Biology subjects

Hoyles, L.

Publications and source records attributed to Hoyles, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-resolved surveillance of African Klebsiella oxytoca species complex genomes reveals resistome-mobilome and biosynthetic gene cluster diversity

The Klebsiella oxytoca species complex (KoSC) comprises taxonomically diverse commensals and opportunistic pathogens, but its genomic diversity remains poorly characterized across Africa. We curated publicly available African KoSC data through raw-read and public-assembly routes and analyzed 163 African genomes together with 282 global comparators. Pangenome, phylogenomic, sequence-typing, surface-locus, antimicrobial-resistance, plasmid-replicon, mobile-element, biosynthetic-gene-cluster, and virulence-component analyses were integrated. The African collection comprised K. michiganensis (112/163), K. oxytoca (36/163), K. pasteurii (8/163), and K. grimontii (7/163) from 13 countries. The African pangenome contained 4,286 core, 4,505 shell, and 18,066 cloud gene families. Official PubMLST sequence types were assigned to 129/163 genomes. Four core/intrinsic antimicrobial-resistance-associated loci (ompA, oqxA, oqxB, and blaOXY) occurred in all genomes, whereas acquired resistance determinants were heterogeneous. Intact til biosynthetic gene clusters occurred in 55/163 genomes and intact leup clusters in 103/163. leup was concentrated in K. michiganensis (102/112), whereas intact til was frequent in K. oxytoca (26/36), K. pasteurii (7/8), and K. grimontii (7/7). Klebsiella-focused Virulence Factor Database screening detected at least one curated component in 112/163 genomes, but no complete curated factor; three K. michiganensis genomes carried complete mrkABCDF structural-operon candidates. These data define an African genome-resolved baseline for KoSC diversity and identify species-structured biosynthetic loci alongside heterogeneous resistance and mobilome profiles.

microbiology↗

bioRxiv2026

Hippurate hydrolases encoded by Klebsiella pneumoniae and Klebsiella oxytoca are functional

Human urine is non-sterile, harbouring its own microbiota (urobiota). Klebsiella species are commensals of the urobiota but can also cause urinary tract infections (UTIs). Gut-microbiota-host co-metabolites (MHCMs) such as hippurate are major components of urine. We previously showed UTI-associated strains of Klebsiella pneumoniae break down hippurate in human urine, though the enzymes responsible for this bioconversion are unknown. Here we sought to confirm that K. pneumoniae hydrolyses hippurate, and to determine whether the enzyme(s) responsible for this bioconversion are present and/or active in other Klebsiella species. Ninhydrin assays confirmed that strains of K. pneumoniae (n=3) and, albeit to a lesser extent, Klebsiella oxytoca (n=4) isolated from human urine could produce glycine from hippurate. Comparative protein (KEGG, UniProt, phylogenetic) and structural (AlphaFold, ChimeraX) analyses were used to predict putative hippurate hydrolases (HHs) encoded by Klebsiella species. Their gene products (n=2 K. pneumoniae; n=3 K. oxytoca) were cloned, expressed and purified. The proteins belonged to three distinct groups: only group 1 and group 2 HHs hydrolysed hippurate under conditions used in this study. Both HHs co-occurred with high prevalence in 15/20 (75 %) Klebsiella species (2885/3012 genomes, 95.8 %). Most Klebsiella species encode two distinct HHs that release glycine and benzoate from hippurate. The functional group 2 HH is predicted to facilitate delivery of amino acids such as glycine to K. pneumoniae cells in the nutrient-limited environment of urine, and its activity demonstrates that members of the urobiota and opportunistic pathogens can contribute to catabolism of MHCMs in human urine.

microbiology↗

bioRxiv2026

A capsule polysaccharide synthesis locus database for the Klebsiella oxytoca Species Complex

