Biology subjects

Mathers, A. J.

Publications and source records attributed to Mathers, A. J..

11 recordsLinked to original sources

bioRxiv2024

Impact of media brand on cefiderocol disk diffusion results

IntroductionCefiderocol is a siderophore cephalosporin that utilizes iron transport systems to cross cell membranes. This unique strategy complicates antimicrobial susceptibility testing (AST) due to variable iron content in media. While guidance for using iron-depleted media exists for broth microdilution (BMD), disk diffusion (DD) with commercial media is a common AST method in the clinical laboratory. We investigated cefiderocol DD result variability using multiple Mueller-Hinton agar (MHA) brands. MethodsDD results using Remel (Thermo Fisher Scientific, San Diego, CA), Hardy (Hardy Diagnostics, Springboro, OH), and BBL (Becton Dickenson, East Rutherford, NJ) MHA were compared to BMD using iron-depleted cation-adjusted Mueller-Hinton broth. BMD reproducibility, BMD trailing endpoints, and DD intra- and inter-brand variability in zones of inhibition were investigated. ResultsForty-seven multidrug-resistant clinical isolates and three Antibiotic Resistance bank isolates composed of Pseudomonas aeruginosa, carbapenemase (CP-) and non-carbapenemase-producing (non-CP-) carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii complex, Stenotrophomonas maltophilia, and Burkholderia cepacia complex were tested. Categorical agreement (CA) [≥]90% was only demonstrated using CLSI breakpoints with BBL agar. Intra- and inter-brand variability in DD were greatest for P. aeruginosa and CRE, with 30% (6/20) and 16.7% (3/18) exhibiting discrepant AST interpretations, respectively. Isolates not susceptible to cefiderocol via BMD were commonly associated with AST interpretation errors and lower CA. ConclusionsUsing commercial MHA for DD resulted in frequent AST interpretation discrepancies, particularly for isolates that were not susceptible to cefiderocol by BMD. Methods and quality control may need to be revisited to ensure the reliability of DD for cefiderocol AST. IMPORTANCE STATEMENTThe novel mechanism of action of cefiderocol overcomes a variety of resistance mechanisms associated with gram-negative bacteria and positions the agent as an attractive option for infections involving multidrug-resistant pathogens. The availability of accurate, timely antimicrobial susceptibility testing methods for cefiderocol in clinical microbiology laboratories is critical as cefiderocol-resistant isolates have been described and may contribute to treatment failure. Iron-depleted broth microdilution testing may not be feasible for many clinical laboratories. While disk diffusion is an appealing, practical method to implement, our data demonstrate reproducibility issues across agar brands, most notably for organisms that do not test susceptible to cefiderocol via broth microdilution. Discrepant errors and misclassifications of resistant isolates as susceptible, and susceptible isolates as resistant, may mislead clinicians and compromise treatment efficacy. More work is needed to standardize practical yet reproducible methods for cefiderocol antimicrobial susceptibility testing.

microbiology↗

bioRxiv2024

Diversity, functional classification and genotyping of SHV β-lactamases in Klebsiella pneumoniae

