Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Predictive Power of Long-Read Whole-Genome Sequencing for Rapid Diagnostics of Multidrug-Resistant Brachyspira hyodysenteriae Strains.

Infections with Brachyspira hyodysenteriae, the etiological agent of swine dysentery, result in major economic losses in the pig industry worldwide. Even though microbial differentiation of various Brachyspira species can be obtained via PCR, no quick diagnostics for antimicrobial susceptibility testing are in place, which is mainly due to the time-consuming (4 to 7 days) anaerobic growth requirements of these organisms. Veterinarians often rely on a clinical diagnosis for initiating antimicrobial treatment. These treatments are not always effective, which may be due to high levels of acquired resistance in B. hyodysenteriae field isolates. By using long-read-only whole-genome sequencing and a custom-trained Bonito base-calling model, 81 complete B. hyodysenteriae genomes with median Q51 scores and 99% completeness were obtained from 86 field strains. This allowed the assessment of the predictive potential of genetic markers in relation to the observed acquired resistance phenotypes obtained via agar dilution susceptibility testing. Multidrug resistance was observed in 77% and 21% of the tested strains based on epidemiological cutoff and clinical breakpoint values, respectively. The predictive power of genetic hallmarks (genes and/or gene mutations) for antimicrobial susceptibility testing was promising. Sensitivity and specificity for tiamulin [tva(A) and 50SL3N148S, 99% and 67%], valnemulin [tva(A), 97% and 92%), lincomycin (23SA2153T/G and lnuC, 94% and 100%), tylvalosin (23SA2153T/G, 99% and 93%), and doxycycline (16SG1026C, 93% and 87%) were determined. The predictive power of these genetic hallmarks is promising for use in sequencing-based workflows to speed up swine dysentery diagnostics in veterinary medicine and determine proper antimicrobial use. IMPORTANCE Diagnostics for swine dysentery rely on the identification of Brachyspira species using molecular techniques. Nevertheless, no quick diagnostic tools are available for antimicrobial susceptibility testing due to extended growth requirements (7 to 14 days). To enable practitioners to tailor antimicrobial treatment to specific strains, long-read sequencing-based methods are expected to lead to rapid methods in the future. Nevertheless, their potential implementation should be validated extensively. This mainly implies assessing sequencing accuracy and the predictive power of genetic hallmarks in relation to their observed (multi)resistance phenotypes.

Assessing evidence of a potential Salmonella transmission across the poultry food chain.

Enhanced Salmonella surveillance programmes in poultry were implemented in all European Member States, with minimum prevalence targets for a list of targeted serotypes to safeguard food and public health. Based on the Belgian Salmonella surveillance programme and focusing on poultry, the overarching aim of this study was to highlight possible Salmonella transmissions across the food chain (FC). For this purpose, firstly, the prevalence patterns of Salmonella (targeted and the most prevalent non-targeted) serotypes along the FC were described over time. Secondly, the effectiveness of the control measures against vertical transmission (breeders to 1-day-old broiler and layer chicks) was indirectly assessed by looking into the odds of targeted serotypes detection. Thirdly, it was appraised if Salmonella prevalence can significantly increase during broilers and layers production. In addition, it was tested if being tested negative at the end of production in broilers when tested positive at the entrance is serotype dependent (targeted vs. non-targeted serotypes). Results showed that, firstly, the prevalence patterns of the listed serotypes were inconstant over time and across the FC. Secondly, the odds of Salmonella targeted serotype detection in 1-day-old broiler and in 1-day-old layer flocks were lower than in breeder flocks while, thirdly, infection during broiler and layer production can lead to significant increase in positivity in subsequent samples. Finally, being infected by a targeted or by non-targeted serotype at the entrance of the flock poorly reflects the Salmonella status at the end of production. Note that this study did not make a distinction between the different sources of contamination and the effects of sampling methods and isolation methods should be subject to further investigation.

Potential of ESBL-producing Escherichia coli selection in bovine feces after intramammary administration of first generation cephalosporins using in vitro experiments.

