Search

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.

Antimicrobial resistance (AMR) is a major public health threat. Plasmids are able to transfer AMR genes among bacterial isolates. Whole genome sequencing (WGS) is a powerful tool to monitor AMR determinants. However, plasmids are difficult to reconstruct from WGS data. This study aimed to improve the characterization, including the localization of AMR genes using short and long read WGS strategies. We used a genetically modified (GM) Bacillus subtilis isolated as unexpected contamination in a feed additive, and therefore considered unauthorized (RASFF 2014.1249), as a case study. In GM organisms, AMR genes are used as selection markers. Because of the concern of spread of these AMR genes when present on mobile genetic elements, it is crucial to characterize their location. Our approach resulted in an assembly of one chromosome and one plasmid, each with several AMR determinants of which five are against critically important antibiotics. Interestingly, we found several plasmids, containing AMR genes, integrated in the chromosome in a repetitive region of at least 53 kb. Our findings would have been impossible using short reads only. We illustrated the added value of long read sequencing in addressing the challenges of plasmid reconstruction within the context of evaluating the risk of AMR spread.

Carbapenemase-producing Enterobacteriaceae (CPE) confer resistance to antibiotics that are of critical importance to human medicine. There have only been a few reported cases of CPEs in the European food chain. We report the first detection of a carbapenemase-producing Escherichia coli (ST 5869) in the Belgian food chain. Our aim was to characterize the origin of the carbapenem resistance in the E. coli isolate. The isolate was detected during the screening of 178 minced pork samples and was shown to contain the carbapenemase gene bla

OBJECTIVES The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted β-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP). METHODS A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(®) platform. We designed 46 xTAG(®)-compatible probes targeting β-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates. RESULTS The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained

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

A collection of 105 colistin-resistant Salmonella isolates collected from 2012 to 2015 in the national surveillance program in Belgium was screened by PCR for the presence of genes mcr-1 and mcr-2. Of these, 1.90% (2/105) and 0.95% (1/105) tested positive for mcr-1 and mcr-2, respectively. The presence of the mcr-1 or mcr-2 determinant has been confirmed by whole genome sequencing and allowed the localization of these two genes on IncX4 type plasmids. We report here the presence of mcr-1 and the first mcr-2 gene in Salmonella ever isolated in the Belgian food chain. Although present at retail since 2012, the occurrence is low and sporadic.

Staphylococcus aureus clonal complex 398 (CC398) isolates colonize livestock and can spread to human contacts. Genetic analysis of isolates epidemiologically associated with human-to-human, but not livestock, transmission in multiple countries and continents identified a common clade that was negative for tet (M) and positive for bacteriophage ϕ3. Another group of human-to-human-transmitted isolates belonged to the common livestock-associated clade but had acquired a unique ϕ7 bacteriophage.

SUMMARYThe prevalence and risk factors associated with livestock-associated MRSA (LA-MRSA) carriage was examined in Danish and Belgian veterinarians. The MRSA and LA-MRSA carriage rates were 9·5% (95% CI 5·3–15·6) and 7·5% (95% CI 3·8–13·1) for MRSA and LA-MRSA, respectively, in Belgium and 1·4% (95% CI: 0·17–5·05) in Denmark (all Danish MRSA isolates belonged to the LA-MRSA genotype). All LA-MRSA isolates were resistant to tetracycline and 53·4% (7/13) showed a multi-resistant phenotype. LA-MRSA was significantly associated with veterinarians in contact with livestock (P=0·046). In the multivariable analysis, working with small animals in a veterinary clinic seems to be negatively associated (OR 0·15, 95% CI 0–1·0,P=0·05) and a strong direct association was found for LA-MRSA acquisition and exposure to live pigs (OR 12·1, 95% CI 1·6–548·5,P=0·01). Since carriage of MRSA ST398 may increase the risk of complications during hospitalization, our results underline that preventive measures may need to be developed for veterinary professionals, particularly for livestock veterinarians.