Search

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.

Acute non-typhoidal salmonellosis (NTS) caused by a Gram-negative bacteriumSalmonella entericaserovar Typhimurium (S.Tm) is one of the most common bacterial foodborne diseases worldwide. Bacteriophages (phages) can specifically target and lyse their host bacteria, including the multidrug-resistant strains, without collateral damage to other bacteria in the community. However, the therapeutic use ofSalmonellaphagesin vivois still poorly investigated.Salmonellaphages ST-W77 and SE-W109 have previously been shown by our group to be useful for biocontrol properties. Here, we tested whether phages ST-W77 and SE-W109 can reduceSalmonellainvasion into cultured human cells and confer a therapeutic benefit for acute NTS in a mammalian host. Human colonocytes, T84 cells, were treated with phages ST-W77, SE-W109, and its combination for 5 min beforeS.Tm infection. Gentamicin protection assays demonstrated that ST-W77 and SE-W109 significantly reducedS.Tm invasion and inflammatory response in human colonocytes. Next, streptomycin-pretreated mice were orally infected withS.Tm (108CFU/mouse) and treated with a single or a combination of ST-W77 and SE-W109 (1010PFU/mouse for 4 days) by oral feeding. Our data showed that phage-treated mice had lowerS.Tm numbers and tissue inflammation compared to the untreated mice. Our study also revealed that ST-W77 and SE-W109 persist in the mouse gut lumen, but not in systemic sites. Together, these data suggested thatSalmonellaphages ST-W77 and SE-W109 could be further developed as an alternative approach for treating an acute NTS in mammalian hosts.

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (n = 52), abattoir (n = 8), and animal markets (n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli. For isolates showing a multi-drug resistance (MDR) phenotype (n = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum β-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset (n = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with blaCTX-M present in all but others in a subset [blaTEM (62.8%) and blaSHV (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying blaCTX-M-55 was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant blaCTX-M variant was blaCTX-M-15 (87.9%), although blaCTX-M-55, blaCTX-M-64, and blaCTX-M-65 were present; it was notable that blaCTX-M-1, common in livestock, was absent. Other predominant AMR genes included: sul2, qnrS1, strB, blaTEM-1b, tetA-v2, and dfrA14, with prevalence varying according to host livestock species. A blaCTX-M-15 harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli; they were likely to be MDR with several harbouring blaCTX-M-15. Surprisingly, human isolates had highest numbers of AMR genes and pigs the least.

Acute non-typhoidal salmonellosis (NTS) caused by a Gram-negative bacterium

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (

OHEJP Project: ARDIG

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

2.AbstractSeveral bioinformatics genotyping algorithms are now commonly used to characterise antimicrobial resistance (AMR) gene profiles in whole genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularly Escherichia coli, is of major importance, because this is a common pathogen. However, robust comparisons of different genotyping approaches on relevant simulated and large real-life WGS datasets are lacking. Here, we used both simulated datasets and a large set of real E. coli WGS data (n=1818 isolates) to systematically investigate genotyping methods in greater detail.Simulated constructs and real sequences were processed using four different bioinformatic programs (ABRicate, ARIBA, KmerResistance, and SRST2, run with the ResFinder database) and their outputs compared. For simulations tests where 3,092 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for all 3,092 simulations, ABRicate for 3,082 (99.7%), ARIBA for 2,927 (94.7%) and SRST2 for 2,120 (68.6%). For simulations tests where two closely related gene variants were inserted into random sequence constructs, ABRicate identified the correct alleles in 11,382/46,279 (25%) of simulations, ARIBA in 2494/46,279 (5%), SRST in 2539/46,279 (5%) and KmerResistance in 38,826/46,279 (84%). In real data, across all methods, 1392/1818 (76%) isolates had discrepant allele calls for at least one gene.Our evaluations revealed poor performance in scenarios that would be expected to be challenging (e.g. identification of AMR genes at 3.Impact statementWhole-genome sequencing is widely used for studying the epidemiology of antimicrobial resistance (AMR) genes in bacteria; however, there is some concern that outputs are highly dependent on the bioinformatics methods used. This work evaluates these concerns in detail by comparing four different, commonly used AMR gene typing methods using large simulated and real datasets. The results highlight performance issues for most methods in at least one of several simulated and real-life scenarios. However most discrepancies between methods were due to differential labelling of the same sequences related to the assumptions made regarding the underlying structure of the reference resistance gene database (i.e. that resistance genes can be easily classified in well-defined groups). This study represents a major advance in quantifying and evaluating the nature of discrepancies between outputs of different AMR typing algorithms, with relevance for historic and future work using these algorithms. Some of the discrepancies can be resolved by choosing methods with fewer assumptions about the reference AMR gene database and manually resolving outputs generated using multiple programs. However, ideally new and better methods are needed.1.5RepositoriesSequencing data are available at the following NCBI BioProject accession number: PRJNA540750.