The Klebsiella oxytoca Species Complex (SC) represents an emerging healthcare-associated group of opportunistic pathogens. The capsular polysaccharide is a virulence determinant and target for novel vaccines, monoclonal antibodies and phage therapy. In the absence of broadly accessible phenotyping techniques, prediction of capsule types from whole-genome sequence data is critical for understanding capsule diversity and epidemiology, and to prioritise capsule types as targets for novel anti-K. oxytoca SC interventions. Here we present the first comprehensive capsule synthesis locus (K locus) database targeted for the K. oxytoca SC, comprising 88 distinct loci defined by gene content and which is compatible with the rapid genome typing tool Kaptive. The database provides high coverage of publicly available K. oxytoca SC genomes (97.6% of 2,244 genomes, dereplicated from a total of 4,055), and the typing rate is significantly higher than that achieved with the pre-existing Klebsiella K locus database (97.6% vs 50.3%, p <0.0001), which primarily targets the Klebsiella pneumoniae SC. We demonstrate the utility of the novel K. oxytoca SC database by application to three diverse clinical K. oxytoca SC isolate collections (n=61 to 102 genomes each), suggesting a high diversity of K types. The novel K. oxytoca SC K locus database (github.com/klebgenomics/KoSC-surface-antigen-loci) will provide a key resource to support larger systematic studies and ongoing genomics surveillance efforts for the K. oxytoca SC. IMPACT STATEMENTMembers of the Klebsiella oxytoca Species Complex (SC) are an emerging cause of infections in humans and are frequently associated with antimicrobial resistance. Klebsiella species produce two key surface antigen sugars (capsular polysaccharide and lipopolysaccharide) that are immunogenic and are targets for novel control strategies such as vaccines and phage therapy. Phenotypic typing of these surface antigen sugars (serotyping) is costly and laborious, with genotyping (predicting the serotype from whole-genome sequence data) a useful alternative. Here, we present a curated capsule (K) locus reference database for the K. oxytoca SC, which represents a useful tool to assist in the epidemiological surveillance of this emerging pathogen. DATA SUMMARYAll Klebsiella oxytoca Species Complex genomes used in this work were publicly available, with accession details listed in Supplementary Tables 1, 2 and 3. The K. oxytoca Species Complex K locus reference database is available under GNU public license at github.com/klebgenomics/KoSC-surface-antigen-loci.

bioinformatics↗

bioRxiv2026

Comparative analyses of Gram-negative bacteria isolated from cancer patients with bacteraemia at the Uganda Cancer Institute

Antimicrobial resistance (AMR) exacerbates bacteraemia in cancer patients, particularly in low-resource settings. At the Uganda Cancer Institute, high rates of Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) have been reported, with DNA-based detection of bla genes limited to PCR. This study aimed to determine whether bacterial genomic DNA shipped at ambient temperature from Uganda to the UK retained sufficient quality for whole-genome sequencing (WGS), to allow in-depth genomic analyses of isolates. Genomic DNA was extracted from Gram-negative bloodstream isolates (n=77) in Uganda and shipped to the UK at ambient temperature. rpoB gene (77/77, 100%) and WGS data (72/77, 93.5%) were generated for isolates, with 66/72 (91.7%) genomes of high-quality (Escherichia coli n=34; Klebsiella spp. n=32). Bioinformatic analyses included species identification, sequence typing, SNP analysis, AMR and virulence gene profiling, and comparison with publicly available genomes of Ugandan isolates. Phenotypic-genotypic concordance was generally high: 7/77 (9.1%) isolates were misidentified by phenotypic testing, and two showed unexplained carbapenem resistance. E. coli isolates showed diverse sequence types, with high prevalence of blaCTX-M (91.2%) and blaOXA-1 (47.1%); carbapenemase genes were rare. Klebsiella isolates lacked hypermucoidy loci and displayed diverse capsule types, with a high prevalence of ESBLs. Genomic clustering suggested limited within-hospital transmission of strains. Genomic data can provide important insights into the dissemination of bacterial subclades of global concern. The widespread AMR genotypes reported here highlight the need for improved diagnostics and updated treatment guidelines for bacteraemia in Ugandan cancer patients. IMPACT STATEMENTBloodstream infections are a major threat to cancer patients, particularly in low-resource settings where access to advanced diagnostics is limited and infection prevention may be challenging. This study shows that it is feasible to transport bacterial DNA at room temperature from Uganda to the UK for high-quality whole-genome sequencing, helping to overcome a logistical barrier to genomic surveillance. By applying genomic analysis to bacteria that had caused bloodstream infections at the Uganda Cancer Institute, we found that traditional laboratory methods can misidentify some bacteria, and that genomic analyses can provide more accurate and detailed insights into the bacteria causing these serious infections. Our work also highlights gaps in current treatment guidelines and demonstrates how genomic data could help inform updates to these, facilitating more effective antibiotic use in situations where urgent treatment is needed and there is no time to wait for laboratory test results. While there was limited evidence of direct transmission of bacteria between patients in our dataset, the genetic diversity and resistance patterns we observed are concerning and emphasise the need for ongoing monitoring and more extensive future studies.

