Rob Davies

Characterization of β-Lactamases Responsible for Resistance to Extended-Spectrum Cephalosporins in Escherichia coli and Salmonella enterica Strains from Food-Producing Animals in the United Kingdom

Nine epidemiologically unrelated isolates [1 Salmonella Bredeney from turkeys, and 8 Escherichia coli [3 environmental isolates (2 from chickens, 1 from pigs), and 5 isolates from cattle with neonatal diarrhea]] were examined both pheno- and genotypically for extended-spectrum beta-lactam (ESBL) resistance. Resistance phenotypes (ampicillin, aztreonam, cefotaxime, cefpodoxime, ceftazidime, and ceftriaxone) suggested the presence of an ESBL enzyme, but cefoxitin MICs (>/= 32 mg/L) suggested the presence of an AmpC-like enzyme. Synergism experiments with benzo(b)thiophene-2-boronic acid (BZBTH2B) and isoelectric focusing (IEF) revealed the presence of an AmpC beta-lactamase with a pI >/= 9. amp C multiplex PCR, sequence, and Southern analyses indicated that only the Salmonella isolate had a plasmid-encoded AmpC beta-lactamase CMY-2 on a nonconjugative 60-MDa plasmid. PCR and sequence analysis of the E. coli ampC promoter identified mutations at positions -88(T), -82(G), -42(T), -18(A), -1(T) and +58(T) in all the isolates. In addition one strain had two extra-mutations at positions +23(A) and +49(G), and another strain had one extra-mutation at position +32(A). DNA fingerprinting revealed that all the E. coli isolates were different clones. It also showed that the U.K. Salmonella isolate was indistinguisable from a Canadian Salmonella isolate from turkeys; both had identical resistance phenotypes and produced CMY-2. This is the first report of a CMY-2 Salmonella isolate in the United Kingdom. These data imply that beta-lactam resistance in animal isolates can be generated de novo as evidenced by the E. coli strains, or in the case of the Salmonella strains be the result of intercontinental transmission due to an acquired resistance mechanism.

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

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

Biosecurity Measures to Control Salmonella and Other Infectious Agents in Pig Farms: A Review

Salmonellosis is the 2nd most common cause of human bacterial food poisoning and can be acquired from meat or eggs, either via direct consumption or cross-contamination in the kitchen. The European Commission has set the criteria to control Salmonella infections within the poultry sector and it is proposed that the swine sector should follow. Pork is considered, after eggs, the major source of infection in humans in the EU, with Salmonella typhimurium, including monophasic strains, being frequently implicated. Good control measures at the farm level are likely to correspond with lower prevalence of Salmonella infection and, subsequently, a reduction of cross-contamination of carcasses processed at the slaughterhouse and a reduction in human salmonellosis. This review focuses on biosecurity measures in pig farms that can help to control important pig diseases at the same time as reducing the within-herd prevalence of Salmonella. This information is likely to provide an economic incentive for farmers to apply improved general standards of farm biosecurity and hygiene management that would have a positive impact in food safety.

Salmonella enterica Serovar Enteritidis, England and Wales, 1945–2011

A focus on eliminating phage type 4 in egg and poultry production has greatly reduced foodborne disease among humans.

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

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

Virulence Characterisation of Salmonella enterica Isolates of Differing Antimicrobial Resistance Recovered from UK Livestock and Imported Meat Samples

Salmonella enterica is a foodborne zoonotic pathogen of significant public health concern. We have characterized 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 h 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.

Whole genome sequencing reveals an outbreak of Salmonella Enteritidis associated with reptile feeder mice in the United Kingdom, 2012-2015

Analysis of whole genome sequencing data uncovered a previously undetected outbreak of Salmonella Enteritidis that had been on-going for four years. Cases were resident in all countries of the United Kingdom and 40% of the cases were aged less than 11 years old. Initial investigations revealed that 30% of cases reported exposure to pet snakes. A case-control study was designed to test the hypothesis that exposure to reptiles or their feed were risk factors. A robust case-definition, based on the single nucleotide polymorphism (SNP) profile, increased the power of the analytical study. Following univariable and multivariable analysis, exposure to snakes was the only variable independently associated with infection (Odds ratio 810 95% CI (85-7715) pxa0<xa00.001). Isolates of S. Enteritidis belonging to the outbreak profile were recovered from reptile feeder mice sampled at the retail and wholesale level. Control measures included improved public health messaging at point of sale, press releases and engagement with public health and veterinary counterparts across Europe. Mice destined to be fed to reptiles are not regarded as pet food and are not routinely tested for pathogenic bacteria. Routine microbiological testing to ensure feeder mice are free from Salmonella is recommended.