Enterobacterales from livestock are potentially important reservoirs for antimicrobial resistance (AMR) to pass through the food chain to humans, thereby increasing the AMR burden and affecting our ability to tackle infections. In this study 168 isolates from four genera of the order Enterobacterales , primarily Escherichia coli , were purified from livestock (cattle, pigs and sheep) faeces from 14 farms in the United Kingdom. Their genomes were resolved using long- and short-read sequencing to analyse AMR genes and their genetic context, as well as to explore the relationship between AMR burden and on-farm antimicrobial usage (AMU), in the three months prior to sampling. Although E. coli isolates were genomically diverse, phylogenetic analysis using a core-genome SNP tree indicated pig isolates to generally be distinct from sheep isolates, with cattle isolates being intermediates. Approximately 28 % of isolates harboured AMR genes, with the greatest proportion detected in pigs, followed by cattle then sheep; pig isolates also harboured the highest number of AMR genes per isolate. Although 90 % of sequenced isolates harboured diverse plasmids, only 11 % of plasmids (n=58 out of 522) identified contained AMR genes, with 91 % of AMR plasmids being from pig, 9 % from cattle and none from sheep isolates; these results indicated that pigs were a principle reservoir of AMR genes harboured by plasmids and likely to be involved in their horizontal transfer. Significant associations were observed between AMU (mg kg−1) and AMR. As both the total and the numbers of different antimicrobial classes used on-farm increased, the risk of multi-drug resistance (MDR) in isolates rose. However, even when AMU on pig farms was comparatively low, pig isolates had increased likelihood of being MDR; harbouring relatively more resistances than those from other livestock species. Therefore, our results indicate that AMR prevalence in livestock is not only influenced by recent AMU on-farm but also livestock-related factors, which can influence the AMR burden in these reservoirs and its plasmid mediated transmission.

Enterobacterales from livestock are potentially important reservoirs for antimicrobial resistance (AMR) to pass through the food chain to humans, thereby increasing the AMR burden and affecting our ability to tackle infections. In this study 168 isolates from four genera of the order Enterobacterales , primarily Escherichia coli , were purified from livestock (cattle, pigs and sheep) faeces from 14 farms in the United Kingdom. Their genomes were resolved using long- and short-read sequencing to analyse AMR genes and their genetic context, as well as to explore the relationship between AMR burden and on-farm antimicrobial usage (AMU), in the three months prior to sampling. Although E. coli isolates were genomically diverse, phylogenetic analysis using a core-genome SNP tree indicated pig isolates to generally be distinct from sheep isolates, with cattle isolates being intermediates. Approximately 28 % of isolates harboured AMR genes, with the greatest proportion detected in pigs, followed by cattle then sheep; pig isolates also harboured the highest number of AMR genes per isolate. Although 90 % of sequenced isolates harboured diverse plasmids, only 11 % of plasmids (n=58 out of 522) identified contained AMR genes, with 91 % of AMR plasmids being from pig, 9 % from cattle and none from sheep isolates; these results indicated that pigs were a principle reservoir of AMR genes harboured by plasmids and likely to be involved in their horizontal transfer. Significant associations were observed between AMU (mg kg−1) and AMR. As both the total and the numbers of different antimicrobial classes used on-farm increased, the risk of multi-drug resistance (MDR) in isolates rose. However, even when AMU on pig farms was comparatively low, pig isolates had increased likelihood of being MDR; harbouring relatively more resistances than those from other livestock species. Therefore, our results indicate that AMR prevalence in livestock is not only influenced by recent AMU on-farm but also livestock-related factors, which can influence the AMR burden in these reservoirs and its plasmid mediated transmission.

