Biology subjects

Woodford, N.

Publications and source records attributed to Woodford, N..

12 recordsLinked to original sources

bioRxiv2025

Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam

This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The {beta}-lactam/{beta}-lactamase inhibitor combinations are widely used to circumvent {beta}-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile--KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital--were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a frameshift mutation in KpMVR1s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summaryIllumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institutes database for K. pneumoniae species complex (bigsdb.pasteur.fr/klebsiella/) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statementThis is the first blaKPC-positive K. pneumoniae isolate referred to the UKs national reference laboratory with resistance to three last-resort {beta}-lactam/{beta}-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne blaKPC-2-like allele blaKPC-157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an ISEc68-induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission.

microbiology↗

bioRxiv2022

Molecular characteristics of phages located in Carbapenemase-Producing Escherichia coli clinical isolates: New Phage-Like Plasmids

Escherichia coli normally inhabits the gastrointestinal tract of humans and animals. Most E. coli bacteria do not cause problems, but the acquisition of different resistance and virulence genes encoded by mobile plasmids or phages by different bacterial isolates has been associated with the appearance of successful high-risk clones of multidrug-resistant (MDR) E. coli such as ST131 or ST405. In the present study, 50 temperate bacteriophages present in 21 clinical isolates of carbapenemase-producing E. coli of sequence types (STs) ST38, ST131, ST167, ST405 and ST410 were analysed. These phages were classified in the three families of the order Caudovirales: 24 within the family Siphoviridae, 23 in Myoviridae and 3 in Podoviridae. The size of the phages studied ranged from 11 to 95 Kb. Phylogenetic analysis of the terminase large subunit allowed us to classify these phages into different groups showing similarity with the phage sequences deposited in the Microbe Versus Phage (MVP) database and which belonged to clusters 229, 604, 2503 and 2725. On the other hand, bioinformatic study revealed that most of the identified proteins exerted a structural function (26.73%) but also functions involved in lysis/lysogeny (6.70%) or regulation (5.20%) among others. In addition, the ParA-ParB partitioning system and the type II toxin-antitoxin Phd-Doc system were also found in two of the phages studied, which could indicate the presence of plasmid-prophages. Host range testing revealed that two isolates were more susceptible to infection than the other isolates. IMPORTANCEEscherichia coli is one of the pathogens that causes most problems in human health, as it presents multiple resistances to different antibiotics. The study of bacteriophages located in different isolates of this species is important for the development of new anti-infective therapies. Currently, antibiotic resistance is a major problem, but more and more studies are pointing to experimental treatments with bacteriophages as a possible solution.

bioinformatics↗

bioRxiv2021

Diverse Genetic Determinants of Nitrofurantoin Resistance in UK Escherichia coli

Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2018. Nitrofurantoin resistance is reported in <6% urinary E. coli isolates in the UK. However, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify genetic determinants of nitrofurantoin resistance in a local E. coli collection and assess their prevalence in a larger dataset of E. coli genomes. Deleterious point mutations and gene-inactivating insertion sequences in both chromosomal nitroreductase genes nfsA and nfsB were identified in genomes of nine nitrofurantoin-resistant urinary E. coli isolates collected from north west London. Eight types of genetic alterations were identified when comparing sequences of nfsA, nfsB, and the associated gene ribE in 12,412 E. coli genomes collected from across the UK. Evolutionary analysis revealed homoplasic mutations and explained the order of stepwise mutations. An algorithm was developed to predict nitrofurantoin susceptibility and predictions for 20 accessible isolates were experimentally validated. Only one genome carrying oqxAB, a mobile gene complex associated with reduced nitrofurantoin susceptibility, was identified. In conclusion, mutations and insertion sequences in nfsA and nfsB are leading causes of nitrofurantoin resistance in UK E. coli. As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored. ImportanceThis study expands knowledge about the genetic basis of nitrofurantoin resistance in E. coli isolates using whole-genome sequencing and genomic analysis. We report novel and previously known deleterious mutations of chromosomal genes nfsA, nfsB, and ribE as well as the interruption of nfsA and nfsB by insertion sequences, recapitulating the roles of oxygen-insensitive nitroreductases in the development of nitrofurantoin resistance in E. coli. We revealed and categorised the genotypic diversity in these three genes in a large collection of UK E. coli genomes. A scoring algorithm is provided to predict nitrofurantoin susceptibility from genotypes. Our predictions suggest that acquired nitrofurantoin resistance is not of immediate concern in the UK. However, experimental validation of predictions suggested the involvement of mechanisms other than alterations in nfsA, nfsB, or ribE in determining nitrofurantoin susceptibility, emphasising the need for monitoring nitrofurantoin resistance amongst E. coli.

