Factors associated with plasmid antibiotic resistance gene carriage revealed using large-scale multivariable analysis

Plasmids are major vectors of bacterial antibiotic resistance, but understanding of factors associated with plasmid antibiotic resistance gene (ARG) carriage is limited. We curated > 14,000 publicly available plasmid genomes and associated metadata. Duplicate and replicate plasmids were excluded; where possible, sample metadata was validated externally (BacDive database). Using Generalised Additive Models (GAMs) we assessed the influence of 12 biotic/abiotic factors (e.g. plasmid genetic factors, isolation source, collection date) on ARG carriage, modelled as a binary outcome. Separate GAMs were built for 10 major ARG types. Multivariable analysis indicated that plasmid ARG carriage patterns across time (collection years), isolation sources (human/livestock) and host bacterial taxa were consistent with antibiotic selection pressure as a driver of plasmid-mediated antibiotic resistance. Only 0.42% livestock plasmids carried carbapenem resistance (compared with 12% human plasmids); conversely, tetracycline resistance was enriched in livestock vs human plasmids, reflecting known prescribing practices. Interpreting results using a timeline of ARG type acquisition (determined by literature review) yielded additional novel insights. More recently acquired ARG types (e.g. colistin and carbapenem) showed increases in plasmid carriage during the date range analysed (1994–2019), potentially reflecting recent onset of selection pressure; they also co-occurred less commonly with ARGs of other types, and virulence genes. Overall, this suggests that following acquisition, plasmid ARGs tend to accumulate under antibiotic selection pressure and co-associate with other adaptive genes (other ARG types, virulence genes), potentially re-enforcing plasmid ARG carriage through co-selection

The prevalences of Salmonella Genomic Island 1 variants in human and animal Salmonella Typhimurium DT104 are distinguishable using a Bayesian approach

Throughout the 1990s, there was an epidemic of multidrug resistant Salmonella Typhimurium DT104 in both animals and humans in Scotland. The use of antimicrobials in agriculture is often cited as a major source of antimicrobial resistance in pathogenic bacteria of humans, suggesting that DT104 in animals and humans should demonstrate similar prevalences of resistance determinants. Until very recently, only the application of molecular methods would allow such a comparison and our understanding has been hindered by the fact that surveillance data are primarily phenotypic in nature. Here, using large scale surveillance datasets and a novel Bayesian approach, we infer and compare the prevalence of Salmonella Genomic Island 1 (SGI1), SGI1 variants, and resistance determinants independent of SGI1 in animal and human DT104 isolates from such phenotypic data. We demonstrate differences in the prevalences of SGI1, SGI1-B, SGI1-C, absence of SGI1, and tetracycline resistance determinants independent of SGI1 between these human and animal populations, a finding that challenges established tenets that DT104 in domestic animals and humans are from the same well-mixed microbial population

Tatajuba: exploring the distribution of homopolymer tracts.

Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialized bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and help predict their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source licence GNU GPLv3

Tatajuba: exploring the distribution of homopolymer tracts.

Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialized bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and help predict their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source licence GNU GPLv3

Whole genome sequencing reveals great diversity of Vibrio spp in prawns at retail

Consumption of prawns as a protein source has been on the rise worldwide with seafood identified as the predominant attributable source of human vibriosis. However, surveillance of non-cholera Vibrio is limited both in public health and in food. Using a population- and market share-weighted study design, 211 prawn samples were collected and cultured for Vibrio spp. Contamination was detected in 46 % of samples, and multiple diverse Vibrio isolates were obtained from 34 % of positive samples. Whole genome sequencing (WGS) and phylogenetic analysis illustrated a comprehensive view of Vibrio species diversity in prawns available at retail, with no known pathogenicity markers identified in Vibrio parahaemolyticus and V. cholerae . Antimicrobial resistance genes were found in 77 % of isolates, and 12 % carried genes conferring resistance to three or more drug classes. Resistance genes were found predominantly in V. parahaemolyticus , though multiple resistance genes were also identified in V. cholerae and V. vulnificus . This study highlights the large diversity in Vibrio derived from prawns at retail, even within a single sample. Although there was little evidence in this study that prawns are a major source of vibriosis in the UK, surveillance of non-cholera Vibrio is very limited. This study illustrates the value of expanding WGS surveillance efforts of non-cholera Vibrios in the food chain to identify critical control points for food safety through the production system and to determine the full extent of the public health impact

ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads.