microbiology↗

bioRxiv2025

The Omp85 family protein, TamA, exhibits characteristics 1 of a suitable drug target against Pseudomonas aeruginosa

The outer membrane (OM) of Gram-negative bacteria is crucial for cell stability and virulence and acts as a permeability barrier. The biogenesis, assembly, and regulation of proteins in the OM are therefore attractive areas of study that could lead to identifying novel drug targets. The Translocation and Assembly Module (TAM), composed of TamA and TamB, facilitates the insertion of some {beta}-barrel proteins into the OM of Escherichia coli and Klebsiella pneumoniae, and has also been implicated in lipid homeostasis. However, its role in Pseudomonas aeruginosa remains mostly uncharacterized. To investigate the TAMs function and drug target potential in P. aeruginosa, we generated both single- and double-gene TAM knockouts and assessed their fitness using competition growth assays against wild-type (WT) strains. The WT significantly outcompeted the TAM mutants, indicating a fitness defect. Proteomic analysis revealed surprisingly similar profiles between WT and the double knockout strains, while single knockouts showed changes in OM proteins and reduced expression of flagellar components consistent with attenuated swimming motility observed in {Delta}tamA. Single mutants exhibited differential levels of expression of lipoproteins of the {beta}-barrel assembly machinery suggesting compensatory OM remodelling. In vivo infection assays using Galleria mellonella larvae demonstrated significantly higher survival rates when infected with TAM mutants, with tamA mutants showing the greatest attenuation in virulence. Our findings demonstrate a role the TAM plays in P. aeruginosa virulence and identify TamA as a potential drug target for the development of new antimicrobial therapies. Data summaryThe RNAseq data reported on in this article are available from ArrayExpress: E-MTAB-15348 The mass spectrometry proteomics data have been deposited to the ProteomeXchange with the dataset identifier PXD06704.

microbiology↗

bioRxiv2025

Growth of Gram-negative uropathogens in human urine under hypoxic conditions produces clinically relevant metabolomic profiles

BackgroundMuch work on uropathogens is done in rich laboratory media that do not reflect the nutrient availability of urine. Also, the environment of the bladder is microaerobic ([~]5 % O2) but routine laboratory work with uropathogens is done under aerobic conditions. AimsTo understand the influence of oxygen conditions (aerobic, microaerobic) and physiologically relevant growth substrates (artificial urine, human urine) on the ability of bacteria isolated from catheter-associated urinary tract infections (CAUTIs) to form biofilms, and to begin to define the CAUTI bacterial metabolome. MethodsBiofilm assays were conducted in rich lab medium (tryptone soy broth supplemented with glucose), and commercially available artificial and pooled human urine for 29 well-characterized uropathogens representing five different genera of Gram-negative bacteria. Spent media were collected and analysed using 400 MHz 1H-NMR, to quantify major metabolites produced by uropathogens in artificial urine and human urine. FindingsThere was a significant decrease in biofilm formation for all uropathogens when grown in physiologically relevant growth substrates. Untargeted metabolomic analyses of artificial and human urine showed the artificial urine used in this study did not recapitulate the complexity of major metabolites present in human urine. Further analyses of spent human urine highlighted significant increases in acetate production by the uropathogens when they were grown under microaerobic compared with aerobic conditions. ConclusionsGrowth of uropathogenic Enterobacteriaceae under physiologically relevant conditions (i.e. human urine, 5 % O2) generates data more relevant to clinical disease and is an important consideration for future work on bacteria causing urinary tract infections.

