To determine the occurrence of mcr-1 and mcr-2 genes in Gram-negative bacteria isolated from healthy pigs in Great Britain.
Gram-negative bacteria (n = 657) isolated from pigs between 2014 and 2015 ...were examined by WGS.
Variants of mcr-1 and mcr-2 were identified in Moraxella spp. isolated from pooled caecal contents of healthy pigs at slaughter collected from six farms in Great Britain. Other bacteria, including Escherichia coli from the same farms, were not detected harbouring mcr-1 or mcr-2. A Moraxella porci-like isolate, MSG13-C03, harboured MCR-1.10 with 98.7% identity to MCR-1, and a Moraxella pluranimalium-like isolate, MSG47-C17, harboured an MCR-2.2 variant with 87.9% identity to MCR-2, from E. coli; the isolates had colistin MICs of 1-2 mg/L. No intact insertion elements were identified in either MSG13-C03 or MSG47-C17, although MSG13-C03 harboured the conserved nucleotides abutting the ISApl1 composite transposon found in E. coli plasmids and the intervening ∼2.6 kb fragment showed 97% identity. Six Moraxella osloensis isolates were positive for phosphoethanolamine transferase (EptA). They shared 62%-64.5% identity to MCR-1 and MCR-2, with colistin MICs from 2 to 4 mg/L. Phylogenetic analysis indicated that MCR and EptA have evolved from a common ancestor. In addition to mcr, the β-lactamase gene, blaBRO-1, was found in both isolates, whilst the tetracycline resistance gene, tetL, was found in MSG47-C17.
Our results add further evidence for the mobilization of the mcr-pap2 unit from Moraxella via composite transposons leading to its global dissemination. The presence of mcr-pap2 from recent Moraxella isolates indicates they may comprise a reservoir for mcr.
Combatting antimicrobial resistant (AMR) using a One-Health approach is essential as various bacteria, including
Escherichia coli
, a common bacteria, are becoming increasingly resistant and ...livestock may be a reservoir. The AMR gene content of 492
E. coli
, isolated from 56 pig farms across Great Britain in 2014–2015, and purified on antibiotic selective and non-selective plates, was determined using whole genome sequencing (WGS). The
E. coli
were phylogenetically diverse harboring a variety of AMR profiles with widespread resistance to “old” antibiotics; isolates harbored up to seven plasmid Inc-types. None showed concurrent resistance to third-generation cephalosporins, fluoroquinolones and clinically relevant aminoglycosides, although ∼3% harbored AMR genes to both the former two. Transferable resistance to carbapenem and colistin were absent, and six of 117
E. coli
STs belonged to major types associated with human disease. Prevalence of genotypically MDR
E. coli
, gathered from non-selective media was 35% and that of extended-spectrum-beta-lactamase
E. coli
was low (∼2% from non-selective). Approximately 72.6% of
E. coli
from ciprofloxacin plates and only 8.5% from the other plates harbored fluoroquinolone resistance due to topoisomerase mutations; the majority were MDR. In fact, multivariable analysis confirmed
E. coli
purified from CIP enrichment plates were more likely to be MDR, and suggested MDR isolates were also more probable from farms with high antibiotic usage, specialist finisher farms, and farms emptying their manure pits only after each batch. Additionally, farms from the South East were more likely to have MDR
E. coli
, whereas farms in Yorkshire and the Humber were less likely. Future investigations will determine whether suggested improvements such as better biosecurity or lower antimicrobial use decreases MDR
E. coli
on pig farms. Although this study focuses on pig farms, we believe the methodology and findings can be applied more widely to help livestock farmers in the United Kingdom and elsewhere to tackle AMR.
Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists ...with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family
, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We
assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.
F-type plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum β-lactamases, particularly in ...Enterobacterales. Organising this plasmid diversity is challenging, and current knowledge is largely based on plasmids from clinical settings. Here, we present a network community analysis of a large survey of F-type plasmids from environmental (influent, effluent and upstream/downstream waterways surrounding wastewater treatment works) and livestock settings. We use a tractable and scalable methodology to examine the relationship between plasmid metadata and network communities. This reveals how niche (sampling compartment and host genera) partition and shape plasmid diversity. We also perform pangenome-style analyses on network communities. We show that such communities define unique combinations of core genes, with limited overlap. Building plasmid phylogenies based on alignments of these core genes, we demonstrate that plasmid accessory function is closely linked to core gene content. Taken together, our results suggest that stable F-type plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The association of F-type plasmids with AMR may reflect their suitability for rapid niche adaptation.
