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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.

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

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.

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.

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.

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.

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.

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

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.

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.

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 increase in bacteria harboring antimicrobial resistance (AMR) is a global problem because there is a paucity of antibiotics available to treat multidrug-resistant bacterial infections in humans and animals. Detection of AMR present in bacteria that may pose a threat to veterinary and public health is routinely performed using standardized phenotypic methods. Molecular methods are often used in addition to phenotypic methods but are set to replace them in many laboratories due to the greater speed and accuracy they provide in detecting the underlying genetic mechanism(s) for AMR. In this article we describe some of the common molecular methods currently used for detection of AMR genes. These include PCR, DNA microarray, whole-genome sequencing and metagenomics, and matrix-assisted laser desorption ionization–time of flight mass spectrometry. The strengths and weaknesses of these methods are discussed, especially in the context of implementing them for routine surveillance activities on a global scale for mitigating the risk posed by AMR worldwide. Based on current popularity and ease of use, PCR and single-isolate whole-genome sequencing seem irreplaceable.

Antibiotic resistance in human bacterial pathogens and commensals is threatening our ability to treat infections and conduct now common medical procedures. As novel antibiotics are discovered and marketed it is important that we understand how resistance to them may arise and know what environments may act as reservoirs for such resistance genes. In this study a tetracycline and tigecycline resistant clone was identified by screening a human saliva metagenomic library in E. coli EPI300 on agar containing 5 μg/ml tetracycline. Sequencing of the DNA insert present within the tetracycline resistant clone revealed it to contain a 7,765 bp fragment harbouring novel ABC half transporter genes, tetAB(60). Mutagenesis studies performed on these genes confirmed that they were responsible for the tetracycline and tigecycline resistance phenotypes. Growth studies performed using E. coli EPI300 clones that harboured either the wild type, the mutated, or none of these genes indicated that there was a fitness cost associated with presence of these genes, with the isolate harbouring both genes exhibiting a significantly slower growth than control strains. Given the emergence of E. coli strains that are sensitive only to tigecycline and doxycycline it is concerning that such a resistance mechanism has been identified in the human oral cavity.

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

The presence of antimicrobial resistance and virulence factors of 174 Escherichia coli strains isolated from healthy Portuguese Gallus gallus was evaluated. Resistance profiles were determined against 33 antimicrobials by microbroth dilution. Resistance was prevalent for tetracycline (70%) and ampicillin (63%). Extended-spectrum beta-lactamase (ESBL) phenotype was observed in 18% of the isolates. Multidrug resistance was found in 56% of isolates. A subset of 74 isolates were screened by DNA microarrays for the carriage of 88 antibiotic resistance genes and 62 virulence genes. Overall, 37 different resistance genes were detected. The most common were tet(A) (72%), blaTEM (68%), and sul1 (47%), while 21% isolates harbored an ESBL gene (blaCTX-M group 1, group 2, or group 9). Of these, 96% carried the increased serum survival (iss) virulence gene, while 89% presented the enterobactin siderophore receptor protein (iroN), 70% the temperature-sensitive hemagglutinin (tsh), and 68% the long polar fimbriae (lpfA) virulence genes associated with extraintestinal pathogenic E. coli. In conclusion, prevalence of antibiotic resistant E. coli from the microbiota of Portuguese chickens was high, including to extended spectrum cephalosporins. The majority of isolates seems to have the potential to trigger extraintestinal human infection due to the presence of some virulence genes. However, the absence of genes specific for enteropathogenic E. coli reduces the risk for human intestinal infection.

Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains.

A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the faeces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being blaTEM, dfr, strB, tet(A) and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of blaTEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harbouring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year.

SUMMARYThe presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157Escherichia coliisolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer'sVstatistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenicE. coliof high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.

Staphylococcus aureusis one of the most important pathogens associated with bovine mastitis. Recent studies have shown thatStaph. aureusstrains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whetherStaph. aureusisolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. AllStaph. aureusisolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20Staph. aureusisolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidinlukD/Egenes were only carried by herd A isolates. Furthermore, all herd A isolates showed β-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate thatStaph. aureusisolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.

METHICILLIN-resistant Staphylococcus aureus (MRSA) has been reported in human medicine as a cause of nosocomial and community-associated infections (Otter and French 2010). In veterinary medicine, MRSA strains have been identified in a wide range of animals and diseases (Leonard and Markey 2008, Fessler and others 2009, Huber and others 2010, Turkyilmaz and others 2010), thus it is considered an emerging threat with a high zoonotic potential (Juhasz-Kaszanyitzky and others 2007). MRSA sequence type (ST) 1, spa type (t) 127 has been mostly isolated from community-associated infections, but t127 has seldom been identified in bovine and swine (Hasman and others 2010). The present report regards a dairy cow with an intramammary infection by MRSA ST1, t127 that was investigated over an entire lactation and submitted to histological evaluation of mammary tissue to explore both bacterial molecular features and host immune response in the milk and mammary tissue. The study was performed because only a few mammary infections of the dairy cow by this particular strain have been reported and no information is available on the interaction between the bacteria and the mammary gland. A five-year-old Holstein-Friesian dairy cow from a closed herd located in Milano province, Italy, consisting of 25 lactating cows, showed an intramammary infection with S aureus in both right quarters. The isolate revealed a multidrug resistance profile by the agar-plate method, and PCR analysis (Costa and Palladini 2005) confirmed the presence of mecA genes in both isolates, which were consequently classified as MRSA. The farmer decided to antibiotic treat and dry off the animal, …