Brachyspira hyodysenteriae is the principal cause of swine dysentery, a disease that threatens economic productivity of pigs in many countries as it can spread readily within and between farms, and only a small number of antimicrobials are authorized for treatment of pigs. In this study, we performed whole-genome sequencing (WGS) of 81 B. hyodysenteriae archived at the Animal and Plant Health Agency (APHA) from diagnostic submissions and herd monitoring in England and Wales between 2004 and 2015. The resulting genome sequences were analyzed alongside 34 genomes we previously published. Multi-locus sequence typing (MLST) showed a diverse population with 32 sequence types (STs) among the 115 APHA isolates, 25 of them identified only in England; while also confirming that the dominant European clonal complexes, CC8 and CC52, were common in the United Kingdom. A core-genome SNP tree typically clustered the isolates by ST, with isolates from some STs detected only within a specific region in England, although others were more widespread, suggesting transmission between different regions. Also, some STs were more conserved in their core genome than others, despite these isolates being from different holdings, regions and years. Minimum inhibitory concentrations to commonly used antimicrobials (Tiamulin, Valnemulin, Doxycycline, Lincomycin, Tylosin, Tylvalosin) were determined for 82 of the genome-sequenced isolates; genomic analysis revealed mutations generally correlated well with the corresponding resistance phenotype. There was a major swine dysentery intervention program in 2009–2010, and antimicrobial survival curves showed a significant reduction in sensitivity to tiamulin and valnemulin in isolates collected in and after 2010, compared to earlier isolates. This correlated with a significant increase in post-2009 isolates harboring the pleuromutilin resistance gene tva(A), which if present, may facilitate higher levels of resistance. The reduction in susceptibility of Brachyspira from diagnostic submissions to pleuromutilins, emphasizes the need for prudent treatment, control and eradication strategies.

AIM: Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhoea, kidney failure and occasionally death. However, identifying the source of infection caused by STEC other than serogroup O157 is hampered by availability of sensitive methods for detecting these pathogens. In this study we developed novel tools for detecting E. coli O55 that is potentially associated with human outbreaks. METHOD AND RESULTS: Overall specificity of immuno-magnetic separation (IMS) beads coated with anti-O55 serum was good with exception of cross reactivity with E. coli O22 and O23, which was eliminated using an O55 specific PCR. Limit of detection for E. coli O55 using O55-IMS-beads in spiked cattle faeces was on average 50 CFU ml-1 (range 1-90), and improved to

Salmonella enterica is a foodborne pathogen showing increasing multidrug resistance (MDR). We characterized the antimicrobial resistance (AMR) genotype using microarrays in a panel of 105 nontyphoidal S. enterica isolated from food animals and foodstuff. Nineteen isolates were chosen on the basis of their MDR and virulence for determination of heavy metal susceptibilities and screened by polymerase chain reaction for heavy metal resistance genes. Whole-genome sequencing (WGS) was performed on three isolates carrying clinically important AMR genes and the cdtB toxin gene to detect other heavy metal resistance mechanisms, and conjugation assays were performed to evaluate transfer of AMR/toxin genes with heavy metal resistance genes. AMR genotyping results showed isolates harbored between 1 and 12 mobile AMR genes, with 58% being classified as MDR. The tested subset of isolates showed reduced susceptibility to zinc (78%), copper (68%), silver (63%), arsenic (47%), and tellurite (26%); phenotypes that could be attributed to zitB (n = 32%), pcoA/pcoD (n = 32%), tcrB (n = 16%), arsB (n = 16%), silA/silE (n = 42%), and terF (n = 26%) genes. WGS confirmed the presence of other heavy metal resistance genes such as copA, cusA, and czcD. Isolates often harbored multiple heavy metal resistance genes. Two strains (Sal25 and Sal368) were able to conjugate with Escherichia coli J53 at a relatively high frequency (∼10

