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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.

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.

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.

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.

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.

[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 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.

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.

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 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.

SAMPLING of poultry carcases is used for monitoring bacterial contaminants. For example, neck flap sampling is used for the detection of Campylobacter on chicken carcases (Bare and others 2013) and studies have found a high prevalence of Salmonella in this sample type (Wu and others 2014). However, with the reported rise in antimicrobial resistance, in this study, chicken carcases were tested for the presence of the extended spectrum β-lactamase (ESBL) bla CTX-M by loop-mediated isothermal amplified (LAMP) and culture. bla CTX-M have been a concern in human health as they confer resistance to third-generation cephalosporins, used for the treatment of human bacterial infections and ESBL bacteria often harbour multi-drug-resistant plasmids (Kirchner and others 2013). Escherichia coli from poultry may carry bla CTX-M genes and caecal sampling has been used to assess carriage rates . In a 2008 broiler survey conducted in the UK, it was found that 3.6 per cent of caeca collected at abattoirs were positive for bla CTX-M-positive E coli (Randall and others 2011). A survey from 2014 to 2015, also in the UK, found the prevalence of bla CTX-M-positive E coli in broiler caeca had increased to 28 per cent (Parker and others 2016). Neck flap and caecal samples used in this study …

Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2 -converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island.

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

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

Escherichia coli have become the enterobacteriaceae species most affected by extended-spectrum β-lactamases (ESBLs) in view of the emergence of CTX-M-type ESBLs. These CTX-M-positive E. coli have been reported in numerous regions worldwide. Virulence determinants of already reported CTX-M-positive E. coli were investigated.To gain insights into the mechanism underlying this phenomenon, we assessed serogroup, susceptibility pattern and diversity of virulence profiles within a collection of nine bla CTX-M-positive E. coli strains and their virulent determinant using miniaturized DNA microarray techniques. The nine ESBL-positive E. coli isolates were from eight male and one female patient(s) selected for study based on previous work. Virulence potential was inferred by detection of 63 virulence factor (VF) genes.Four (44.4%) of the 9 E. coli isolates exhibited the same set of core characteristics: serotype O8:Hnt, while all were positive for OXA-1, ciprofloxacin resistance. Five of the isolates exhibited highly similar (91% to 100%) VF profiles.The findings describe a broadly disseminated, bla CTX-M-positive and virulent E. coli serogroup with highly homogeneous virulence genotypes, suggesting recent emergence in this zone. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority.

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

Salmonella enterica is a foodborne zoonotic pathogen of significant public health concern. We have characterised the virulence and antimicrobial resistance gene content of 95 Salmonella isolates from 11 serovars by DNA microarray recovered from UK livestock or imported meat. Genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tet(A), tet(B)), streptomycin (strA, strB), aminoglycoside (aadA1, aadA2), beta-lactam (blaTEM), and trimethoprim (dfrA17) were common. Virulence gene content differed between serovars; S. Typhimurium formed two subclades based on virulence plasmid presence. Thirteen isolates were selected by their virulence profile for pathotyping using the Galleria mellonella pathogenesis model. Infection with a chicken invasive S. Enteritidis or S. Gallinarum isolate, a multidrug resistant S. Kentucky, or a S. Typhimurium DT104 isolate resulted in high mortality of the larvae; notably presence of the virulence plasmid in S. Typhimurium was not associated with increased larvae mortality. Histopathological examination showed that infection caused severe damage to the Galleria gut structure. Enumeration of intracellular bacteria in the larvae 24 hours post-infection showed increases of up to 7 log above the initial inoculum and transmission electron microscopy (TEM) showed bacterial replication in the haemolymph. TEM also revealed the presence of vacuoles containing bacteria in the haemocytes, similar to Salmonella containing vacuoles observed in mammalian macrophages; although there was no evidence from our work of bacterial replication within vacuoles. This work shows that microarrays can be used for rapid virulence genotyping of S. enterica and that the Galleria animal model replicates some aspects of Salmonella infection in mammals. These procedures can be used to help inform on the pathogenicity of isolates that may be antibiotic resistant and have scope to aid the assessment of their potential public and animal health risk.

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.