Interpreting phenotypes of blaSHV alleles in Klebsiella pneumoniae genomes is complex. While all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal blaSHV alleles or additional plasmid-borne blaSHV alleles that have extended-spectrum {beta}-lactamase (ESBL) activity and/or {beta}-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/amino acid changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases (e.g., NCBIs Reference Gene Catalog and the {beta}-lactamase database (BLDB)) over the specific functionality of individual SHV protein variants. Therefore, identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific amino-acid substitutions. We then systematically assigned SHV alleles to functional classes (wildtype, ESBL, BLI-resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with current assignments in NCBIs Reference Gene Catalog and/or BLDB (21 to wildtype, 12 to ESBL, 4 to BLI-resistant). Phylogenetic and comparative genomic analyses support that; i) SHV-1 (encoded by blaSHV-1) is the ancestral chromosomal variant; ii) ESBL and BLI-resistant variants have evolved multiple times through parallel substitution mutations; iii) ESBL variants are mostly mobilised to plasmids; iv) BLI-resistant variants mostly result from mutations in chromosomal blaSHV. We used matched genome-phenotype data from the KlebNET-GSP Genotype-Phenotype Group to identify 3,999 K. pneumoniae isolates carrying one or more blaSHV alleles but no other acquired {beta}-lactamases, with which we assessed genotype-phenotype relationships for blaSHV. This collection includes human, animal, and environmental isolates collected between 2001 to 2021 from 24 countries across six continents. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, while most omega-loop substitutions do not. Our data also provide direct support for wildtype assignment of 67 protein variants, including eight that were noted in public databases as ESBL. We reclassified these eight variants as wildtype, because they lack ESBL-associated mutations, and our phenotype data support susceptibility to 3GCs (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164, SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae from genome assemblies), whereby known and novel blaSHV alleles are classified based on causative mutations. Kleborate v2.4.1 was also updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve understanding of resistance mechanisms. Impact statementSince every K. pneumoniae genome has an intrinsic SHV {beta}-lactamase and may also carry additional mobile forms, the correct interpretation of blaSHV genes detected in genome data can be challenging and can lead to K. pneumoniae being misclassified as ESBL-producing. Here, we use matched K. pneumoniae genome and drug susceptibility data contributed from dozens of studies, together with systematic literature review of experimental evidence, to improve our understanding of blaSHV allele variation and mapping of genotype to phenotype. This study shows the value of coordinated data sharing, in this case via the KlebNET-GSP Genotype-Phenotype Group, to improve our understanding of the evolutionary history and functionality of blaSHV genes. The results are captured in an open-source AMR dictionary utilised by the Kleborate genotyping tool, that could easily be incorporated into or used to update other tools and AMR gene databases. This work is part of the wider efforts of the KlebNET-GSP group to develop and support a unified platform tailored for the analysis and interpretation of K. pneumoniae genomes by a wide range of stakeholders. Data summaryBlaSHV allele sequences and class assignments are distributed with Kleborate, v2.4.1, DOI:10.5281/zenodo.10469001. Table S1 provides a summary of blaSHV alleles, including primary accessions, class-modifying mutations, and supporting evidence for class assignments that differ from NCBIs Reference Gene Catalog or BLDB. Whole genome sequence data are publicly available as reads and/or assemblies, individual accessions are given in Table S2; corresponding genotypes and antibiotic susceptibility phenotypes and measurements are available in Tables S3 and S4, respectively.

genomics↗

bioRxiv2023

Tailpiece Heating device for effective prevention of biofilm growth in sink plumbing

In recent years, numerous hospitals have linked patient infections to Klebsiella pneumoniae producing Enterobacterales (KPCE) and other resistant bacterial species in their wastewater systems and handwashing sinks. Wastewater plumbing provides a reservoir for bacteria, making them incredibly difficult to eliminate through traditional disinfection methods. Data suggests that patients become infected when bacteria grow or migrate up the proximal wastewater plumbing and into the sink basin, and are subsequently dispersed onto surrounding surfaces. Therefore, a novel electronic device was developed that acts at the highest risk area, just below the sink drain, to heat and dry out the biofilm and creating a biofilm barrier and prevent upward growth from the sink trap. The efficacy of the first prototype of a tailpiece heater (TPH) in preventing drain colonization was tested using GFP-expressing Escherichia coli (GFP-E. coli) as the challenge organism. In control sinks without the TPH, GFP-E. coli biofilm grew from the p-trap upwards to colonize the drain within 7 days. Sinks with a TPH set to 75{degrees}C were found to prevent sink drain colonization. In contrast, 65{degrees}C was not adequate to prevent drain colonization. Using KPCE the TPH was more effective than no heat control in preventing drain colonization in sinks over time. Lastly, when challenged with seeding from above, the TPH also effectively prevented KPCE colonization at the drain level. Heating of the tailpiece may offer a safe, effective, and economically attractive approach to preventing the spread of resistant bacterial species from contaminated drain biofilm to patients.

microbiology↗

bioRxiv2020

The unintended consequences of a surge in antibiotic resistance when removing wastewater drain biofilm