Enterohemorrhagic Escherichia coli (EHEC) are a cause of bloody diarrhea, hemorrhagic colitis (HC) and the potentially fatal hemolytic uremic syndrome (HUS). While O157:H7 is the dominant EHEC serotype, non-O157 EHEC have emerged as serious causes of disease. In Germany, the most important non-O157 O-serogroups causing one third of EHEC infections, including diarrhea as well as HUS, are O26, O103, O111 and O145. Interestingly, we identified EHEC O-serogroups O26 and O111 in one single sequence type complex, STC29, that also harbours atypical enteropathogenic E. coli (aEPEC). aEPEC differ from typical EHEC merely in the absence of stx-genes. These findings inspired us to unravel a putative microevolutionary scenario of these non-O157 EHEC by whole genome analyses. Analysis of single nucleotide polymorphisms (SNPs) of the maximum common genome (MCG) of 20 aEPEC (11 human/ 9 bovine) and 79 EHEC (42 human/ 36 bovine/ 1 food source) of STC29 identified three distinct clusters: Cluster 1 harboured strains of O-serogroup O111, the central Cluster 2 harboured only O26 aEPEC strains, while the more heterogeneous Cluster 3 contained both EHEC and aEPEC strains of O-serogroup O26. Further combined analyses of accessory virulence associated genes (VAGs) and insertion sites for mobile genetic elements suggested a parallel evolution of the MCG and the acquisition of virulence genes. The resulting microevolutionary model suggests the development of two distinct EHEC lineages from one common aEPEC ancestor of ST29 by lysogenic conversion with stx-converting bacteriophages, independent of the host species the strains had been isolated from. In conclusion, our cumulative data indicate that EHEC of O-serogroups O26 and O111 of STC29 originate from a common aEPEC ancestor and are bona fide zoonotic agents. The role of aEPEC in the emergence of O26 and O111 EHEC should be considered for infection control measures to prevent possible lysogenic conversion with stx-converting bacteriophages as major vehicle driving the emergence of EHEC lineages with direct Public Health consequences.

Brachyspira hyodysenteriae is the principal cause of swine dysentery, a disease that threatens economic productivity of pigs in many countries as it can spread readily within and between farms, and only a small number of antimicrobials are authorized for treatment of pigs. In this study, we performed whole-genome sequencing (WGS) of 81 B. hyodysenteriae archived at the Animal and Plant Health Agency (APHA) from diagnostic submissions and herd monitoring in England and Wales between 2004 and 2015. The resulting genome sequences were analyzed alongside 34 genomes we previously published. Multi-locus sequence typing (MLST) showed a diverse population with 32 sequence types (STs) among the 115 APHA isolates, 25 of them identified only in England; while also confirming that the dominant European clonal complexes, CC8 and CC52, were common in the United Kingdom. A core-genome SNP tree typically clustered the isolates by ST, with isolates from some STs detected only within a specific region in England, although others were more widespread, suggesting transmission between different regions. Also, some STs were more conserved in their core genome than others, despite these isolates being from different holdings, regions and years. Minimum inhibitory concentrations to commonly used antimicrobials (Tiamulin, Valnemulin, Doxycycline, Lincomycin, Tylosin, Tylvalosin) were determined for 82 of the genome-sequenced isolates; genomic analysis revealed mutations generally correlated well with the corresponding resistance phenotype. There was a major swine dysentery intervention program in 2009–2010, and antimicrobial survival curves showed a significant reduction in sensitivity to tiamulin and valnemulin in isolates collected in and after 2010, compared to earlier isolates. This correlated with a significant increase in post-2009 isolates harboring the pleuromutilin resistance gene tva(A), which if present, may facilitate higher levels of resistance. The reduction in susceptibility of Brachyspira from diagnostic submissions to pleuromutilins, emphasizes the need for prudent treatment, control and eradication strategies.

The pangenome of Escherichia coli from nearby farms shows how livestock host and local geography shape bacterial populations., Escherichia coli and other Enterobacteriaceae are diverse species with “open” pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

International audience; Livestock data on antimicrobial resistance (AMR) are commonly collected from bacterial populations of clinical and non-clinical isolates. In contrast to data on non-clinical isolates from livestock, data on clinical isolates are not harmonized in Europe. The Normalized Resistance Interpretation (NRI) method was applied to overcome the lack of harmonization of laboratory methods and interpretation rules between monitoring systems. Statistical analyses were performed to identify associations between the isolate type (clinical vs. non-clinical) and resistance to four antimicrobials (ampicillin, tetracycline, gentamicin, and nalidixic acid) per animal category in Germany and France. Additional statistical analyses comparing clinical and non-clinical isolates were performed with the available data on the same antimicrobial panel and animal categories from the UK and Norway. Higher resistance prevalence was found in clinical isolates compared to non-clinical isolates from calves to all antimicrobials included in Germany and France. It was also found for gentamicin in broilers from France. In contrast, in broilers and turkeys from Germany and France and in broilers from the UK, a higher resistance level to ampicillin and tetracycline in non-clinical isolates was encountered. This was also found in resistance to gentamicin in isolates from turkeys in Germany. Resistance differed within countries and across years, which was partially in line with differences in antimicrobial use patterns. Differences in AMR between clinical and non-clinical isolates of Escherichia coli are associated with animal category (broiler, calf, and turkey) and specific antimicrobials. The NRI method allowed comparing results of non-harmonized AMR systems and might be useful until international harmonization is achieved.