We have developed a Salmonella genoserotyping array (SGSA) which rapidly generates an antigenic formula consistent with the White-Kauffmann-Le Minor scheme, currently the gold standard for Salmonella serotyping. A set of 287 strains representative of 133 Salmonella serovars was assembled to validate the array and to test the array probes for accuracy, specificity, and reproducibility. Initially, 76 known serovars were utilized to validate the specificity and repeatability of the array probes and their expected probe patterns. The SGSA generated the correct serovar designations for 100% of the known subspecies I serovars tested in the validation panel and an antigenic formula consistent with that of the White-Kauffmann-Le Minor scheme for 97% of all known serovars tested. Once validated, the SGSA was assessed against a blind panel of 100 Salmonella enterica subsp. I samples serotyped using traditional methods. In summary, the SGSA correctly identified all of the blind samples as representing Salmonella and successfully identified 92% of the antigens found within the unknown samples. Antigen- and serovar-specific probes, in combination with a pepT PCR for confirmation of S. enterica subsp. Enteritidis determinations, generated an antigenic formula and/or a serovar designation consistent with the White-Kauffmann-Le Minor scheme for 87% of unknown samples tested with the SGSA. Future experiments are planned to test the specificity of the array probes with other Salmonella serovars to demonstrate the versatility and utility of this array as a public health tool in the identification of Salmonella .

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

Salmonella are closely related to commensal Escherichia coli but have gained virulence factors enabling them to behave as enteric pathogens. Less well studied are the similarities and differences that exist between the metabolic properties of these organisms that may contribute toward niche adaptation of Salmonella pathogens. To address this, we have constructed a genome scale Salmonella metabolic model (iMA945). The model comprises 945 open reading frames or genes, 1964 reactions, and 1036 metabolites. There was significant overlap with genes present in E. coli MG1655 model iAF1260. In silico growth predictions were simulated using the model on different carbon, nitrogen, phosphorous, and sulfur sources. These were compared with substrate utilization data gathered from high throughput phenotyping microarrays revealing good agreement. Of the compounds tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless a number of differences were identified both between Salmonella and E. coli and also within the Salmonella strains included. These differences provide valuable insight into differences between a commensal and a closely related pathogen and within different pathogenic strains opening new avenues for future explorations.

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E. fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusoniiprfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E. fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E. coli, and support existing evidence that strains of E. fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored.

The influence of geographical origin, host animal and presence of the stx gene on the virulence of Escherichia coli O26 strains from ruminants was determined in this study. A clear association was found between the virulence profile and geographical origin of Shiga-toxigenic E. coli (STEC) O26 strains, with UK STEC O26 strains harbouring virtually identical profiles, whilst central European strains showed considerable heterogeneity in plasmid-encoded genes. The former group were also more likely to be non-motile and katP gene positive. Comparison of UK STEC and atypical enteropathogenic E. coli (aEPEC) O26 strains showed that the presence of the stx1 gene was positively correlated with the presence of espP and katP genes and negatively associated with the presence of the yagP–yagT region and with rhamnose fermentation. In contrast to the uniform profiles of STEC O26 strains from ruminants in the UK, aEPEC O26 strains of bovine and ovine origin showed diverse profiles both within and between groups, and could not be separated into discrete groups. These results indicate that the characteristics of UK O26 strains from ruminants are distinct from those of O26 strains from ruminants and humans in other regions in central Europe. Such differences are expected to influence the zoonotic potential of this pathogen and the subsequent incidence of O26-associated human disease.

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.

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.

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa . Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons ( fim , bcf , and stb ) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S.enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.

The Salmonella enterica serovar Typhi CT18 (S.Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S.Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S.Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S.Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S.Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S.enterica.

