Biology subjects

Dingle, K. E.

Publications and source records attributed to Dingle, K. E..

5 recordsLinked to original sources

bioRxiv2022

Penicillin Binding Protein Substitutions Co-occur with Fluoroquinolone Resistance in 'Epidemic Lineages of Multi Drug-Resistant Clostridioides difficile

Clostridioides difficile remains a key cause of healthcare-associated infection, with multi-drug-resistant (MDR) lineages causing high mortality ([≥]20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species resistance is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins, PBPs). Here, we investigated five C. difficile transpeptidases (PBP1-5) for recent substitutions. Previously published genome assemblies (n=7096) were obtained, representing sixteen geographically widespread lineages, including healthcare-associated MDR ST1(027), ST3(001) and ST17(018). Recent amino acid substitutions were found within PBP1 (n=50) and PBP3 (n=48), ranging from 1-10 substitutions per genome. {beta}-lactam MICs were measured for closely related pairs of wild-type and PBP substituted isolates separated by 20-273 SNPs. Recombination-corrected, dated phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1-4 doubling dilutions >wild-type up to [≤]1506g/ml. Substitution patterns varied by lineage and clade, showed geographic structure, and notably occurred post-1990, coincident with the acquisition of gyrA/B substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. The co-occurrence of resistance to cephalosporins and fluoroquinolones hinders attempts to understand their relative importance in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCEFluoroquinolone and cephalosporin prescribing in healthcare settings have triggered outbreaks of high-mortality, multi-drug resistant C. difficile infection. Here, we identify a mechanism of acquired cephalosporin resistance in C. difficile, comprising amino acid substitutions in two cell-wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that resistance to both cephalosporins and fluoroquinolones was co-acquired immediately before clinically important, outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes conferring resistance likely impart a fitness-cost after antibiotic withdrawal. Our study identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of cephalosporin and fluoroquinolone resistance, further work is needed to determine the relative importance of each.

microbiology↗

bioRxiv2020

The Clostridioides difficile species problem: global phylogenomic analysis uncovers three ancient, toxigenic, genomospecies

Clostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work impacts the diagnosis of CDI worldwide.

microbiology↗

bioRxiv2020

DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

Mycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99{degrees}C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes [≥]1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

microbiology↗

bioRxiv2018

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised mock clinical samples was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

microbiology↗

bioRxiv2018

A role for tetracycline selection in the evolution of Clostridium difficile PCR-ribotype 078

Farm animals have been identified as reservoirs of Clostridium difficile PCR-ribotype 078 (RT078). Since 2005, the incidence of human clinical cases (frequently severe), with this genotype has increased. We aimed to understand this change, by studying the recent evolutionary history of RT078. Phylogenetic analysis of international genomes (isolates from 2006-2014) revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired different alleles of the tetracycline resistance gene tetM. Consequently, an unusually high proportion of RT078 genomes were tetM positive (76.5%). Additional tetracycline resistance determinants were also identified, some for the first time in C. difficile (efflux pump tet40). Each tetM-clonal expansion lacked geographic structure, indicating rapid international spread. Resistance determinants for C. difficile-infection-triggering antimicrobials including fluoroquinolones and clindamycin were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid spread via the food-chain may explain the increased RT078 prevalence in humans.

microbiology↗