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.

Salmonella spp. is a leading cause of gastrointestinal enteritis in humans where it is largely contracted via contaminated poultry and pork. Phages can be used to control Salmonella infection in the animals, which could break the cycle of infection before the products are accessible for consumption. Here, the potential of 21 myoviruses and a siphovirus to eliminate Salmonella in vitro and in vivo was examined with the aim of developing a biocontrol strategy to curtail the infection in poultry and swine. Together, the phages targeted the twenty-three poultry and ten swine prevalent Salmonella serotype isolates tested. Although individual phages significantly reduced bacterial growth of representative isolates within 6 h post-infection, bacterial regrowth occurred 1 h later, indicating proliferation of resistant strains. To curtail bacteriophage resistance, a novel three-phage cocktail was developed in vitro, and further investigated in an optimized Galleria mellonella larva Salmonella infection model colonized with representative swine, chicken and laboratory strains. For all the strains examined, G. mellonella larvae given phages 2 h prior to bacterial exposure (prophylactic regimen) survived and Salmonella was undetectable 24 h post-phage treatment and throughout the experimental time (72 h). Administering phages with bacteria (co-infection), or 2 h post-bacterial exposure (remedial regimen) also improved survival (73–100% and 15–88%, respectively), but was less effective than prophylaxis application. These pre-livestock data support the future application of this cocktail for further development to effectively treat Salmonella infection in poultry and pigs. Future work will focus on cocktail formulation to ensure stability and incorporation into feeds and used to treat the infection in target animals.

An important element in the control of antimicrobial resistance (AMR) is reduction in antimicrobial usage. In the veterinary sector individual antimicrobial treatment of livestock, rather than the use of group treatment, can help achieve this goal. The aim of this study was to investigate how cessation of group antimicrobial treatment impacted the prevalence of AMR in commensal Escherichia coli in pigs at one farm over an 11-month period. Minimum inhibitory concentrations of eight antimicrobials were determined for 259 E. coli isolates collected during the study. A significant reduction in the prevalence of multidrug resistance and a significant increase in the proportion of full susceptibility to the panel of nine antimicrobials tested was seen after 11 months. Whole genome sequencing of 48 multidrug resistant isolates revealed E. coli clones that persisted across multiple visits and provided evidence for the presence of plasmids harbouring AMR genes shared across multiple E. coli lineages. E. coli were also isolated from on-farm environmental samples. Whole genome sequencing of one multidrug resistant isolate obtained from cleaning tools showed it was clonal to pig-derived E. coli that persisted on the farm for 11 months. In this study we provide evidence that withdrawal of group antimicrobial use leads to significant reductions in key indicators for AMR prevalence and the importance of the farm environment as a reservoir of resistant bacteria. These findings support policy makers and producers in the implementation of measures to control AMR and reduce antimicrobial use.

This study was performed to investigate the occurrence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in batches of pigs at slaughter and at different stages along the slaughter line. Nasal and ear skin swabs were collected from 105 batches of 10 pigs at six abattoirs. Cultures (pooled or individual) were performed for MRSA using selective media; presumptive MRSA were confirmed by mecA and nuc gene detection and a selection was spa-typed. MRSA was detected in 46 batches. All spa-types detected were those associated with LA-MRSA clonal complex 398. The proportion of positive batches varied among abattoirs (0–100%). Two abattoirs were subsequently further investigated, with samples taken at post-stunning, chiller and either at lairage or post-singe. Results suggested cross-contamination occurred between the lairage and point of post-stunning, but the slaughter processes appeared effective at reducing contamination before carcases entered the chiller. One abattoir provided only negative samples in the initial study and in the subsequent study along the slaughter line (26 batches in total), suggesting differences possibly in the MRSA status of pigs on arrival from supply farms or in its abattoir practices affecting the MRSA status of pigs at the sampling points. This study highlights that in the investigated abattoirs, MRSA was detected in 43.8% of batches of pigs at slaughter using sensitive selective culture methods.

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.

The ST307 multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae is an emerging pathogen, which has become disseminated worldwide in humans but is rarely reported from other reservoirs. We report the first isolation of K. pneumoniae from an animal in Europe and also from a reptile, a captive tortoise, whose death it probably caused. Detection of this clone from an animal adds to evidence of niche expansion in non-human environments, where it may amplify, recycle and become of greater public health concern.