Selection and spread of Extended Spectrum Beta-Lactamase (ESBL) -producing Enterobacteriaceae within animal production systems and potential spillover to humans is a major concern. Intramammary treatment of dairy cows with first-generation cephalosporins is a common practice and potentially selects for ESBL-producing Enterobacteriaceae, although it is unknown whether this really occurs in the bovine fecal environment. We aimed to study the potential effects of intramammary application of cephapirin (CP) and cefalonium (CL) to select for ESBL-producing Escherichia coli in the intestinal content of treated dairy cows and in manure slurry, using in vitro competition experiments with ESBL and non-ESBL E. coli isolates. No selection of ESBL-producing E. coli was observed at or below concentrations of 0.8 µg/ml and 4.0 µg/ml in bovine feces for CP and CL, respectively, and at or below 8.0 µg/ml and 4.0 µg/ml, respectively, in manure slurry. We calculated that the maximum concentration of CP and CL after intramammary treatment with commercial products will not exceed 0.29 µg/ml in feces and 0.03 µg/ml in manure slurry. Therefore, the results of this study did not find evidence supporting the selection of ESBL-producing E. coli in bovine feces or in manure slurry after intramammary use of commercial CP or CL-containing products.

First detection of a plasmid located carbapenem resistant blaVIM-1 gene in E. coli isolated from meat products at retail in Belgium in 2015.

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 blaVIM-1 by PCR and Sanger sequencing. Whole genome short and long read sequencing (MiSeq and MinION) was performed to characterize the isolate. With a hybrid assembly we reconstructed a 190,205 bp IncA/C2 plasmid containing blaVIM-1 (S15FP06257_p), in addition to other critically important resistance genes. This plasmid showed only low similarity to plasmids containing blaVIM-1 previously reported in Germany. Moreover, no sequences existed in the NCBI nucleotide database that completely covered S15FP06257_p. Analysis of the blaVIM-1 gene cassette demonstrated that it likely originated from an integron of a Klebsiella plasmid reported previously in a clinical isolate in Europe, suggesting that the meat could have been contaminated by human handling in one of the steps of the food chain. This study shows the relevance of fully reconstructing plasmids to characterize their genetic content and to allow source attribution. This is especially important in view of the potential risk of antimicrobial resistance gene transmission through mobile elements as was reported here for the of public health concern blaVIM-1.

Antimicrobial resistance patterns and molecular resistance markers of Campylobacter jejuni isolates from human diarrheal cases.

The aim of this study is to characterize the antimicrobial resistance of Campylobacter jejuni recovered from diarrheal patients in Belgium, focusing on the genetic diversity of resistant strains and underlying molecular mechanisms of resistance among Campylobacter jejuni resistant strains isolated from diarrheal patients in Belgium. Susceptibility profile of 199 clinical C. jejuni isolates was determined by minimum inhibitory concentrations against six commonly-used antibiotics (ciprofloxacin, nalidixic acid, tetracycline, streptomycin, gentamicin, and erythromycin). High rates of resistance were observed against nalidixic acid (56.3%), ciprofloxacin (55.8%) and tetracycline (49.7%); these rates were similar to those obtained from different national reports in broilers intended for human consumption. Alternatively, lower resistance rates to streptomycin (4.5%) and erythromycin (2%), and absolute sensitivity to gentamicin were observed. C. jejuni isolates resistant to tetracycline or quinolones (ciprofloxacin and/or nalidixic acid) were screened for the presence of the tetO gene and the C257T mutation in the quinolone resistance determining region (QRDR) of the gyrase gene gyrA, respectively. Interestingly, some of the isolates that displayed phenotypic resistance to these antimicrobials lacked the corresponding genetic determinants. Among erythromycin-resistant isolates, a diverse array of potential molecular resistance mechanisms was investigated, including the presence of ermB and mutations in the 23S rRNA gene, the rplD and rplV ribosomal genes, and the regulatory region of the cmeABC operon. Two of the four erythromycin-resistant isolates harboured the A2075G transition mutation in the 23S rRNA gene; one of these isolates exhibited further mutations in rplD, rplV and in the cmeABC regulatory region. This study expands the current understanding of how different genetic determinants and particular clones shape the epidemiology of antimicrobial resistance in C. jejuni in Belgium. It also reveals many questions in need of further investigation, such as the role of other undetermined molecular mechanisms that may potentially contribute to the antimicrobial resistance of Campylobacter.

Validation of EN ISO method 10273 - Detection of pathogenic Yersinia enterocolitica in foods.

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ILS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52-92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on CIN was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.

Molecular epidemiology and antimicrobial resistance mechanisms of Campylobacter coli from diarrhoeal patients and broiler carcasses in Belgium.

