Jon Rogers

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain

OBJECTIVES The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured β-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory

Matrix-Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) Mass Spectrometry with Bruker MALDI Biotyper software was evaluated as a method for identifying veterinary bacteria. For 620 isolates (~100 bacterial species), identification by MALDI-ToF and non-16S rDNA methods (mainly phenotypic/biochemical) agreed to species-level (95.3%) and to species/genus-level (100%), but in the absence of 16S rDNA as a gold standard. For a further panel of 107 anaerobes and 234 aerobes (~100 bacteria species) using 16S rDNA results as the gold standard, MALDI-ToF/biochemical tests showed 97.8/96.6% species-level and 99.6/93.5% genus-level agreement for aerobes and 95.3/93.6% species-level and 100/95.3% genus-level agreement for anaerobes compared to the gold standard. Where results were obtained from direct spots, direct spots overlaid with formic acid and extracts, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-ToF. MALDI-ToF was shown to be a rapid and reliable method to identify veterinary bacteria.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.

Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

Highlights • First complete sequence of a floR plasmid from Actinobacillus pleuropneumoniae• Extended similarity to floR plasmids in other Pasteurellaceae species• Conjugal transfer between between species confirmed

Livestock-Associated Methicillin Resistant Staphylococcus aureus (LA-MRSA) Clonal Complex (CC) 398 Isolated from UK Animals belong to European Lineages

In recent years, there has been an increase in the number of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 recovered from S. aureus isolated animals in the UK. To determine possible origins of 12 LA-MRSA CC398 isolates collected after screening more than a thousand S. aureus animal isolates from the UK between 2013 and 2015, whole genome sequences (WGS) of CC398 European, including UK, and non-European isolates from diverse animal hosts were compared. Phylogenetic reconstruction applied to WGS data to assess genetic relatedness of all 89 isolates, clustered the 12 UK CC398 LA-MRSA within the European sub-lineages, although on different nodes; implicating multiple independent incursions into the UK, as opposed to a single introduction followed by clonal expansion. Three UK isolates from healthy pigs and one from turkey clustered within the cassette chromosome recombinases ccr C S. aureus protein A (spa)-type t011 European sub-lineage and three UK isolates from horses within the ccrA2B2 t011 European sub-lineage. The remaining UK isolates, mostly from pigs, clustered within the t034 European lineage. Presence of virulence, antimicrobial (AMR), heavy metal (HMR), and disinfectant (DR) resistance genes were determined using an in-house pipeline. Most, including UK isolates, harbored resistance genes to ≥3 antimicrobial classes in addition to β-lactams. HMR genes were detected in most European ccrC positive isolates, with >80% harboring czrC, encoding zinc and cadmium resistance; in contrast ~60% ccrC isolates within non-European lineages and 6% ccrA2B2 isolates showed this characteristic. The UK turkey MRSA isolate did not harbor φAVβ avian prophage genes (SAAV_2008 and SAAV_2009) present in US MSSA isolates from turkey and pigs. Absence of some of the major human-associated MRSA toxigenic and virulence genes in the UK LA-MRSA animal isolates was not unexpected. Therefore, we can conclude that the 12 UK LA-MRSA isolates collected in the past 2 years most likely represent separate incursions into the UK from other European countries. The presence of zinc and cadmium resistance in all nine food animal isolates (pig and poultry), which was absent from the 3 horse isolates may suggest heavy metal use/exposure has a possible role in selection of some MRSA.

Whole Genome Sequencing for Surveillance of Antimicrobial Resistance in Actinobacillus pleuropneumoniae

The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.

ICEApl1, an integrative conjugative element related to ICEHin1056, identified in the pig pathogen Actinobacillus pleuropneumoniae

ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015

Objectives: To determine the occurrence of mcr-1-harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods: Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT–PCR. mcr-1-harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results: Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%–1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1-positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1-plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1-plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%–99%. Conclusions: mcr-1-harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1-positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

MRSA spa type t899 from food animals in the UK

We report on the detection of a strain of antimicrobial-resistant bacteria with zoonotic potential identified in the UK for the first time. Livestock-associated meticillin resistant Staphylococcus aureus (LA-MRSA), clonal complex (CC) 398 is predominantly asymptomatic in livestock, with infrequent reports of opportunistic infections in farm workers.1 LA-MRSA CC398, spa type t899, was detected from UK animals, harbouring prophage φSa3-immune evasion cluster (IEC) genes, sak , scn and chp , markers for human adaptation, which have been absent from previously reported UK CC398 animal strains. 2, 3 In 2016, …

Emergence of Klebsiella pneumoniae subspecies pneumoniae as a cause of septicaemia in pigs in England

Between 2011 and 2014 outbreaks of septicaemia due to Klebsiella pneumoniae subspecies pneumoniae (Kpp) were diagnosed on thirteen English pig farms. The most consistent features were rapid deaths of pigs from ten-days-old to weaning, seasonal occurrence (May to September), affected farms being outdoor breeding herds and the location of all but one of the outbreaks in the East Anglia region in Eastern England. Molecular characterisation of the outbreak Kpp isolates showed that by multilocus sequencing all were sequence type 25 (ST25) of K2 capsular type with a combination of a 4.3kb plasmid (pKPMC25), three phage sequences and the rmpA virulence gene. No archived Kpp isolates of porcine origin pre-dating 2011 were identified as ST25. In 2013 there was the first detection of an outbreak Kpp isolate showing antimicrobial resistance to six antibiotics. Human infection with Kpp ST25 has not been reported in the UK.