IncF plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum β-lactamases, particularly inEnterobacteriaceae. 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 IncF 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 IncF plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The recent association of IncF plasmids with AMR likely reflects their suitability for rapid niche adaptation.

Escherichia coli and other Enterobacteriaceae are highly diverse species with ‘open’ pangenomes1,2, where genes move intra- and inter-species via horizontal gene transfer3. These species can cause clinical infections4,5 as well as persist environmentally6,7. Environmental populations have been suggested as important reservoirs of antimicrobial resistance (AMR) genes. However, as most analyses focus on clinical isolates8,9, the pangenome dynamics of natural populations remain understudied, particularly the role of plasmids. Here, we reconstructed near-complete genomes for 828 Enterobacteriaceae, including 553 Escherichia spp. and 275 non-Escherichia species with 2,293 circularised plasmids in total, collected from nineteen locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30km radius at three timepoints over the course of a year. We find different dynamics for the chromosomal and plasmid-borne components of the pangenome, showing that plasmids have a higher burden of both AMR genes and insertion sequences, and AMR plasmids show evidence of being under stronger selective pressure. Focusing on E. coli, we observe that plasmid dynamics are more strongly dominated by niche and local geography, rather than phylogeny or season. Our results highlight the diversity of the AMR reservoir in these species and niches, and the importance of local strategies for controlling the emergence and spread of AMR.

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes, most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons, demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

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.

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

Octavio Mesa Varona,1 Katerina Chaintarli,2 Berit Muller-Pebody,3 Muna F Anjum,2 Tim Eckmanns,4 Madelaine Norström,5 Ides Boone,4 Bernd-Alois Tenhagen1 1Department of Biological Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany; 2Department of Bacteriology, Animal and Plant Health Agency (APHA), Addlestone, Surrey, UK; 3Healthcare-Associated Infections & Antimicrobial Resistance Division, National Infection Service, Public Health England (PHE), London, UK; 4Department for Infectious Disease Epidemiology, Robert Koch Institute (RKI), Berlin, Germany; 5Department of Analysis and Diagnostics, Section of Epidemiology, Norwegian Veterinary Institute (NVI), Oslo, NorwayCorrespondence: Octavio Mesa Varona Diedersdorfer Weg 1, Berlin 12277, GermanyTel +49 30 1841224338Email Octavio.Mesa-Varona@bfr.bund.deIntroduction: Antimicrobial resistance (AMR), associated with antimicrobial use (AMU), is a major public concern. Surveillance and monitoring systems are essential to assess and control the trends in AMU and AMR. However, differences in the surveillance and monitoring systems between countries and sectors make comparisons challenging. The purpose of this article is to describe all surveillance and monitoring systems for AMU and AMR in the human and livestock sectors, as well as national surveillance and monitoring systems for AMR in food, in six European countries (Spain, Germany, France, the Netherlands, the United Kingdom and Norway) as a baseline for developing suggestions to overcome current limitations in comparing AMU and AMR data.Methods: A literature search in 2018 was performed to identify relevant peer-reviewed articles and national and European grey reports as well as AMU/AMR databases.Results: Comparison of AMU and AMR systems across the six countries showed a lack of standardization and harmonization with different AMU data sources (prescription vs sales data) and units of AMU and AMR being used. The AMR data varied by sample type (clinical/non-clinical), laboratory method (disk diffusion, microdilution, and VITEK, among others), data type, ie quantitative (minimum inhibition concentration (MIC) in mg/L/inhibition zone (IZ) in mm) vs qualitative data (susceptible-intermediate-resistant (SIR)), the standards used (EUCAST/CLSI among others), and/or the evaluation criteria adopted (epidemiological or clinical).Discussion: A One Health approach for AMU and AMR requires harmonization in various aspects between human, animal and food systems at national and international levels. Additionally, some overlap between systems of AMU and AMR has been encountered. Efforts should be made to improve standardization and harmonization and allow more meaningful analyses of AMR and AMU surveillance data under a One Health approach.Keywords: AMR, AMU, food-producing animals, harmonization, monitoring, surveillance

Background The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans. Objectives To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5 month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ∼20 months. Methods mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined. Results E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5 months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ∼20 months after colistin cessation. Conclusions We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.