The RpoE and CpxR regulated envelope stress responses are extremely important for Salmonella Typhimurium to cause infection in a range of hosts. Until now the role for BaeSR in both the Salmonella Typhimurium response to stress and its contribution to infection have not been fully elucidated. Here we demonstrate stationary phase growth, iron and sodium tungstate as novel inducers of the BaeRregulon, with BaeR critically required for Salmonella resistance to sodium tungstate. We show that functional overlap between the resistance nodulation-cell division (RND) multidrug transporters, MdtA, AcrD and AcrB exists for the waste disposal of tungstate from the cell. We also point to a role for enterobactinsiderophores in the protection of enteric organisms from tungstate, akin to the scenario in nitrogen fixing bacteria. Surprisingly, BaeR is the first envelope stress response pathway investigated in S. Typhimurium that is not required for murine typhoid in either ity(S) or ity(R) mouse backgrounds. BaeR is therefore either required for survival in larger mammals such as pigs or calves, an avian host such as chickens, or survival out with the host altogether where Salmonella and related enterics must survive in soil and water.

Salmonella enterica is a bacterial pathogen that causes enteric fever and gastroenteritis in humans and animals. Although its population structure was long described as clonal, based on high linkage disequilibrium between loci typed by enzyme electrophoresis, recent examination of gene sequences has revealed that recombination plays an important evolutionary role. We sequenced around 10% of the core genome of 114 isolates of enterica using a resequencing microarray. Application of two different analysis methods (Structure and ClonalFrame) to our genomic data allowed us to define five clear lineages within S. enterica subspecies enterica, one of which is five times older than the other four and two thirds of the age of the whole subspecies. We show that some of these lineages display more evidence of recombination than others. We also demonstrate that some level of sexual isolation exists between the lineages, so that recombination has occurred predominantly between members of the same lineage. This pattern of recombination is compatible with expectations from the previously described ecological structuring of the enterica population as well as mechanistic barriers to recombination observed in laboratory experiments. In spite of their relatively low level of genetic differentiation, these lineages might therefore represent incipient species., Author Summary Salmonella enterica is a species of bacteria that causes severe diseases in humans and animals. We sequenced about a tenth of the genome from a broadly sampled collection of S. enterica. By comparing these genetic sequences, we were able to partially reconstruct the ancestry of this sample. We identified five lineages within S. enterica, one of which is almost as old as the common ancestor of our sample. We also found evidence for frequent homologous recombination in the ancestry of S. enterica, where fragments of genes from one individual bacterium are acquired by a distinct individual. These recombination events make the ancestry harder to reconstruct in its entirety, but also contain interesting information. We found in particular that recombination had happened more often between strains belonging to the same lineage than across lineage boundaries. This observation is compatible with the lineages of S. enterica becoming progressively isolated from each other, which could lead to their gradual splintering into new species.

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and β-lactams, including extended-spectrum β-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations.

The CpxAR (Cpx) two-component regulator controls the expression of genes in response to a variety of environmental cues. The Cpx regulator has been implicated in the virulence of several gram-negative pathogens, although a role for Cpx in vivo has not been demonstrated directly. Here we investigate whether positive or negative control of gene expression by Cpx is important for the pathogenesis of Salmonella enterica serotype Typhimurium. The Cpx signal pathway in serotype Typhimurium was disrupted by insertional inactivation of the cpxA and cpxR genes. We also constitutively activated the Cpx pathway by making an internal in-frame deletion in cpxA (a cpxA* mutation). Activation of the Cpx pathway inhibited induction of the envelope stress response pathway controlled by the alternative sigma factor σ E (encoded by rpoE ). Conversely, the Cpx pathway was highly up-regulated (>40-fold) in a serotype Typhimurium rpoE mutant. The cpxA * mutation, but not the cpxA or the cpxR mutation, significantly reduced the capacity of serotype Typhimurium to adhere to and invade eucaryotic cells, although intracellular replication was not affected. The cpxA and cpxA* mutations significantly impaired the ability of serotype Typhimurium to grow in vivo in mice. To our knowledge, this is the first demonstration that the Cpx system is important for a bacterial pathogen in vivo.

The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), bla TEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.

In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

Objectives WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. Methods The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. Results Genotype–phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype–phenotype concordance was Conclusions WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.