microbiology↗

bioRxiv2020

Effective in-vitro inactivation of SARS-CoV-2 by commercially available mouthwashes

Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate [≥]4.1 to [≥]5.5 log10 reduction in SARS-CoV-2 titre following a one minute treatment with commercially available mouthwashes containing 0.01-0.02% stabilised hypochlorous acid or 0.58% povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5% hydrogen peroxide or 0.2% chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.

microbiology↗

bioRxiv2020

Emergence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

ObjectivesTo characterise the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. MethodsLinezolid-resistant E. faecalis were identified in three Scottish Health Boards and confirmed to carry the optrA gene at the national reference laboratory. WGS was performed with short read (Illumina MiSeq) and long read (Oxford Nanopore MinION) technologies to generate complete genome assemblies. Illumina reads for 94 E. faecalis bloodstream isolates were used to place the optrA-positive isolates in a larger UK phylogeny. ResultsSix optrA-positive linezolid-resistant E. faecalis were isolated from urogenital samples in three Scottish Health Boards (2014-2017). No epidemiological links were identified between the patients, four were community-based, and only one had recent linezolid exposure. Reference-based mapping confirmed the isolates were genetically distinct (>13,900 core SNPs). optrA was located on a plasmid in each isolate and these plasmids showed limited nucleotide similarity. There was variable presence of transposable elements surrounding optrA, (including IS1216, IS3, and Tn3) and not always as a recognisable gene cassette. OptrA amino acid sequences were also divergent, resulting in four protein variants differing in 1-20 residues. One isolate belonged to ST16 and clustered with three other isolates in the UK collection (76-182 SNPs), otherwise the optrA-positive isolates were genetically distinct from the bloodstream isolates (>6,000 SNPs). ConclusionsWe report multiple variants of the linezolid resistance gene optrA in diverse E. faecalis strain and plasmid backgrounds, suggesting multiple introductions of the gene into the E. faecalis population and selection driving recent emergence.

microbiology↗

bioRxiv2019

Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: An inter-laboratory study

BackgroundAntimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a one-stop test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data sequenced from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants and identify problem cases and factors that lead to discordant results.\n\nMethodsWe produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ( participants) were provided these sequence data without any other contextual information. Each participant used their own pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime.\n\nResultsIndividual participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment a different antibiotic would have been recommended for each isolate by at least one participant.\n\nConclusionsWe found that participants produced discordant predictions from identical WGS data. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases and standardisation in the comparisons between genotype and resistance phenotypes will be fundamental before AST prediction using WGS can be successfully implemented in standard clinical microbiology laboratories.

microbiology↗

bioRxiv2019

Genomic epidemiology of a complex, multi-species plasmid-borne blaKPC carbapenemase outbreak in Enterobacterales in the UK, 2009-2014

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the commonest transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC has historically been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the UK, blaKPC has caused a large-scale, persistent outbreak focused on hospitals in North-West England. This outbreak has evolved to be polyclonal and poly-species, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterisation. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 (97%) isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments amongst plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates) and IncR replicons (252/604 isolates). The subset of reconstructed plasmid sequences also highlighted modular exchange amongst non-blaKPC and blaKPC plasmids, and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales, for the implementation of adequate surveillance approaches, and for control.\n\nIMPORTANCEAntimicrobial resistance is a major threat to the management of infections, and resistance to carbapenems, one of the \"last line\" antibiotics available for managing drug-resistant infections, is a significant problem. This study used large-scale whole genome sequencing over a five-year period in the UK to highlight the complexity of genetic structures facilitating the spread of an important carbapenem resistance gene (blaKPC) amongst a number of bacterial species that cause disease in humans. In contrast to a recent pan-European study from 2012-2013(1), which demonstrated the major role of spread of clonal blaKPC-Klebsiella pneumoniae lineages in continental Europe, our study highlights the substantial plasticity in genetic mechanisms underpinning the dissemination of blaKPC. This genetic flux has important implications for: the surveillance of drug resistance (i.e. making surveillance more difficult); detection of outbreaks and tracking hospital transmission; generalizability of surveillance findings over time and for different regions; and for the implementation and evaluation of control interventions.

genomics↗

bioRxiv2019

Diversity of Escherichia coli from the enteric microbiota in patients with Escherichia coli bacteraemia