Aims In 2015, colistin-resistant Escherichia coli and Salmonella with the mcr-1 gene were isolated from a pig farm in Great Britain. Pigs were subsequently monitored over a ~20-month period for the occurrence of mcr-1-mediated colistin resistance and the risk of mcr-1 E. coli entering the food chain was assessed. Methods and results Pig faeces and slurry were cultured for colistin-resistant E. coli and Salmonella, tested for the mcr-1 gene by PCR and selected isolates were further analysed. Seventy-eight per cent of faecal samples (n = 275) from pigs yielded mcr-1 E. coli after selective culture, but in positive samples only 0·2-1·3% of the total E. coli carried mcr-1. Twenty months after the initial sampling, faecal samples (n = 59) were negative for E. coli carrying mcr-1. Conclusions The risk to public health from porcine E. coli carrying mcr-1 was assessed as very low. Twenty months after cessation of colistin use, E. coli carrying mcr-1 was not detected in pig faeces on a farm where it was previously present. Significance and impact of the study The results suggest that cessation of colistin use may help over time to reduce or possibly eliminate mcr-1 E. coli on pig farms where it occurs.

Objectives To determine the occurrence of mcr-1 and mcr-2 genes in Gram-negative bacteria isolated from healthy pigs in Great Britain. Methods Gram-negative bacteria (n = 657) isolated from pigs between 2014 and 2015 were examined by WGS. Results 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. Conclusions 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.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines)., Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

[EN] Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging problem in many parts of the world. Although animal-adapted LA-MRSA has been known for many years, recent reports suggest a possible increasing trend in the zoonotic transmission of LA-MRSA in Europe. Since its emergence in the early 2000¿s, several investigations have indicated that persons in prolonged, repeated contact with affected livestock are at a higher risk of becoming colonized with LA-MRSA. LA-MRSA monitoring in livestock is voluntary under current EU legislation, and not all member states, including the UK, participate. UK LA-MRSA isolates have been detected through scanning surveillance, where samples are submitted from clinically diseased livestock for diagnostic investigation, and research studies. Surveys conducted on retail beef, pig and poultry meat on sale in the UK have also detected LA-MRSA. Taken together these results suggest that LA-MRSA is present in the UK, possibly at low prevalence level, as suggested by available evidence. In this review, we examine the data available from UK livestock and animal products, and make recommendations for future. We also review the findings from whole genome sequencing (WGS) of the possible lineage of some UK livestock isolates., We are grateful to the Veterinary Medicines Directorate in the UK for funding this work through VMD0533. FM-J contributed during a sabbatical to the APHA with a grant from Consellería de Educación y Ciencia of Generalitat Valenciana (BEST/2017/050). CM contributed during a sabbatical to the APHA which was supported by a Lecturer research grant from the Santander bank (programme XIII Convocatoria de ayudas a la movilidad investigadora CEU-Banco Santander).

Background Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ‘ResPipe’. Results Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment. Conclusions Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred on the Isle of Wight between August and October 2017. Of the seven cases linked to the outbreak, five were identified through the statutory notification process and two were identified through national surveillance of whole genome sequencing data. Enhanced surveillance questionnaires established a common link to a farm, and link to the likely food vehicle, raw drinking milk (RDM). Microbiological investigations, including PCR, identified the presence of STEC O157:H7 in samples of RDM. Analysis of core genome single nucleotide polymorphism (SNP) data of STEC O157:H7 from human stool specimens, animal faecal samples and RDM demonstrated a one SNP difference between isolates, and therefore close genetic relatedness. Control measures that were put in place included suspension of sales and recall of RDM, as well as restrictions on public access to parts of the farm. Successful integration of traditional epidemiological surveillance and advanced laboratory methods for the detection and characterisation of STEC O157:H7 from human, animal and environmental samples enabled prompt identification of the outbreak vehicle and provided evidence to support the outbreak control team’s decision-making, leading to implementation of effective control measures in a timely manner.