Efficacy of disinfectants and detergents intended for a pig farm environment where Salmonella is present

Disinfection is a useful component of disease control, although products and chemical groups vary in their activity against different pathogens. This study investigated the ability of fifteen disinfectants to eliminate pig-associated Salmonella. Active compounds of products included chlorocresol, glutaraldehyde/formaldehyde, glutaraldehyde/quaternary ammonium compounds (QAC), iodine, peracetic acid and potassium peroxomonosulphate. Six detergents were also tested for their ability to dislodge faecal material, and interactions with specific disinfectants. Eight serovars were screened against all products using dilution tests and a monophasic Salmonella Typhimurium strain was selected for further testing. The disinfectants were tested using models to replicate boot dip (faecal suspension) and animal housing (surface contamination) disinfection respectively at the Department for Environment, Food and Rural Affairs Approved Disinfectant General Orders (GO) concentration, half GO and twice GO. Stability over time and ability to eliminate Salmonella in biofilm was also assessed. The most effective products were then field tested. Most products at GO concentration eliminated Salmonella in the faecal suspension model. One glutaraldehyde/QAC and one glutaraldehyde/formaldehyde-based product at GO concentration eliminated Salmonella in the surface contamination model. Chlorocresol-based products were more stable in the faecal suspension model. One chlorocresol and the glutaraldehyde/formaldehyde-based product were most successful in eliminating Salmonella from biofilms. All products tested on farm reduced bacterial log counts; the glutaraldehyde/QAC based product produced the greatest reduction. The type of product and the application concentration can impact on efficacy of farm disinfection; therefore, clearer guidance is needed to ensure the appropriate programmes are used for specific environments.

Observations on the introduction and dissemination of Salmonella in three previously low prevalence status pig farms in the United Kingdom

In the United Kingdom a serological Salmonella surveillance scheme was run in pigs up to 2012. Farms that maintained a low seroprevalence (<10%) were recognised as Platinum pig farms. The aim of this study was to investigate the occurrence and distribution of Salmonella in three farms (17P, 18P and 46P) that had lost their Platinum status. Four visits to each farm were made over a period of 15 months. The sampling was carried out by collecting pooled pen floor faecal swab and environmental samples. All samples were tested for Salmonella by a modification of ISO6579 Annex D, and serovars were determined for all isolates. The Salmonella prevalence peaked in the Summer/Autumn months and all farms were still positive at the end of the study. The overall sample prevalence was higher in farm 17P (46%) and 18P (35%) than 46P (19%). Monophasic S. Typhimurium (mST) represented 77.8% of the Salmonella isolates, mainly from farms 17P and 46P. The mST isolated at the initial visit may have been introduced via other livestock present on farm or introduction into the herd of infected animals. The results of this study suggest that incursion of mST was likely to be the main cause of the loss of Platinum status and confirm that mST can persist in pigs and their environment.

Observations on Salmonella contamination of commercial duck farms before and after cleaning and disinfection

ABSTRACT In the European Union, statutory control of Salmonella is in place in the chicken and turkey sectors, but not in the duck sector. In this study, 14 Salmonella-positive duck farms were sampled before and after cleaning and disinfection, and once the houses had been restocked with a new flock. The cleaning and disinfection programmes used were subdivided into two main categories: ones in which a final formaldehyde disinfection step was included (1) and ones in which it was not included (2). Several types of samples were collected during the study, and faecal samples were those more frequently positive (62% of faecal samples were positive for Salmonella in comparison to 2–23% of samples from all the other sample categories) (Pu2009<u20090.001). Independently of the cleaning and disinfection programme used, there was a statistically significant (Pu2009<u20090.001) reduction in the percentage of Salmonella-positive samples between before cleaning and disinfection (41.1%) and after cleaning and disinfection (3.1%). After restocking, the number of Salmonella-positive samples increased significantly (Pu2009<u20090.001), with 65.3% of the samples tested being positive for Salmonella. Farms in which disinfection programme 1 was used were 5.34 times less likely to have samples positive for Salmonella after cleaning and disinfection than farms which implemented programme 2. Formaldehyde acts effectively against Salmonella even in the presence of some residual organic matter. Limited residual contamination on farms after cleaning and disinfection represents a risk of infection for young ducklings, and thorough cleaning and disinfection procedures should be implemented to reduce the carry-over of infection between flocks.