Javier Nunez-Garcia

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla CTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla CTX-M-group-1 and bla OXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, bla OXA-1-like and bla CTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

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

OBJECTIVES The 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. METHODS Phenotypic 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. RESULTS Two 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. CONCLUSIONS Identification 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.

Mycobacterium bovis-BCG vaccination induces specific pulmonary transcriptome biosignatures in mice.

Background In the present study, we applied microarray technology to define biosignatures by microarray transcriptome analysis in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, these biosignatures should be detectable without in vitro antigenic challenge. Principal Findings After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection related to neutrophil biology and inflammation. Conclusions This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are potentially associated with protective immune responses that will be useful to evaluate future vaccine candidates.

mcr-1 and mcr-2 (mcr-6.1) variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015

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.

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses.

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport.

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized l-histidine, l-glutamine, l-proline, l-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin

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 feces 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 harboring 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.

Meta-analyses of the sensitivity and specificity of ante-mortem and post-mortem diagnostic tests for bovine tuberculosis in the UK and Ireland

Bovine Tuberculosis (bTB) in cattle is a global health problem and eradication of the disease requires accurate estimates of diagnostic test performance to optimize their efficiency. The objective of this study was, through statistical meta-analyses, to obtain estimates of sensitivity (Se) and specificity (Sp), for 14 different ante-mortem and post-mortem diagnostic tests for bTB in cattle. Using data from a systematic review of the scientific literature (published 1934-2009) diagnostic Se and Sp were estimated using Bayesian logistic regression models adjusting for confounding factors. Random effect terms were used to account for unexplained heterogeneity. Parameters in the models were implemented using Markov Chain Monte Carlo (MCMC), and posterior distributions for the diagnostic parameters with adjustment for covariates (confounding factors) were obtained using the inverse logit function. Estimates for Se and/or Sp of the tuberculin skin tests and the IFN-γ blood test were compared with estimates published 2010-2015. Median Se for the single intradermal comparative cervical tuberculin skin (SICCT) test (standard interpretation) was 0.50 and Bayesian credible intervals (CrI) were wide (95% CrI 0.26, 0.78). Median Sp for the SICCT test was 1.00 (95% CrI 0.99, 1.00). Estimates for the IFN-γ blood test Bovine Purified Protein Derivative (PPD)-Avian PPD and Early Secreted Antigen target 6 and Culture Filtrate Protein 10 (ESAT-6/CFP10) ESAT6/CFP10 were 0.67 (95% CrI 0.49, 0.82) and 0.78 (95% CrI 0.60, 0.90) respectively for Se, and 0.98 (95% CrI 0.96, 0.99) and 0.99 (95% CrI 0.99, 1.00) for Sp. The study provides an overview of the accuracy of a range of contemporary diagnostic tests for bTB in cattle. Better understanding of diagnostic test performance is essential for the design of effective control strategies and their evaluation.

Livestock-Associated Methicillin Resistant Staphylococcus aureus (LA-MRSA) Clonal Complex (CC) 398 Isolated from UK Animals belong to European Lineages

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 φAVβ 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.

Differential transcriptome profiles of attenuated and hypervirulent strains of Mycobacterium bovis

The identification of factors involved in the interaction of Mycobacterium bovis with the hosts will lead to new strategies to control bovine tuberculosis. In this study we compared the transcriptional profile of an attenuated M. bovis strain and a virulent M. bovis strain as a means to elucidate the molecular basis for their differential phenotype. Microarray and RT-qPCR results demonstrated that the expression of mce4D, Mb2607/Mb2608 and Mb3706c were up-regulated in the virulent strain whereas alkB, Mb3277c and Mb1077c were expressed at higher levels in the attenuated strain. These differential expression profiles were confirmed for Mb2607/Mb2608, mce4D, Mb1077c, alkB and Mb3277c during the replication of bacteria inside macrophages.