Biology subjects

McCartney, A. L.

Publications and source records attributed to McCartney, A. L..

9 recordsLinked to original sources

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

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

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↗

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↗

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

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↗

bioRxiv2020

The kleboxymycin biosynthetic gene cluster is encoded by several species belonging to the Klebsiella oxytoca complex

As part of ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. blaOXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis. Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to speciate members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii. The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7,170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four (K. oxytoca, K. pasteurii, K. grimontii, K. michiganensis) of the six species of the complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs. IMPACT STATEMENTMembers of the Klebsiella oxytoca complex are difficult to speciate using phenotypic and chemotaxonomic methods. Consequently, many genomes deposited in public databases are misclassified as K. oxytoca. Here we demonstrate that the current multi-locus sequence typing (MLST) system for the complex can be used to accurately speciate many strains, which will be of use to clinical laboratories in resource-limited settings which rely on the MLST scheme for typing and epidemiological tracking of isolates. In addition, extended analyses of the genomes of Klebsiella spp. have revealed the kleboxymycin biosynthetic gene cluster (BGC) is restricted to species of the Klebsiella oxytoca complex (K. oxytoca, K. michiganensis, K. pasteurii and K. grimontii). Species- and/or gene-specific differences in the clusters sequences may be relevant to virulence of K. oxytoca and related species. The finding of the kleboxymycin BGC in the preterm infant gut microbiota may have implications for disease presentation in a subset of neonates.

microbiology↗

bioRxiv2020

Succession of Bifidobacterium longum strains in response to the changing early-life nutritional environment reveals specific adaptations to distinct dietary substrates.

Diet-microbe interactions play a crucial role in infant development and modulation of the early-life microbiota. The genus Bifidobacterium dominates the breast-fed infant gut, with strains of B. longum subsp. longum (B. longum) and B. longum subsp. infantis (B. infantis) particularly prevalent. Although transition from milk to a more diversified diet later in infancy initiates a shift to a more complex microbiome, specific strains of B. longum may persist in individual hosts for prolonged periods of time. Here, we sought to investigate the adaptation of B. longum to the changing infant diet. Genomic characterisation of 75 strains isolated from nine either exclusively breast- or formula-fed (pre-weaning) infants in their first 18 months revealed subspecies- and strain-specific intra-individual genomic diversity with respect to glycosyl hydrolase families and enzymes, which corresponded to different dietary stages. Complementary phenotypic growth studies indicated strain-specific differences in human milk oligosaccharide and plant carbohydrate utilisation profiles of isolates between and within individual infants, while proteomic profiling identified active polysaccharide utilisation loci involved in metabolism of selected carbohydrates. Our results indicate a strong link between infant diet and B. longum subspecies/strain genomic and carbohydrate utilisation diversity, which aligns with a changing nutritional environment: i.e. moving from breast milk to a solid food diet. These data provide additional insights into possible mechanisms responsible for the competitive advantage of this Bifidobacterium species and its long-term persistence in a single host and may contribute to rational development of new dietary therapies for this important developmental window.

microbiology↗

bioRxiv2017

Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota

BACKGROUNDThe dietary methylamines choline, carnitine and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.\n\nRESULTSA feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did not produce notable quantities of TMA from TMAO in pure culture.\n\nCONCLUSIONSReduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium.\n\nCorrelation of metabolomic and abundance data from mixed microbiota fermentation systems did not give a true picture of which members of the gut microbiota were responsible for converting TMAO to TMA; only by supplementing the study with pure culture work and additional metabolomics was it possible to increase our understanding of TMAO bioconversions by the human gut microbiota.

microbiology↗