The aim of this study was to better understand the molecular epidemiology of Campylobacter coli isolated from multiple sources in Belgium, by studying the genotypic diversity and antimicrobial resistance phenotypes and resistance mechanisms of 59 C. coli isolates. Isolates from broiler carcasses and human cases were genotyped using multilocus sequence typing (MLST), porA typing, flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, and by PCR binary typing (P-BIT). Thirty-two MLST sequence types, 24 flaA types, 31 porA alleles, and 29 P-BIT types were identified among the screened isolates. Some types and alleles were shared among strains recovered from both broiler carcasses and diarrhoeal patients. Both porA typing and MLST revealed a similar discriminatory power (0.969), which was the highest discriminatory power when compared to other methods. Minimum inhibitory concentrations against seven different antibiotics (ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, tetracycline, gentamicin, and erythromycin) were analysed. Strains were most frequently resistant to tetracycline (81.4%), followed by: ciprofloxacin and nalidixic acid (76.3%); streptomycin (33.9%); erythromycin (27.1%); and chloramphenicol (3.4%). All isolates were sensitive to gentamicin. Multidrug resistance was observed in 24 of 59 C. coli isolates (40.7%). Molecular screening of antimicrobial resistance mechanisms revealed the predominance of the gyrA T86I substitution among ciprofloxacin- and nalidixic acid-resistant isolates, the tet(O) gene among tetracycline-resistant isolates and the 23S rRNA A2075G mutation among erythromycin- resistant isolates. Furthermore, some erythromycin-resistant isolates harboured a diverse array of resistance mechanisms, including the presence of ermB, 23S rRNA A2074G mutation, and point mutations the rplD and rplV ribosomal genes, and the cmeABC multidrug efflux pump genes.

Genetic Basis and Clonal Population Structure of Antibiotic Resistance in Campylobacter jejuni Isolated From Broiler Carcasses in Belgium.

Human campylobacteriosis is the leading food-borne zoonosis in industrialized countries. This study characterized the clonal population structure, antimicrobial resistance profiles and occurrence of antimicrobial resistance determinants of a set of Campylobacter jejuni strains isolated from broiler carcasses in Belgium. Minimum inhibitory concentrations (MICs) against five commonly-used antibiotics (ciprofloxacin, nalidixic acid, tetracycline, gentamicin, and erythromycin) were determined for 204 C. jejuni isolates. More than half of the isolates were resistant to ciprofloxacin or nalidixic acid. In contrast, a lower percentage of screened isolates were resistant to gentamicin or erythromycin. C. jejuni isolates resistant to ciprofloxacin and/or nalidixic acid were screened for the substitution T86I in the quinolone resistance determining region (QRDR) of the gyrA gene, while C. jejuni isolates resistant to tetracycline were screened for the presence of the tet(O) gene. These resistance determinants were observed in most but not all resistant isolates. Regarding resistance to erythromycin, different mutations occurred in diverse genetic loci, including mutations in the 23S rRNA gene, the rplD and rplV ribosomal genes, and the intergenic region between cmeR and cmeABC. Interestingly, and contrary to previous reports, the A2075G transition mutation in the 23S rRNA gene was only found in one strain displaying a high level of resistance to erythromycin. Ultimately, molecular typing by multilocus sequence typing revealed that two sequence types (ST-824 and ST-2274) were associated to quinolones resistance by the presence of mutations in the gene gyrA (p = 0.01). In addition, ST-2274 was linked to the CIP-NAL-TET-AMR multidrug resistant phenotype. In contrast, clonal complex CC-45 was linked to increased susceptibility to the tested antibiotics. The results obtained in this study provide better understanding of the phenotypic and the molecular basis of antibiotic resistance in C. jejuni, unraveling some the mechanisms which confer antimicrobial resistance and particular clones associated to the carriage and spread of resistance genes.

Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness.

Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006-2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly associated with clinical infections occurring after travelling abroad. Ultimately, the data generated from this study will help to better understand the molecular epidemiology of C. jejuni infection.

Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system.

A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

Molecular surveillance of norovirus, 2005-16: an epidemiological analysis of data collected from the NoroNet network.

BACKGROUND: The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. METHODS: We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. FINDINGS: We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2-3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16-GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17-GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015-16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2-53·9], as did GII.17 [median 6·3%, range 0-44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe-GII.4 or GII.P4-GII.4). INTERPRETATION: Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. FUNDING: European Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

Detection of Plasmid-Mediated Colistin Resistance, mcr-1 and mcr-2 Genes, in Salmonella spp. Isolated from Food at Retail in Belgium from 2012 to 2015.

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.

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection.

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.

Multicenter evaluation of the revised RIDA® QUICK test (N1402) for rapid detection of norovirus in a diagnostic laboratory setting.