microbiology↗

bioRxiv2025

Phenotype-genotype discordance in antimicrobial resistance profiles of Gram-negative uropathogens recovered from catheter-associated urinary tract infections in Egypt

Catheter-associated urinary tract infections (CAUTIs) are among the most common healthcare-associated infections in low- and middle-income countries (LMICs). In this study, phenotypic (EUCAST AMR profiles) and genomic data were generated for 132 isolates (67 Escherichia coli, 14 Pseudomonas aeruginosa, 11 Klebsiella pneumoniae, 9 Proteus mirabilis, 8 Providencia spp., 5 Enterobacter hormaechei, and 18 rare uropathogens) recovered from CAUTIs in an Egyptian hospital. Among the Escherichia coli isolates, phylogroup B2 was most represented (53.7 %), followed by B1 (19.4 %), A (11.9 %), D (7.4 %), F (5.9 %) and C (1.4 %). Several (22/132, 16.6 %) isolates were multidrug-resistant, while almost half (62/132, 46.9 %) were extensively drug-resistant. Comparison of phenotypic data with genotypic data from three different AMR-profiling tools (ResFinder, CARD, AMRFinder) highlighted phenotype-genotype discordance as an important consideration in resistome studies in Egypt, with a total concordance of 91.1 %, 85.7 %, and 80.3 % for ResFinder, CARD and AMRFinder, respectively. Pseudomonas, at the species level, exhibited the greatest discordance. At the antimicrobial level, meropenem was subject to greatest discordance. In addition to the findings from our comparative analyses, new AMR variants are reported for Egypt for Pseudomonas (OXA-486, OXA-488, OXA-905, IMP-43, PDC-35, PDC-45, PDC-201) and Escherichia coli (TEM-176, TEM-190). In summary, we show that there is phenotype-genotype discordance in AMR profiling among CAUTI isolates, highlighting the need for comprehensive approaches in resistome studies. We also show the genomic diversity of Gram-negative uropathogens contributing to disease burden in a little-studied LMIC setting.

microbiology↗

bioRxiv2024

Menadione reduces the expression of virulence- and colonisation-associated genes in Helicobacter pylori

Novel treatment options are needed for the gastric pathogen Helicobacter pylori due to its increasing antibiotic resistance. The vitamin K analogue menadione has been extensively studied due to interest in its antibacterial and anti-cancer properties. Here, we investigated the effects of menadione on H. pylori growth, viability, antibiotic resistance, motility and gene expression using clinical isolates. The minimum inhibitory concentration (MIC) of menadione was 313 {micro}M for 11/13 isolates and 156 {micro}M for 2/13 isolates. The minimum bactericidal concentrations were 1.25-2.5 mM, indicating that concentrations in the micromolar range were bacteriostatic rather than bactericidal. We were not able to experimentally evolve resistance to menadione in vitro. Sub-MIC menadione (16 {micro}M for 24 h) did not significantly inhibit bacterial growth but significantly (p < 0.05) changed the expression of 1291/1615 (79.9 %) genes encoded by strain 322A. Expression of the virulence factor genes cagA and vacA was downregulated in the presence of sub-MIC menadione, while genes involved in stress responses were upregulated. Sub-MIC menadione significantly (p < 0.0001) inhibited the motility of H. pylori, consistent with the predicted effects of the observed significant (p < 0.05) downregulation of cheY, upregulation of rpoN, and changes in expression of flagellar assembly pathway genes seen in the transcriptomic analysis. Through in-depth interrogation of transcriptomics data, we concluded that sub-MIC menadione elicits a general stress response in H. pylori with survival in the stationary phase likely mediated by upregulation of surE and rpoN. Sub-MIC menadione caused some modest increases in H. pylori susceptibility to antibiotics, but the effect was variable with strain and antibiotic type and did not reach statistical significance. Menadione (78 {micro}M) was minimally cytotoxic to human gastric adenocarcinoma (AGS) cells after 4 hours but caused significant loss of cell viability after 24 hours. Given its inhibitory effects on bacterial growth, motility and expression of virulence and colonisation- associated genes, menadione at low micromolar concentrations may have potential utility as a virulence-attenuating agent against H. pylori. Data summaryRNAseq data reported on in this study are available to download from ArrayExpress under accession E-MTAB-14439. Supplementary materials associated with this publication are available from https://figshare.com/projects/Menadione_reduces_the_expression_of_virulence-_and_colonisation-associated_genes_in_Helicobacter_pylori/219631.