ObjectiveThe increase in Escherichia coli bloodstream infections mandates better characterisation of the relationship between commensal and invasive isolates. This study established a simple approach to characterize diversity of E. coli in the gut reservoir from patients with either E. coli bacteraemia, other Gram-negative bacteraemia, or patients without bacteraemia not receiving antibiotics. Stool or rectal swabs from patients in the three groups were cultured on selective chromogenic agar. Genetic diversity of E. coli in gut microbiota was estimated by RAPD-PCR. ResultsEnteric samples from E. coli bacteraemia patients yielded a median of one E. coli RAPD pattern (range 1-4) compared with two (range 1-5) from groups without E. coli bacteraemia. Of relevance to large-scale clinical studies, observed diversity of E. coli among E. coli bacteraemia patients was not significantly altered by sample type (rectal swab or stool), nor by increasing the colonies tested beyond ten. Overall, hospitalised patients demonstrated an apparently limited diversity of E. coli in the enteric microbiota and this was further reduced in those with E. coli bacteraemia. The reduced diversity of E. coli within the gut during E. coli bacteraemia raises the possibility that dominant strains may outcompete other lineages in patients with bloodstream infection.

microbiology↗

bioRxiv2019

When do big problems far away become smaller than the problems closer to home: Lessons for antimicrobial resistance from Father Ted

Infection prevention and control strategies aimed at reducing the occurrence of Carbapenemase-Producing Enterobacteriaceae (CPE) and other antimicrobial-resistant organisms often include advice about screening patients coming from hospitals with a known resistance problem, to prevent introductions into new hospitals by shared patients. We argue that, despite being an efficient method of identifying cases, admission screening for introduction prevention is only effective if the absolute number of imported cases from other hospitals outnumbers the cases coming from the hospitals own patient population, and therefore is only a feasible control strategy during the start of an epidemic. When determining whether import screening is still advisable, we therefore need to be continuously reminded of how Father Ted so eloquently summarised the principles of perspective: "These are small, but the ones out there are far away".

epidemiology↗

bioRxiv2019

Using hospital network-based surveillance for antimicrobial resistance as a more robust alternative to self-reporting

Hospital performance is often measured using self-reported statistics, such as the incidence of hospital-transmitted micro-organisms or those exhibiting antimicrobial resistance (AMR), encouraging hospitals with high levels to improve their performance. However, hospitals that increase screening efforts will appear to have a higher incidence and perform poorly, undermining comparison between hospitals and disincentivising testing, thus hampering infection control. We propose a surveillance system in which hospitals test patients previously discharged from other hospitals and report observed cases. Using NHS Hospital Episode Statistics data, we analysed patient movements across England and assessed the number of hospitals required to participate in such a reporting scheme to deliver robust estimates of incidence. With over 1.2 million admissions to English hospitals previously discharged from other hospitals annually, even when only a fraction of hospitals (41/155) participate (each screening at least 1000 of these admissions), the proposed surveillance system can estimate incidence across all hospitals. By reporting on other hospitals, the reporting of incidence is separated from the task of improving own performance. Therefore the incentives for increasing performance can be aligned to increase (rather than decrease) screening efforts, thus delivering both more comparable figures on the AMR problems across hospitals and improving infection control efforts.

epidemiology↗

bioRxiv2019

Reconciling the potentially irreconcilable? Genotypic and phenotypic amoxicillin-clavulanate resistance in Escherichia coli

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity 23% (78/339)) but improved when genetic features associated with penicillinase hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. We investigated two potential causes; the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement ({+/-}1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

microbiology↗

bioRxiv2018

Different impacts of naturally occurring variants of the globally disseminated carbapenemase-encoding plasmid pKpQIL on the biology of host strains

SynopsisKlebsiella-associated plasmid pKpQIL and its variant have been isolated globally. Our study aimed to determine whether a naturally occurring variant has altered host range and impacts on the fitness of different bacterial host strains. The plasmids pKpQIL-UK and pKpQIL-D2 were transferred from the original clinical isolate host strains of Klebsiella pneumoniae into Escherichia coli, Salmonella Typhimurium, Enterobacter cloacae and Serratia marcescens strains by filter-mating and conjugation frequencies determined and compared. The fitness of the resulting transconjugants was assessed by determining growth kinetics, ability to form a biofilm and persistence of the plasmids in each host was also measured. Transfer of either plasmid into Salmonella and S. marcescens was similar. However, pKpQIL-UK transferred into E. coli at a higher rate than did pKpQIL-D2; the reverse was found for E. cloacae. Both plasmids were rapidly lost from the E. coli population. Plasmid pKpQIL-UK, but not -D2, was able to persist in Salmonella. Although pKpQIL-UK imposed a greater fitness cost (inferred from an increased generation time) than -D2 on E. cloacae, it was able to persist as well as pKpQIL-D2 in this host. The pKpQIL-D2 plasmid did not confer any fitness benefit on any of the hosts under the conditions tested. Variants of the globally important pKpQIL plasmid have arisen in patients due to recombination. The impacts of the pKpQIL-UK plasmid and the -D2 variant in various Enterobacteriaceae are host-dependent. Continuing evolution of pKpQIL may alter its host range in the future.

microbiology↗