Application of a strain-level shotgun metagenomics approach on food samples: resolution of the source of a Salmonella food-borne outbreak.

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.

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.

Evaluation of the ISO 10273:2003 method for the isolation of human pathogenic Yersinia enterocolitica from pig carcasses and minced meat.

Pig carcass swabs (n = 254) and minced meat samples (n = 82) were examined for pathogenic Yersinia enterocolitica using different routinely used enrichment protocols. All samples were obtained in the context of the official Yersinia monitoring program in Belgium. In total, 28 carcasses (11.0%) were contaminated with Y. enterocolitica bioserotype 4/O:3 and one (0.4%) with bioserotype 2/O:9. Four minced meat samples (4.9%) tested positive for Y. enterocolitica bioserotype 4/O:3. Using the ISO 10273:2003 method, eight out of the 29 Yersinia-positive carcasses (27.6%) and none of the contaminated minced meat samples (0.0%) were detected. Reducing the enrichment time in PSB from 5 to 2 days increased the number of positive samples. Overall, enrichment in PSB at 25 °C recovered more positive carcasses and minced meat samples than selective enrichment and cold enrichment. As the exclusive use of the ISO 10273:2003 method results in a strong underestimation of Y. enterocolitica positive carcasses and minced meats, efforts are needed to optimize the current version of the ISO method. In addition, isolation of pathogenic Y. enterocolitica requires experience and the use of a stereomicroscope to avoid false negative results.

Trend analysis of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from Belgian pork and poultry meat products using surveillance data of 2004-2009.

The purpose of this study was to analyze and compare antimicrobial resistance in Campylobacter spp. isolated from pork and poultry carcasses, and pork and poultry meat (at slaughterhouse level, during meat cutting, and at retail) in Belgium, using available surveillance data over the period 2004-2009. The susceptibilities of 1724 Campylobacter isolates for ampicillin, ciprofloxacin, nalidixic acid, tetracycline, erythromycin, and gentamicin were tested by E-test. Gentamicin resistance was low (near 0%) until 2007, with an increase to over 20% by 2009 for all species-matrix combinations. Resistance to tetracycline fluctuated around the same level during the entire study period and was significantly higher (p-value of <0.05) in C. coli than in C. jejuni. Erythromycin resistance was low and showed a slight decrease between 2004 and 2007, but increased from 2007 until 2009. Fluoroquinolone and ampicillin resistance was significantly higher in isolates derived from poultry, compared to pork-related isolates. This correlates with the higher use of these antimicrobials in poultry husbandry. A total of 25% of C. coli isolates from poultry showed the most apparent multiresistance (resistance to four or more antimicrobials). Approximately 1% of the poultry-derived isolates (both C. coli and C. jejuni) showed resistance to all tested antimicrobials, while none was found in pork products.

Lessons learned from the management of a national outbreak of Salmonella ohio linked to pork meat processing and distribution.

During the summer of 2005, an increase in reports of human cases of Salmonella enterica serovar Ohio infection was observed in Belgium. During 11 weeks, between 1 July and 13 September, 60 cases of laboratory-confirmed Salmonella Ohio infection were reported to the National Reference Centre for Salmonella, with a peak onset of symptoms in the third week of July. All clinical isolates caused self-limiting gastroenteritis; both genders (32 males and 28 females) and all age groups (three children <5 years of age, three children 5 to 14 years of age, 32 adults 15 to 64 years of age, and 22 adults >65 years of age) were affected. The isolates were distributed throughout Belgium but a cluster of several cases was observed around Brussels. At the same time, an increase in the incidence of this serovar was observed in the Salmonella isolates originating from the official surveillance campaign conducted by the Federal Agency for the Safety of the Food Chain, which identified pork as a likely source of the outbreak strain. Pulsed-field gel electrophoresis typing confirmed the clonal relationship between the human isolates, the isolates from samples collected in the cutting plants, and the isolates from pork meat in distribution. Further epidemiological investigations indicated that one particular slaughterhouse was involved. In that slaughterhouse, the carcasses were contaminated during the evisceration process because of contaminated equipment and uncontrolled environmental conditions. This study highlights the importance of a centralized surveillance laboratory in the management of outbreaks and the need of strict implementation of hygienic rules to avoid this type of outbreak.