Background Surveillance of commensal Escherichia coli, a possible reservoir of antimicrobial resistance (AMR) genes, is important as they pose a risk to human and animal health. Most surveillance activities rely on phenotypic characterisation, but whole genome sequencing (WGS) presents an alternative. Aim In this retrospective study, we tested 515 E. coli isolated from pigs to evaluate the use of WGS to predict resistance phenotype. Methods Minimum inhibitory concentration (MIC) was determined for nine antimicrobials of clinical and veterinary importance. Deviation from wild-type, fully-susceptible MIC was assessed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off (ECOFF) values. Presence of AMR genes and mutations were determined using APHA SeqFinder. Statistical two-by-two table analysis and Cohen’s kappa (k) test were applied to assess genotype and phenotype concordance. Results Overall, correlation of WGS with susceptibility to the nine antimicrobials was 98.9% for test specificity, and 97.5% for the positive predictive value of a test. The overall kappa score (k = 0.914) indicated AMR gene presence was highly predictive of reduced susceptibility and showed excellent correlation with MIC. However, there was variation for each antimicrobial; five showed excellent correlation; four very good and one moderate. Suggested ECOFF adjustments increased concordance between genotypic data and kappa values for four antimicrobials. Conclusion WGS is a powerful tool for accurately predicting AMR that can be used for national surveillance purposes. Additionally, it can detect resistance genes from a wider panel of antimicrobials whose phenotypes are currently not monitored but may be of importance in the future.

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.

Aims This study investigated the occurrence and genetic diversity of Enterobacteriaceae with extended-spectrum β-lactamase (ESBL)-, AmpC- and carbapenemase-mediated resistance in British beef cattle, and related risk factors. Methods and results Faecal samples (n = 776) were obtained from farms in England and Wales (n = 20) and Scotland (n = 20) in 2015. Isolates from selective agars were identified by MALDI ToF mass spectrometry. Selected isolates were characterized by multiplex PCR (blaCTX -M, blaOXA , blaSHV and blaTEM genes), whole-genome sequencing (WGS), minimum inhibitory concentrations and pulsed-field gel electrophoresis. None of the faecal samples yielded carbapenem-resistant Escherichia coli. Ten (25%) of the farms tested positive for ESBL-producing CTX-M Enterobacteriaceae, 15 (37·5%) of the farms were positive for AmpC phenotype E. coli and none were positive for carbapenem-resistant E. coli. WGS showed a total of 30 different resistance genes associated with E. coli, Citrobacter and Serratia from ESBL agars, and colocation of resistance genes with blaCTX -M1 . Buying bulls and bringing in fattening cattle from another farm were identified as significant risk factors for positive samples harbouring CTX-M Enterobacteriaceae or AmpC phenotype E. coli respectively. Conclusions Beef cattle on a proportion of farms in GB carry ESBL-producing Enterobacteriaceae. Factors, such as operating as a closed herd, may have an important role in reducing introduction and transmission of resistant Enterobacteriaceae. The results indicate management factors may play an important role in impacting ESBL prevalence. In particular, further study would be valuable to understand the impact of maintaining a closed herd on reducing the introduction of resistant Enterobacteriaceae. Significance and impact of the study This is the first study showing the presence of ESBL-producing Enterobacteriaceae in British beef cattle.

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.

A total of 502 Campylobacter jejuni isolates from poultry in 12 different European countries (10 of them the largest poultry production countries in Europe) were whole genome sequenced to examine the genomic diversity of fluoroquinolone resistant (FQ‐R) and susceptible (FQ‐S) C. jejuni across the poultry producing European countries and to determine whether the emergence of fluoroquinolone resistance among C. jejuni is related to the transmission through countries or to the selection through fluoroquinolone use in the individual countries. A high genomic diversity was observed. The isolates clustered in four main clusters. All trees revealed that the isolates were clustered according to the presence/absence of the gyrA mutations causing fluoroquinolone resistance and ST‐types. The cgMLST trees of only FQ‐R and FQ‐S isolates showed that isolates from the same country of origin were distributed into multiple clusters similarly to the trees combining FQ‐R and FQ‐S isolates. The different phylogenetic methods, ranging from single nucleotide polymorphisms analysis to gene‐by‐gene approaches such as rMLST, cgMLST, wgMLST and core genome tree, provided concordant results, but it is not known which is the most accurate method for identifying the country of origin of the isolates. Allele frequency analysis of isolates under this study and a selection of previously published C. jejuni genomes in ENA showed association of geographical origin of poultry C. jejuni populations between Romania‐Poland, Italy‐Germany‐England, Portugal‐The Netherlands and USA‐Luxemburg. Allele frequency and phylogenetic analysis indicated that the isolates from Finland were genetically different from C. jejuni populations from other European countries included in this study. Trade pattern and antimicrobial use in livestock were not significantly associated with allele frequency or populations of C. jejuni, but data available to investigate these associations were limited.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene ( stx ), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx -negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum β-lactamase bla CTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein.

The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1. We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo. It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies., IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.

The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase bla CTX-M1 . We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo . It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies. IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.