The updated RIDA® QUICK (N1402) immunochromatographic assay (R-Biopharm) for detection of norovirus was evaluated during a prospective, multicenter study using 771 stool samples from patients with gastroenteritis. Compared to real-time reverse transcriptase polymerase chain reaction (RT-rtPCR) as gold standard, the RIDA® QUICK had an overall sensitivity of 72.8% (91/125) and a specificity of 99.5% (640/643). Genotype analysis of the polymerase (ORF1) and capsid (ORF2) region of the genome indicated that the RIDA® QUICK assay could detect a broad range of genotypes including new variants (15 of 125 positive samples) which were detected by an in-house SYBR®Green RT-rtPCR, but not by the RIDA® GENE PCR PG1415 (R-Biopharm) and mostly not by the RIDA® GENE PCR PG1405 and the Xpert® Norovirus assay (Cepheid). The RIDA® QUICK can be used to reliably confirm norovirus in stool samples, but a negative result does not definitively exclude the presence of norovirus.

Development of a Luminex xTAG® assay for cost-effective multiplex detection of ß-lactamases in Gram-negative bacteria.

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 ß-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 ß-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 <€5 per sample. CONCLUSIONS: We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.

Discriminative power of Campylobacter phenotypic and genotypic typing methods.

The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpson's index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass.

Evaluation of detection methods for non-O157 Shiga toxin-producing Escherichia coli from food.

Shiga toxin-producing Escherichia coli (STEC) remains a major foodborne pathogen of concern across the globe. Rapid detection and isolation of this pathogen is of great importance for public health reasons. In this study the detection and isolation of four non-O157 STEC strains (O26, O103, O111, O145) from different artificially contaminated matrices, namely ground (minced) beef, cattle carcass swab, lettuce mix and sprouted soy beans, were evaluated. Low amounts of STEC were used (0.25-1.40 cfu/g) to spike the samples. All samples were enriched in parallel in Buffered Peptone Water (BPW) and Brila broth. After enrichment, detection was performed using real-time PCR (qPCR), and isolation using two chromogenic agar media, CHROMagar™ STEC and ChromID™ EHEC. Inoculation on the agar media was performed either directly after enrichment or after the use of an acid treatment procedure. Furthermore, the use of this procedure was also tested on naturally contaminated food products, using 150 stx-positive samples. Although the qPCR Cycle Threshold (Ct) values were lower after enrichment in Brila broth, no significant differences in recovery were observed between both enrichment broths. Both agar media were equally suitable for the isolation of STEC, although a significantly higher recovery was obtained when using both agar media in parallel. For samples with a Ct value above 25, an acid treatment step prior to isolation ensured a significant improvement in the recovery of STEC due to the reduction in background microbiota. This acid treatment procedure proved especially useful for the isolation of STEC from sprouted soy bean samples.

Comparison of sample types and analytical methods for the detection of highly campylobacter-colonized broiler flocks at different stages in the poultry meat production chain.

Exclusion of broiler batches, highly colonized with Campylobacter (>7.5 log10 colony-forming units/g), from the fresh poultry meat market might decrease the risk of human campylobacteriosis. The objective of this study was to compare different sample types (both at the farm and the slaughterhouse) and methods (direct culture, quantitative real-time polymerase chain reaction [qPCR], propidium monoazide [PMA]-qPCR) applied for the quantification of the Campylobacter colonization level. In addition, the applicability of the lateral flow-based immunoassay, Singlepath(®) Direct Campy Poultry test (Singlepath(®) test), was evaluated as a rapid method for the qualitative detection of Campylobacter in highly colonized broiler batches. Campylobacter counts differed significantly between sample types collected at farm level (cecal droppings, feces, boot swabs) and at slaughterhouse level (cecal content, fecal material from crates). Furthermore, comparison of Campylobacter counts obtained by different methods (direct culture, qPCR, PMA-qPCR) in cecal droppings revealed significant differences, although this was not observed for cecal-content samples. Evaluation of the Singlepath(®) test on cecal droppings and cecal-content samples revealed an acceptable level of sensitivity and specificity. In conclusion, cecal droppings and cecal content are proposed as the most representative sample types for quantification of Campylobacter colonization level of broilers at farm and slaughterhouse, respectively. Direct culture and qPCR are equally sensitive for quantification of Campylobacter in fresh cecal-content samples. PMA treatment before qPCR inhibits the signal from dead Campylobacter cells. Consequently, when samples are extensively stored and/or transported, qPCR is preferred to direct culture and PMA-qPCR. Furthermore, the Singlepath(®) test offers a convenient alternative method for rapid detection of Campylobacter in highly colonized broiler batches.