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To tackle the problem of antimicrobial resistance (AMR) surveillance programmes are in place within Europe applying phenotypic methods, but there are plans for implementing whole genome sequencing (WGS). We tested the benefits of WGS using Escherichia coli collected from pig surveillance performed between 2013 to 2017. WGS was performed on 498 E. coli producing ESBL and AmpC enzymes, recovered from pig caeca on MacConkey + cefotaxime (McC + CTX) agar, as recommended by the European Commission, or ESBL agar, used additionally by United Kingdom. Our results indicated WGS was extremely useful for monitoring trends for specific ESBL genes, as well as a plethora of AMR genotypes, helping to establish their prevalence and co-linkage to certain plasmids. Recovery of isolates with multi-drug resistance (MDR) genotypes was lower from McC + CTX than ESBL agar. The most widespread ESBL genes belonged to the blaCTX-M family. blaCTX-M-1 dominated all years, and was common in two highly stable IncI1 MDR plasmids harbouring (blaCTX-M-1,sul2, tetA) or (blaCTX-M-1, aadA5, sul2, dfrA17), in isolates which were phylogenetically dissimilar, suggesting plasmid transmission. Therefore, WGS provided a wealth of data on prevalence of AMR genotypes and plasmid persistence absent from phenotypic data and, also, demonstrated the importance of culture media for detecting ESBL E. coli.

Background The beta-lactamase enzyme OXA-48 has spread widely in recent years in Enterobacteriaceae associated with man, disseminated primarily on incompatibility group L/M plasmids. OXA-48 confers resistance to carbapenems, important antimicrobials for treating highly resistant bacterial infections in humans. This enzyme has rarely been detected in bacteria from animals. Furthermore, the use of carbapenem compounds is not permitted in food-producing animals in Europe and to our knowledge has not been reported in food-producing animals globally. Methods Bacterial isolates from lesions in stranded, free-living, juvenile common seals (Phoca vitulina) were identified. Antimicrobial susceptibility testing and whole genome sequencing analysis were used to characterise antimicrobial resistance genes carried by the bacteria. Results Here, we report the detection of Klebsiella pneumoniae subspecies pneumoniae carrying the blaOXA-48 gene on an incompatibility group L/M plasmid from an infection in a common seal. Conclusion Evidence is accruing that marine mammals may be infected with bacteria originating from anthropogenic sources, such as human sewage, contaminating the environment.

Brachyspira hyodysenteriae 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 Brachyspira hyodysenteriae 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 Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(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 rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, 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 tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(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.

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.

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 fAVβ 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., SCOPUS: ar.j, info:eu-repo/semantics/published

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, …

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 frompigs 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.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium. M. T. G. H. was supported by the Wellcome Trust (grant number 098051). The MIC work was funded from the former AHVLA’s Research and Development Internal Investment Fund (grant number RD0030c). 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. Publisher PDF

KLEBSIELLA pneumoniae subspecies pneumoniae (Kpp) was isolated from three, free-living, juvenile common seals ( Phoca vitulina ) found, independently, in ill-health or abandoned ashore on the east coast of England. The seals were hospitalised at two rehabilitation centres. Postmortem examinations revealed lesions of neck abscessation, pleurisy and pyothorax in one seal, and omphalitis and peritonitis in a second seal. The third seal had a ruptured optic globe but survived (and was eventually released). Kpp was recovered from lesion swabs by culture on blood agar under standard aerobic conditions and further characterisation of isolates was by multilocus sequence typing (MLST). Sequence type (ST)398 was identified in one seal and ST11 in two seals. All three Kpp isolates showed multiple antibiotic resistance in broth microdilution susceptibility tests. ST11 represents an epidemic clone of Kpp which is found worldwide in man, often with extended-spectrum β-lactamase (ESBL) or, more recently, carbapenemase resistance. The resistance patterns and molecular typing of the Kpp isolates suggest that microbial marine pollution of human origin, possibly from human sewage, may have been the source of these infections in juvenile seals. Klebsiella species are members of the family Enterobacteriaceae and are ubiquitous in the environment, occurring in surface water, sewage and soil, as well as being found as commensals of the nasopharynx and intestine of human beings and other animals (Podschun and Ullmann 1998). Klebsiella pneumoniae is a common human nosocomial pathogen causing urinary, respiratory or generalised infections. The gastrointestinal tract of patients is considered one of the main reservoirs of the organism in cases of human infection (Podschun and Ullmann 1998). Klebsiella pneumoniae in human healthcare settings has developed increasing resistance to …

The authors regret that the BraDP1T consortium were not named in the author listing of their original paper. The correct listing appears above.