals while limiting pathogenic organisms [3]. In this context, ThymoQuin(R)--a cold-pressed, standardized black cumin (Nigella sativa) seed oil developed by TriNutra Ltd. and defined by [≥]3% thymoquinone (TQ), controlled p-cymene levels, and low free fatty acids ([≤]1.25%)--was evaluated for its microbiome-relevant activity. In vitro minimum bactericidal concentration (MBC) assays across three independent batches demonstrated a biphasic, dose-dependent response. At intermediate concentrations (0.25-0.5%), Streptococcus thermophilus was strongly stimulated (up to 53-fold) and Lactiplantibacillus plantarum fully preserved, while Klebsiella pneumoniae was effectively reduced (>94%). Akkermansia muciniphila exhibited stable viability at concentrations below 1%, with reductions only observed at 1%. This is notable given its role as a mucin-degrading commensal that has been linked to metabolic health, but whose abundance may vary across physiological and disease contexts [4,5]. At concentrations [≥]1%, selective effects diminished, resulting in broader antimicrobial activity and reduced specificity. These findings indicate a defined concentration range in which selective microbiome modulation is maintained, whereas higher thymoquinone levels may increase the risk of non-selective detrimental effect on microbes.

BackgroundThe global rise of multidrug-resistant (MDR) bacteria represents a critical public health threat, and Romania ranks amongst the most affected countries in Europe. As conventional therapy increasingly fails, bacteriophage therapy has re-emerged as a promising alternative to antibiotics. Urban rivers, contaminated with resistant bacterial strains, represent an underexplored and accessible reservoir for the isolation of lytic phages with therapeutic potential. MethodsTwo bacteriophages, 17M_Ec17_D and 22C_Ec22_D, were isolated from the Dambovita River, Bucharest, Romania, using MDR E. coli as host bacteria. Phage characterization included plaque morphology, transmission electron microscopy, and host range assessment by spot assay against 30 MDR E. coli isolates. Whole genome sequencing was performed on Illumina MiSeq and Oxford Nanopore Technologies MinION platforms, followed by bioinformatic analysis including taxonomic classification, lifestyle prediction, and functional annotation. ResultsBoth phages formed clear plaques and were classified as Kayfunavirus (17M_Ec17_D, Podoviridae-like) and Kagunavirus (22C_Ec22_D, Siphoviridae-like) with nucleotide similarities of 89.2% and 71.4% to their closest relatives, respectively, suggesting both are candidates for novel species. Host range analysis revealed lytic activity against 13% and 10% of tested MDR isolates, with complementary infection profiles. Genomic analysis confirmed a strictly lytic lifestyle for both phages, supported by the presence of holin and spanin genes and the absence of lysogenic modules, antibiotic resistance genes, and virulence factors. ConclusionsTo the best of our knowledge, this is the first study conducted in Romania to isolate and genomically characterize lytic bacteriophages targeting MDR E. coli. The characterized phages represent safe therapeutic candidates whose complementary host ranges suggest potential application as part of phage cocktail to broaden antimicrobial coverage against MDR infections.