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.

Foodborne pathogens in unpasteurized milk in Sweden.

Raw milk may be a risk for public health if it is contaminated with zoonotic pathogens. To study the prevalence in unpasteurized milk from Swedish farms, bovine and small ruminant dairy farms were sampled. Since the sampling method and transport conditions may influence the outcome of analyses, efforts were made to optimize the methodology. Culturing of bacteria was done from in-line milk filters collected from the milk pipe at the point where it enters the milk bulk tank at the farms and this way of sampling was compared to sampling bulk tank milk (BTM) directly. Analysing milk filters were found to be superior to analysing BTM directly. Conditions for transport of milk filter samples were further improved by the addition of Cary Blair transport medium, which significantly increased the number of positive samples for pathogenic bacteria. The isolation of several foodborne pathogens from milk filters was demonstrated. The prevalence of samples with Staphylococcus aureus was 71% and 64%, and Listeria spp. 21% and 29% from dairy cow and goat/sheep farms, respectively. Campylobacter jejuni, Yersinia enterocolitica and verotoxigenic Escherichia coli (VTEC) O157 were detected in 9%, 2% and 2% of samples from bovine milk, respectively. We conclude that the choice of sampling method and sample handling influence the results of bacterial culturing. From the results of this study, we strongly recommend to sample in-line milk filters instead of BTM directly and to use Cary Blair medium during transport, especially if the samples are to be analysed for Campylobacter spp. and/or Listeria spp. The findings also show that unpasteurized milk from Swedish farms occasionally contain bacteria with zoonotic potential.

The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis.

Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n=354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.

Network Experiences from a Cross-Sector Biosafety Level-3 Laboratory Collaboration: A Swedish Forum for Biopreparedness Diagnostics.

The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness.

A nationwide outbreak of listeriosis associated with cold-cuts, Sweden 2013-2014.

In January 2014, the Public Health Agency of Sweden noticed an increase in listeriosis cases. Isolates from 10 cases had identical pulsed field gel electrophoresis (PFGE) profiles, suggesting a common source. We investigated the outbreak to identify the source and stop transmission. We looked for cases in 2013-2014 and also compared cases notified after February 2014 to randomly selected controls. We surveyed food items consumed two weeks prior to symptom onset. Listeria monocytogenes isolates found by food producers were PFGE-typed. Patient and food isolates with the outbreak PFGE profile were whole-genome sequenced and 51 cases with identical PFGE profile were identified; 12/20 cases and 108/186 controls responded to the survey. All cases were exposed to cold-cuts, compared with 72% of controls (p = 0.034). Five isolates of L. monocytogenes with the outbreak PFGE profile were found in cold-cuts from a food producer which stopped production in February 2014, but cases appeared until October 2014. Whole-genome sequencing showed that cold-cut and patient isolates differed by eight single nucleotide polymorphisms. Three patient isolates differed more and were probably not part of the outbreak. Epidemiological and microbiological results indicated cold-cuts as a possible source of the outbreak.

Foodborne Bacterial Pathogens in Retail Prepacked Ready-to-Eat Mixed Ingredient Salads.

Prepacked ready-to-eat mixed ingredient salads (RTE salads) are readily available whole meals that include a variety of ingredients such as raw vegetables, cooked meat, and pasta. As part of a trend toward healthy convenience foods, RTE salads have become an increasingly popular product among consumers. However, data on the incidence of foodborne pathogens in RTE salads are scarce. In this study, the microbiological safety of 141 RTE salads containing chicken, ham, or smoked salmon was investigated. Salad samples were collected at retail and analyzed using standard methods for Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), pathogenic Yersinia enterocolitica, Salmonella, and Campylobacter spp.L. monocytogenes was isolated from two (1.4%) of the RTE salad samples. Seven (5.0%) of the samples were positive for the ail gene (present in all human pathogenic Y. enterocolitica isolates) and three (2.1%) of the samples were positive for the Shiga toxin genes stx1 and/or stx2. However, no strains of pathogenic Y.enterocolitica or STEC were isolated. Thus, pathogens were found or suspected in almost 1 of 10 RTE salads investigated, and pathogenic bacteria probably are present in various RTE salads from retail premises in Sweden. Because RTE salads are intended to be consumed without heat treatment, control of the ingredients and production hygiene is essential to maintain consumer safety. The recommended maximum storage temperature for RTE salads varies among countries but can be up to 8°C (e.g., in Sweden). Even during a short shelf life (3 to 5 days), storage at 8°C can enable growth of psychrotrophs such as L. monocytogenes and Y. enterocolitica. The maximum storage temperature should therefore be reduced.

Yersinia enterocolitica in sheep--a high frequency of biotype 1A.

BACKGROUND: Pigs are regarded as the main reservoir for human pathogenic Yersinia enterocolitica, which is dominated by bioserotype 4/O:3. Other animals, including sheep, have occasionally been reported as carriers of pathogenic strains of Y. enterocolitica. To our knowledge, this is the first study performed in the Nordic countries in which the presence of Y. enterocolitica in sheep is investigated. METHODS: Tonsils and faecal samples collected from sheep slaughtered on the island Gotland (Sweden) from September 2010 through January 2011 were analysed for presence of Y. enterocolitica. In an attempt to maximize recovery, several cultural strategies were applied. Various non-selective media were used and different temperatures and durations of the enrichment were applied before subculturing on Cefsulodin Irgasan Novobiocin (CIN) agar. Presumptive Y. enterocolitica colonies were subjected to urease, API 20E and agglutination test. Yersinia enterocolitica isolates were biotyped, serotyped, and tested for pathogenicity using a TaqMan PCR directed towards the ail-gene that is associated with human pathogenic strains of Y. enterocolitica. RESULTS: The samples collected from 99 sheep yielded 567 presumptive Y. enterocolitica colonies. Eighty urease positive isolates, from 35 sheep, were identified as Y. enterocolitica by API 20E. Thirty-four of 35 further subtyped Y. enterocolitica isolates, all from faecal samples, belonged to biotype 1A serotype O:5, O:6. O:13,7 and O:10. One strain was Yersinia mollaretii serotype O:62. No human pathogenic strains of Y. enterocolitica were found in the investigated sheep. Other species identified were Y. kristensenii (n = 4), Y. frederiksenii/intermedia (n = 3), Providencia rettgeri (n = 2), Serratia marcescens (n = 1) and Raoultella ornithinolytica (n = 1). CONCLUSIONS: This study does not support the hypothesis that sheep play an important role in transmission of the known human pathogenic Y. enterocolitica in the studied geographical region. However, because there are studies indicating that some strains of Y. enterocolitica biotype 1A may cause disease in humans, the relative importance of sheep as carriers of human pathogenic strains of Y. enterocolitica remains unclear. Tonsils do not appear to be favourable sites for Y. enterocolitica biotype 1A in sheep.