The yeiL open reading frame located at 48.5 min (2254 kb) in the nfo-fruA region of the Escherichia coli chromosome was predicted to encode a CRP and FNR paralogue capable of forming inter- or intra-molecular disulphide bonds and incorporating one iron-sulphur centre per 25 kDa subunit. Purified MBP-YeiL (a maltose-binding-protein-YeiL fusion protein) was a high-molecular-mass oligomer or aggregate which released unstable monomers (68 kDa) under reducing conditions. The MBP-YeiL protein contained substoichiometric amounts of iron and acid-labile sulphide, and an average of one disulphide bond per monomer. The iron and sulphide contents increased consistent with the acquisition of one [4Fe-4S] cluster per monomer after anaerobic NifS-catalysed reconstitution. By analogy with FNR and FLP (the FNR-like protein of Lactobacillus casei) it was suggested that the transcription-regulatory activity of YeiL might be modulated by a sensory iron-sulphur cluster and/or by reversible disulphide bond formation. A yeiL-lacZ transcriptional fusion showed that aerobic yeiL expression increases at least sixfold during stationary phase, requires RpoS, and is positively autoregulated by YeiL, positively activated by Lrp (and IHF in the absence of FNR) and negatively regulated by FNR. A regulatory link between the synthesis of YeiK (a potential nucleoside hydrolase) and YeiL was inferred by showing that the yeiK and yeiL genes are divergently transcribed from overlapping promoters. A 10-15% deficiency in aerobic growth yield and an enhanced loss of viability under nitrogen starvation conditions were detected with a yeiL::kan(R) mutant, suggesting that YeiL might function as a post-exponential-phase nitrogen-starvation regulator.

Escherichia coli possesses a flavohemoglobin (Hmp), product of hmp, the first microbial globin gene to be sequenced and characterized at the molecular level. Although related proteins occur in numerous prokaryotes and eukaryotic microorganisms, the function(s) of these proteins have been elusive. Here we report construction of a defined hmp mutation and its use to probe Hmp function. As anticipated from up-regulation of hmp expression by nitric oxide (NO), S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents. The hmp promoter is more sensitive to SNP and S-nitroso-N-penicillamine (SNAP) than is the soxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species. Additional functions for Hmp are indicated by (a) parallel sensitivity of the hmp mutant to the redox-cycling agent, paraquat, (b) inability of the mutant to up-regulate fully the soxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c) failure of the mutant to accumulate reduced paraquat radical after anoxic growth. We conclude that Hmp plays a role in protection from nitrosating agents and NO-related species and oxidative stress. This protective role probably involves direct detoxification of those species and sensing of NO-related and oxidative stress.

The Escherichia coli flavohaemoglobin (Hmp) has a globin-like N-terminal domain and a ferredoxin-NADP-reductase-like C-terminal domain. We show here that purified Hmp oxidises both NADH and NADPH with K m values of 1.8 and 19.6 μM, respectively. Prolonged incubation of a hmp-lacZ fusion strain with the redox cycling agent paraquat resulted in a 28-fold induction of hmp gene expression, nearly 3-fold higher than after short periods of exposure. A strain overproducing Hmp was significantly more sensitive to paraquat than was the wild-type strain but, in vitro, purified Hmp was not an effective NADPH-paraquat diaphorase. Prolonged incubation of a wild-type strain with paraquat increased intracellular Hmp to spectrally detectable levels.

Summary: The ndh gene of Escherichia coli encodes the non-proton-translocating NADH dehydrogenase II. Expression of the ndh gene is subject to a complex network of regulatory controls at the transcriptional level. Under anaerobic conditions ndh is repressed by the regulator of fumarate and nitrate reduction (FNR). However, in the absence of FNR, ndh expression is activated by the amino acid response regulator (Arr) during anaerobic growth in rich medium. Expression of the ndh gene varies during the growth cycle in response to the intracellular concentration of the heat-stable DNA-binding protein, Fis. In this work two additional heat-stable proteins, integration host factor (IHF) and the histone-like protein HU were found to interact with the ndh promoter. IHF was shown to bind at three sites centred at +26, -17 and -58 in the ndh promoter (K d = 10−8 M), to prevent open-complex formation and to repress ndh transcription in vitro. Studies with an ndh-lacZ fusion confirmed that IHF represses ndh expression in vivo. Two putative binding sites for Arr, which overlap the two FNR boxes in the ndh promoter, were identified. Studies with the FNR-activated and amino-acid-inducible asparaginase II gene (ansB) showed that IHF and a component of the Arr-containing fraction (but not HU) interact with the corresponding ansB promoter.

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.