microbiology↗

bioRxiv2024

Weberviruses are gut-associated phages that infect Klebsiella spp.

Weberviruses are bacteriophages (phages) that can infect and lyse clinically relevant, multidrug-resistant (MDR) strains of Klebsiella. They are an attractive therapeutic option to tackle Klebsiella infections due to their high burst sizes, long shelf life and associated depolymerases. In this study we isolated and characterized seven new lytic phages and compared their genomes with those of their closest relatives. Gene-sharing network, ViPTree proteome and terL gene-sequence-based analyses incorporating all publicly available webervirus genomes [n=258 from isolates, n=65 from metagenome-assembled genome (MAG) datasets] confirmed the seven phages as members of the genus Webervirus and identified a novel genus (Defiantjazzvirus) within the family Drexlerviridae. Using our curated database of 265 isolated phage genomes and 65 MAGs (n=330 total), we found that weberviruses are distributed globally and primarily associated with samples originating from the gut: sewage (154/330, 47 %), wastewater (83/330, 25 %) and human faeces (66/330, 20 %). We identified three distinct clusters of potential depolymerases encoded within the 330 genomes. Due to their global distribution, frequency of isolation and lytic activity against the MDR clinical Klebsiella strains used in this study, we conclude that weberviruses and their depolymerases show promise for development as therapeutic agents against Klebsiella spp.

microbiology↗

bioRxiv2024

The plasmid-borne hipBA operon of Klebsiella michiganensis encodes a potent plasmid stabilization system

AimsKlebsiella michiganensis is a medically-important bacterium that has been subject to relatively little attention in the literature. Interrogation of sequence data from K. michiganensis strains in our collection has revealed the presence of multiple large plasmids encoding type II toxin-antitoxin (TA) systems. Such TA systems are responsible for mediating a range of phenotypes including plasmid stability ("addiction") and antibiotic persistence. In this work, we characterize the hipBA TA locus found within the Klebsiella oxytoca species complex (KoSC). Methods and ResultsThe HipBA TA system is encoded on a plasmid carried by K. michiganensis PS_Koxy4, isolated from an infection outbreak. Employing viability and plasmid stability assays, we demonstrate that PS_Koxy4 HipA is a potent antibacterial toxin and that HipBA is a functional TA module contributing substantially to plasmid maintenance. Further, we provide in silico data comparing HipBA modules across the entire KoSC. ConclusionsWe provide the first evidence of the role of a plasmid-encoded HipBA system in stability of mobile genetic elements and analyze the presence of HipBA across the KoSC. These results expand our knowledge of both a common enterobacterial TA system and a highly medically-relevant group of bacteria. Impact StatementThe HipBA TA system is typically encoded on bacterial chromosomes where it contributes to antimicrobial tolerance by interfering with translation during cellular stress. Here, we show that plasmid-encoded HipBA from a disease isolate of Klebsiella michiganensis is responsible for highly effective plasmid addiction; the first such evidence of a HipBA module contributing to plasmid stability. This has important implications for enteric pathogen evolution and horizontal gene transfer in the era of multidrug resistance.

microbiology↗

bioRxiv2023

Lelliottia amnigena recovered from the lung of a harbour porpoise, and comparative analyses with Lelliottia spp.