Antimicrobial resistance (AMR) is one of the major threats to human and animal health worldwide, yet few high-throughput tools exist to analyse and predict the resistance of a bacterial isolate from sequencing data. Here we present a new tool, ARIBA, that identifies AMR-associated genes and single nucleotide polymorphisms directly from short reads, and generates detailed and customizable output. The accuracy and advantages of ARIBA over other tools are demonstrated on three datasets from Gram-positive and Gram-negative bacteria, with ARIBA outperforming existing methods

Factors determining human-to-human transmissibility of zoonotic pathogens via contact.

The pandemic potential of zoonotic pathogens lies in their ability to become efficiently transmissible amongst humans. Here, we focus on contact-transmitted pathogens and discuss the factors, at the pathogen, host and environmental levels that promote or hinder their human-to-human transmissibility via the following modes of contact transmission: skin contact, sexual contact, respiratory contact and multiple route contact. Factors common to several modes of transmission were immune evasion, high viral load, low infectious dose, crowding, promiscuity, and co-infections; other factors were specific for a pathogen or mode of contact transmission. The identification of such factors will lead to a better understanding of the requirements for human-to-human spread of pathogens, as well as improving risk assessment of newly emerging pathogens

Determining antimicrobial susceptibility in Salmonella enterica serovar Typhimurium through whole genome sequencing: a comparison against multiple phenotypic susceptibility testing methods

Background : UK public health organisations perform routine antimicrobial susceptibility tests (ASTs) to characterise the potential for antimicrobial resistance in Salmonella enterica serovars. Genetic determinants of these resistance mechanisms are detectable by whole genome sequencing (WGS), however the viability of WGS-based genotyping as an alternative resistance screening tool remains uncertain. We compared WGS-based genotyping, disk diffusion and agar dilution to the broth microdilution reference AST for 102 Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates across 11 antimicrobial compounds. Results : Genotyping concordance, interpreted using epidemiological cut-offs (ECOFFs), was 89.8% (1007/1122) with 0.83 sensitivity and 0.96 specificity. For seven antimicrobials interpreted using Salmonella clinical breakpoints, genotyping produced 0.84 sensitivity and 0.88 specificity. Although less accurate than disk diffusion (0.94 sensitivity, 0.93 specificity) and agar dilution (0.83 sensitivity, 0.98 specificity), genotyping performance improved to 0.89 sensitivity and 0.97 specificity when two antimicrobials with relatively high very major error rates were excluded (streptomycin and sulfamethoxazole). Conclusions : An 89.8% concordance from WGS-based AST predictions using ECOFF interpretations suggest that WGS would serve as an effective screening tool for the tracking of antimicrobial resistance mechanisms in S. Typhimurium. For use as a standalone clinical diagnostic screen, further work is required to reduce the error rates for specific antimicrobials

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly

Repeated cross-sectional study identifies differing risk factors associated with microbial contamination in common food products in the United Kingdom

All foods carry microbes, many of which are harmless, but foods can also carry pathogens and/or microbial indicators of contamination. Limited information exists on the co-occurrence of microbes of food safety concern and the factors associated with their presence. Here, a population-based repeated cross-sectional design was used to determine the prevalence and co-occurrence of Escherichia coli, Klebsiella spp., Salmonella spp. and Vibrio spp. in key food commodities - chicken, pork, prawns, salmon and leafy greens. Prevalence in 1369 food samples for these four target bacterial genera/species varied, while 25.6% of all samples had at least two of the target bacteria and eight different combinations of bacteria were observed as co-occurrence profiles in raw prawns. Imported frozen chicken was 6.4 times more likely to contain Salmonella than domestic chicken, and imported salmon was 5.5 times more likely to be contaminated with E. coli. Seasonality was significantly associated with E. coli and Klebsiella spp. contamination in leafy greens, with higher detection in summer and autumn. Moreover, the odds of Klebsiella spp. contamination were higher in summer in chicken and pork samples. These results provide insight on the bacterial species present on foods at retail, and identify factors associated with the presence of individual bacteria, which are highly relevant for food safety risk assessments and the design of surveillance programmes