The prevalence and proliferation of antimicrobial resistant bacteria is considered one of the critical issues of our time. Wastewater is a habitat for complex microbial communities where bacteria share genes of antimicrobial resistance through horizontal gene transfer. Hospital wastewater drainage systems are an ideal reservoir for environmental and pathogenic bacteria to interface and exchange genes of antimicrobial resistance1. Highly resistant Enterobacterales are of the greatest concern because they not only cause human infections but can also thrive in the environment2,3 and have shown to transmit from sink drains to patients4. Replacement of contaminated plumbing may be the most intuitive and widely deployed response once highly resistant potentially pathogenic bacteria are identified in a sink drain. Here we show using shotgun metagenomic sequencing of sink drain biofilms (in 6 intensive care patient rooms, 36 subsamples) paired with microbial culture analysis, an evident shift of biofilm community structure towards increased abundance of Enterobacteriaceae in the newly replaced sink plumbing. Significant increase in resistome load and abundance of clinically relevant resistance and typically mobile genes in the newly replaced plumbing was also observed. Findings from this study demonstrate that exchanging contaminated plumbing for new plumbing may actually have the unexpected consequence of increased abundance of Enterobacterales and genes of antimicrobial resistance in the sink drains. Disruption of preexisting complex environmental biofilms may result in an unintended increase in the amount of antimicrobial resistant Enterobacterales which are targeted for elimination. Temporal variability is an important attribute of ecosystems affecting colonization dynamics and biotic interactions (composition of introduced and resident communities) are key to functional outcomes. Absence of competition within an ecosystem favors invasion.

microbiology↗

bioRxiv2020

Multi-omic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonised with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole genome sequencing (WGS) of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies which may vary as a result of different inputs and selection pressures. WGS of 46 contemporaneous patient isolates identified one (2%; 95% CI 0.05-11%) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10% of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including blaCTX-M, blaSHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention. IMPORTANCEEscherichia coli and Klebsiella spp. cause a wide range of bacterial infections, including bloodstream, urine and lung infections. Previous studies have shown that sink drains in hospitals may be part of transmission chains in outbreaks of antimicrobial-resistant E. coli and Klebsiella spp., leading to colonisation and clinical disease in patients. We show that even in non-outbreak settings, contamination of sink drains by these bacteria is common across hospital wards, and that many antimicrobial resistance genes can be found and potentially exchanged in these sink drain sites. Our findings demonstrate that the colonisation of handwashing sink drains by these bacteria in hospitals is likely contributing to some infections in patients, and that additional work is needed to further quantify this risk, and to consider appropriate mitigating interventions.

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

The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates

With multidrug resistant (MDR) Enterobacteriales on the rise, a non-toxic agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-E. coli in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with multiple strains and species of KPC-producing clinical isolates collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole genome sequencing (WGS) data using NCBIs AMRFinder and custom HMM search. Susceptibility testing was performed using glucose-6-phosphate supplemented fosfomycin E-Test and Kirby-Bauer disk diffusion (DD) assays, and compared to agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by E-Test, 70% (67/96) by DD, and 88% (84/96) by agar dilution. FosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA-positive compared to fosA-negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory when compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

microbiology↗

bioRxiv2018

Klebsiella quasipneumonaiae provides a window into carbapenemase gene transfer, plasmid rearrangements and nosocomial acquisition from the hospital environment

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

microbiology↗

bioRxiv2018

Droplet rather than Aerosol Mediated Dispersion is the Primary Mechanism of Bacterial transmission from Contaminated Hand Washing Sink Traps

An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilm in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events amending earlier theory that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods.\n\nIMPORTANCEAmong the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as potential reservoir of multidrug resistant healthcare-associated pathogens to hospitalized patients. With increasing antimicrobial resistance limiting therapeutic options for patients, better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria colonizing sink drains.

microbiology↗

bioRxiv2018

TETyper: a bioinformatic pipeline for classifying variation and genetic contexts of transposable elements from short-read whole-genome sequencing data

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of >3000 publicly available Illumina datasets containing blaKPC. This revealed surprising diversity, with >200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high copy number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

bioinformatics↗

bioRxiv2017

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common human infections. blaKPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10kb). Here we characterize the genetic features of the emergence of blaKPC in global E. coli, 2008-2013, using both long-and short-read whole genome sequencing.\n\nAmongst 43/45 successfully sequenced blaKPC-E. coli strains, we identified high strain (n=21 sequence types, 18% of annotated genes in the core genome); plasmid ([≥]9 replicon types); and blaKPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of interspecies, regional and international plasmid spread. In several cases blaKPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures.\n\nE. coli is a common human pathogen, but also a commensal in a multiple environmental and animal reservoirs, and easily transmissible. The association of blaKPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. blaTEM, blaCTX-M) suggests that it is likely to become similarly prevalent.

microbiology↗