Strain M1325/93/1 (= GFKo1) of Lelliottia amnigena was isolated from the lung of a harbour porpoise in 1993. The genome sequence and antimicrobial resistance profile (genomic, phenotypic) of the strain were generated, with the genomic data compared with those from closely related bacteria. We demonstrate the recently described chromosomally-encoded AmpC {beta}-lactamase blaLAQ is a core gene of L. amnigena, and suggest new variants of this class of lactamase are encoded by other members of the genus Lelliottia. Although presence of blaLAQ is ubiquitous across the currently sequenced members of L. amnigena, we highlight that strain GFKo1 is sensitive to ampicillin and cephalosporins. These data suggest blaLAQ may act as a useful genetic marker for identification of L. amnigena strains, but its presence may not correlate with expected phenotypic resistances. Further studies are required to determine the regulatory mechanisms of blaLAQ in L. amnigena.

microbiology↗

bioRxiv2023

Phenotypic and genomic characterization of Pseudomonas aeruginosa isolates recovered from catheter-associated urinary tract infections in an Egyptian hospital

Catheter-associated urinary tract infections (CAUTIs) represent one of the major healthcare-associated infections, and Pseudomonas aeruginosa is a common Gram-negative bacterium associated with catheter infections in Egyptian clinical settings. The present study describes the phenotypic and genotypic characteristics of 31 P. aeruginosa isolates recovered from CAUTIs in an Egyptian hospital over a 3-month period. Genomes of isolates were of good quality and were confirmed to be P. aeruginosa by comparison to the type strain (average nucleotide identity, phylogenetic analysis). Clonal diversity among the isolates was determined; eight different sequence types were found (STs 244, 357, 381, 621, 773, 1430, 1667 and 3765), of which 357 and 773 are considered high-risk clones. Antimicrobial resistance (AMR) testing according to EUCAST guidelines showed the isolates were highly resistant to quinolones [ciprofloxacin (12/31, 38.7 %) and levofloxacin (9/31, 29 %) followed by tobramycin (10/31, 32.5 %)], and cephalosporins (7/31, 22.5 %). Genotypic analysis of resistance determinants predicted all isolates to encode a range of AMR genes, including those conferring resistance to aminoglycosides, {beta}-lactamases, fluoroquinolones, fosfomycin, sulfonamides, tetracyclines and chloramphenicol. One isolate was found to carry a 422,938 bp pBT2436-like megaplasmid encoding OXA-520, the first report from Egypt of this emerging family of clinically important mobile genetic elements. All isolates were able to form biofilms, and were predicted to encode virulence genes associated with adherence, antimicrobial activity, antiphagocytosis, phospholipase enzymes, iron uptake, proteases, secretion systems, and toxins. The present study shows how phenotypic analysis alongside genomic analysis may help us understand the AMR and virulence profiles of P. aeruginosa contributing to CAUTIs in Egypt.

microbiology↗

bioRxiv2023

Phage vB_KmiS-Kmi2C infects members of the Klebsiella oxytoca complex and represents a novel genus of lytic bacteriophage

AIMSThis study aimed to characterise the lytic phage vB_KmiS-Kmi2C, isolated from sewage water on a GES-positive strain of Klebsiella michiganensis. METHODS AND RESULTSComparative phylogenetic and network-based analyses were used to characterise the genome of phage vB_KmiS-Kmi2C (circular genome of 42,234 bp predicted to encode 55 genes), demonstrating it shared little similarity with other known phages. The phage was lytic on clinical strains of K. oxytoca (n=2) and K. michiganensis (n=4), and was found to both prevent biofilm formation and disrupt established biofilms produced by these strains. CONCLUSIONSWe have identified a phage capable of killing clinically relevant members of the Klebsiella oxytoca complex (KoC). The phage represents a novel virus family (proposed name Dilsviridae) and genus (proposed name Dilsvirus). SIGNIFICANCE AND IMPACT OF THE STUDYIdentification a novel lytic phage active against clinically relevant strains of the KoC provides an alternative to antibiotics to treat these increasingly antimicrobial-resistant opportunistic pathogens. The unusual way in which the phage can disrupt established biofilms may allow us to identify novel phage-based approaches for biofilm remediation in the future.

microbiology↗

bioRxiv2022

Cerebrovascular damage caused by the gut microbe-derived uraemic toxin p-cresol sulfate isprevented by blockade of the epidermal growth factor receptor

