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.

Genomic Epidemiology of Complex, Multispecies, Plasmid-Borne blaKPC Carbapenemase in Enterobacterales in the United Kingdom from 2009 to 2014.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, blaKPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of blaKPC has evolved to be polyclonal and polyspecies, 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 characterization. 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 among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among 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.

Hotspot mutations and ColE1 plasmids contribute to the fitness of Salmonella Heidelberg in poultry litter.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry. Investigations of a large, multistate outbreak in the USA in 2013 identified poultry litter (PL) as an important extra-intestinal environment that may have selected for specific S. Heidelberg strains. Poultry litter is a mixture of bedding materials and chicken excreta that contains chicken gastrointestinal (GI) bacteria, undigested feed, feathers, and other materials of chicken origin. In this study, we performed a series of controlled laboratory experiments which assessed the microevolution of two S. Heidelberg strains (SH-2813 and SH-116) in PL previously used to raise 3 flocks of broiler chickens. The strains are closely related at the chromosome level, differing from the reference genome by 109 and 89 single nucleotide polymorphisms/InDels, respectively. Whole genome sequencing was performed on 86 isolates recovered after 0, 1, 7 and 14 days of microevolution in PL. Only strains carrying an IncX1 (37kb), 2 ColE1 (4 and 6kb) and 1 ColpVC (2kb) plasmids survived more than 7 days in PL. Competition experiments showed that carriage of these plasmids was associated with increased fitness. This increased fitness was associated with an increased copy number of IncX1 and ColE1 plasmids. Further, all Col plasmid-bearing strains had hotspot mutations in 37 loci on the chromosome and in 3 loci on the IncX1 plasmid. Additionally, we observed a decrease in susceptibility to tobramycin, kanamycin, gentamicin, neomycin and fosfomycin for Col plasmid-bearing strains. Our study demonstrates how positive selection from poultry litter can change the evolutionary path of S. Heidelberg.

A curated dataset of complete Enterobacteriaceae plasmids compiled from the NCBI nucleotide database.

Thousands of plasmid sequences are now publicly available in the NCBI nucleotide database, but they are not reliably annotated to distinguish complete plasmids from plasmid fragments, such as gene or contig sequences; therefore, retrieving complete plasmids for downstream analyses is challenging. Here we present a curated dataset of complete bacterial plasmids from the clinically relevant Enterobacteriaceae family. The dataset was compiled from the NCBI nucleotide database using curation steps designed to exclude incomplete plasmid sequences, and chromosomal sequences misannotated as plasmids. Over 2000 complete plasmid sequences are included in the curated plasmid dataset. Protein sequences produced from translating each complete plasmid nucleotide sequence in all 6 frames are also provided. Further analysis and discussion of the dataset is presented in an accompanying research article: "Ordering the mob: insights into replicon and MOB typing…" (Orlek et al., 2017) [1]. The curated plasmid sequences are publicly available in the Figshare repository.

Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. ß-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes.

Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology.

Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of 'accessory genes,' such as antibiotic resistance genes, as well as 'backbone' loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made.