Surveillance is vital for monitoring the increasing risk of antimicrobial resistance (AMR) in bacteria leading to failures in humans and animals to treat infections. In a One Health context, AMR ...bacteria from livestock and food can transfer through the food chain to humans, and vice versa, which can be characterized in detail through genomics. We investigated the critical aspects of AMR and the dynamics of AMR in poultry in the UK.
In this study, we performed whole genome sequencing for genomic characterization of 761 extended-spectrum cephalosporinases (ESCs) harboring Escherichia coli isolated from poultry caeca and meat through EU harmonized monitoring of AMR in zoonotic and commensal bacteria from 2016 and 2018 and UK national monitoring in 2020.
The most common ESC in 2016 and 2018 was blaCTX-M-1; however, 2020 had a greater diversity of ESCs with blaCTX-M-55 dominant in chickens and blaCTX-M-15 more prevalent in turkeys. Co-resistance to sulphonamides, tetracycline, and trimethoprim was widespread, and there were several positive correlations between the sequence types (STs) and ESC genes. We identified certain AMR genotypes and STs that were frequent each year but not as successful in subsequent years, e.g., ST350 harboring blaCTX-M-1, sul2, and tetA-v4.Phylogenetic comparison of isolates prevalent in our panel with global ones from the same STs available in public databases showed that isolates from the UK generally clustered together, suggesting greater within-country than between-country transmission.
We conclude that future genomic surveillance of indicator organisms will be invaluable as it will enable detailed comparisons of AMR between and within neighboring countries, potentially identifying the most successful sequence types, plasmids, or emerging threats.
Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major ...Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.
There are concerns that antimicrobial usage (AMU) is driving an increase in multi-drug resistant (MDR) bacteria so treatment of microbial infections is becoming harder in humans and animals. The aim ...of this study was to evaluate factors, including usage, that affect antimicrobial resistance (AMR) on farm over time.
A population of 14 cattle, sheep and pig farms within a defined area of England were sampled three times over a year to collect data on AMR in faecal Enterobacterales flora; AMU; and husbandry or management practices. Ten pooled samples were collected at each visit, with each comprising of 10 pinches of fresh faeces. Up to 14 isolates per visit were whole genome sequenced to determine presence of AMR genes.
Sheep farms had very low AMU in comparison to the other species and very few sheep isolates were genotypically resistant at any time point. AMR genes were detected persistently across pig farms at all visits, even on farms with low AMU, whereas AMR bacteria was consistently lower on cattle farms than pigs, even for those with comparably high AMU. MDR bacteria was also more commonly detected on pig farms than any other livestock species.
The results may be explained by a complex combination of factors on pig farms including historic AMU; co-selection of AMR bacteria; variation in amounts of antimicrobials used between visits; potential persistence in environmental reservoirs of AMR bacteria; or importation of pigs with AMR microbiota from supplying farms. Pig farms may also be at increased risk of AMR due to the greater use of oral routes of group antimicrobial treatment, which were less targeted than cattle treatments; the latter mostly administered to individual animals. Also, farms which exhibited either increasing or decreasing trends of AMR across the study did not have corresponding trends in their AMU. Therefore, our results suggest that factors other than AMU on individual farms are important for persistence of AMR bacteria on farms, which may be operating at the farm and livestock species level.
is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig ...health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In
pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in
. Genome-wide association studies identified a new pleuromutilin resistance gene,
(A) (
iamulin
alnemulin
ntibiotic resistance), encoding a predicted ABC-F transporter.
culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that
(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and
, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in
, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that
(A) contributed to development of tiamulin resistance
in a manner consistent with that seen experimentally
. The
studies further showed that
(A) broadened the mutant selection window and raised the mutant prevention concentration above reported
antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of
(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.
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