Circulating levels of the gut microbe/host co-metabolite p-cresol sulfate (pCS) correlate with cerebrovascular event risk in individuals with chronic kidney disease, but whether this relationship is mechanistic is unclear. We hypothesised that pCS would impair function of the blood-brain barrier (BBB), the primary brain-vasculature interface. We report that pCS exposure impairs BBB integrity in human cells in vitro and both acutely and chronically in mice, enhancing tracer extravasation, disrupting barrier-regulating tight junction components and ultimately affecting whole-brain transcriptomic activity. In vitro and in vivo mechanistic studies showed that pCS activated epidermal growth factor receptor (EGFR) signalling, resulting in matrix metalloproteinase mobilisation and BBB damage. Furthermore, blockade of EGFR prevented the permeabilising effects of serum from haemodialysis patients upon cerebromicrovascular endothelia in vitro. Our results define a pathway linking the co- metabolite pCS with BBB damage and suggest targeting the EGFR may mitigate against cerebrovascular damage in CKD.

systems biology↗

bioRxiv2022

Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes

High levels of antimicrobial resistance among members of the Klebsiella oxytoca complex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positive Klebsiella michiganensis strains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genus Slopekvirus. rpoB gene-based sequence analysis of 108 presumptive K. oxytoca isolates (n=59 clinical, n=49 veterinary) found K. michiganensis to be more prevalent (46 % clinical and 43 % veterinary, respectively) than K. oxytoca (40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several putative homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at [≥]95 % identity and [≥]70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.

microbiology↗

bioRxiv2022

A host-gut microbial co-metabolite of aromatic amino acids, p-cresol glucuronide, promotes blood-brain barrier integrity in vivo

PurposeThe sequential activity of gut microbial and host processes can exert a powerful modulatory influence on dietary components, as exemplified by the metabolism of the amino acids tyrosine and phenylalanine to p-cresol by gut microbes, and then to p-cresol glucuronide (pCG) by host enzymes. Although such glucuronide conjugates are classically thought to be biologically inert, there is accumulating evidence that this may not always be the case. We investigated the activity of pCG, studying its interactions with the cerebral vasculature and the brain in vitro and in vivo. MethodsMale C57Bl/6J mice were used to assess blood-brain barrier (BBB) permeability and whole brain transcriptomic changes in response to pCG treatment. Effects were then further explored using the human cerebromicrovascular endothelial cell line hCMEC/D3, assessing paracellular permeability, transendothelial electrical resistance and barrier protein expression. ResultsMice exposed to pCG showed reduced BBB permeability and significant changes in whole brain transcriptome expression. Surprisingly, treatment of hCMEC/D3 cells with pCG had no notable effects until co-administered with bacterial lipopolysaccharide, at which point it was able to prevent the permeabilising effects of endotoxin. Further analysis suggested that pCG acts as an antagonist at the principal lipopolysaccharide receptor TLR4. ConclusionThe amino acid phase II metabolic product pCG is biologically active at the BBB, highlighting the complexity of gut microbe to host communication and the gut-brain axis.

systems biology↗

bioRxiv2021

Regulation of blood-brain barrier integrity and cognition by the microbiome-associated methylamines trimethylamine N-oxide and trimethylamine

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied. ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.

systems biology↗

bioRxiv2020

An Integrated Workflow for Enhanced Taxonomic and Functional Coverage of the Mouse Faecal Metaproteome

Intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behaviour. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomics is well suited for analysis of individual microbes, metaproteomics of faecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of microorganisms. Furthermore, there is a lack of consensus regarding preparation of faecal samples, as well as downstream bioinformatic analyses following metaproteomics data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse faeces in a typical mass spectrometry-based metaproteomic experiment. We show that subtle changes in sample preparation protocols may influence interpretation of biological findings. Two-step database search strategies led to significant underestimation of false positive protein identifications. Unipept software provided the highest sensitivity and specificity in taxonomic annotation of the identified peptides of unknown origin. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomics when studying complex microbiome samples.

microbiology↗