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, despite the …

Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene. A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the phi (hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source. Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression. Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation. Anaerobic, but not aerobic, expression of phi (hmp-lacZ)1 was stimulated three- to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter. Iron depletion of rich broth medium by the chelator 2'2'-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr. At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically. Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite. Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes. Nitric oxide (10 to 20 microM) stimulated aerobic phi (hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect. We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability. Hmp is implicated in reactions with small nitrogen compounds.

Summary The ndh gene that encodes the non-proton-translocating NADH dehydrogenase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins that may be associated with this activation have previously been detected, Arr (amino acid response regulator) and Nbp (ndh-bind'mg protein). Studies with the heat-stable Nbp have now shown that it is present in E. coli grown both aerobicaliy and anaerobically in rich and minimal media, indicating that it is not specifically associated with the anaerobic enhancement of ndh expression. The Nbp activity of aerobic cultures was maximal during exponential growth phase (when ndh promoter activity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor for inversion stimulation). Three major and two minor Nbp (Fis)-binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at -123, 72 and +51, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whereas at higher concentrations it prevented RNA polymerase (RNAP) binding and open complex formation. Consequently, Nbp (Fis) can both activate and repress transcription from the ndh promoter. The results suggest that Nbp (Fis) serves to ensure that the energetically efficient proton-translocating NADH dehydrogenase I is used in preference to the non-proton translocating NADH dehydrogenase II during periods of rapid growth, by repressing expression of the ndh gene.

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.

Plasmid-mediated quinolone resistance (PMQR) of enterobacteria encoded by qnr genes is an emerging concern in human and veterinary medicine. Here we aimed to study PMQR of porcine Escherichia coli in two large piggeries in Romania and Hungary. The studies identified PMQR E. coli strains in 34% of piglets in the Romanian farm. Clonality of six qnrS1 E. coli strains representing the Romanian pig farm was established by multilocus sequence typing (MLST), and the qnrS1 plasmids were characterized by plasmid transfer and PCR-based replicon typing. The six tested strains were assigned to three different MLST types. All proved to carry IncN plasmids, representing the first IncN-borne qnrS1 gene to be identified in E. coli from food-producing animals. DNA sequences flanking the qnrS1 gene showed ≥99% homology with the corresponding resistance region of the pINF5 plasmid from Salmonella Infantis isolated from chicken carcass and of IncN plasmids from human clinical E. coli strains. Thus, our data suggest that transfer of qnrS1 plasmids occurs between Salmonella and E. coli of animal and human origin, with pigs representing one of the potential reservoirs. Further, we report on identification and characterization of the qnrS1 gene in porcine E. coli for the first time in Europe.

Recent data from the European and Hungarian Antimicrobial Resistance Monitoring Systems have indicated that the routine use of gentamicin in human and veterinary medicine frequently leads to the selection of gentamicin resistance in Escherichia coli. The aim of this study was to provide molecular characterization of gentamicin resistance in clinical and commensal E. coli strains representing humans and food producing animals by genotyping for antimicrobial resistance and virulence using a miniaturized microarray. All 50 strains tested proved to be multidrug resistant defined as resistance to three or more antimicrobial classes. Antimicrobial resistances genes such as aadA1-like, strB, bla(TEM), sul1 and tet(A) or tet(B), and corresponding phenotypes (streptomycin-, ampicillin-, sulfamethoxazole- and tetracycline resistance) were detected in >50% of isolates regardless of the host or clinical background. However, certain genes encoding gentamicin resistance such as aac(6')-Ib and ant(2″)-Ia as well as catB3-like genes for phenicol resistance were only detected in human isolates. Among virulence genes, the increased serum survival gene iss was predominant in all host groups. Although the majority of gentamicin resistant E. coli strains were characterized by diverse antimicrobial resistance, and virulence gene patterns, accentuated links between catB3-like, aac(6')-Ib, bla(CTX-M-1) and sat genes could be detected in human strains. Further resistance/virulence gene associations (tet(A) with iroN and iss) were detected in poultry strains. In conclusion, the simultaneous characterization of antimicrobial resistance and virulence genotypes of representative clinical and commensal strains of E. coli should be useful for the identification of emerging genotypes with human and or animal health implications.