Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Staphylococcus aureus strains associated with food poisoning outbreaks in France: comparison of different molecular typing methods, including MLVA.

Staphylococcal food poisoning outbreaks (SFPOs) are frequently reported in France. However, most of them remain unconfirmed, highlighting a need for a better characterization of isolated strains. Here we analyzed the genetic diversity of 112 Staphylococcus aureus strains isolated from 76 distinct SFPOs that occurred in France over the last 30 years. We used a recently developed multiple-locus variable-number tandem-repeat analysis (MLVA) protocol and compared this method with pulsed field gel electrophoresis (PFGE), spa-typing and carriage of genes (se genes) coding for 11 staphylococcal enterotoxins (i.e., SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEP, SER). The strains known to have an epidemiological association with one another had identical MLVA types, PFGE profiles, spa-types or se gene carriage. MLVA, PFGE and spa-typing divided 103 epidemiologically unrelated strains into 84, 80, and 50 types respectively demonstrating the high genetic diversity of S. aureus strains involved in SFPOs. Each MLVA type shared by more than one strain corresponded to a single spa-type except for one MLVA type represented by four strains that showed two different-but closely related-spa-types. The 87 enterotoxigenic strains were distributed across 68 distinct MLVA types that correlated all with se gene carriage except for four MLVA types. The most frequent se gene detected was sea, followed by seg and sei and the most frequently associated se genes were sea-seh and sea-sed-sej-ser. The discriminatory ability of MLVA was similar to that of PFGE and higher than that of spa-typing. This MLVA protocol was found to be compatible with high throughput analysis, and was also faster and less labor-intensive than PFGE. MLVA holds promise as a suitable method for investigating SFPOs and tracking the source of contamination in food processing facilities in real time.

Pulsed-field gel electrophoresis, conventional, and molecular serotyping of Listeria monocytogenes from food proficiency testing trials toward an harmonization of subtyping at European level.

The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.

Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping.

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

Screening food raw materials for the presence of the world's most frequent clinical cases of Shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157.

This work aims to provide a strategy for rapidly screening food raw materials of bovine origin for the presence of the most frequent O-serogroups of Shiga toxin-encoding Escherichia coli (STEC) involved in food poisoning outbreaks. The prevalence of highly pathogenic serogroups of STEC was surveyed in 25 g portions of minced meat and raw milk using PCR-ELISA and multiplex real-time PCR assays. The prevalence of STEC in raw milk (n=205) and meat samples (n=300) was 21% and 15%, respectively. Contamination by the main pathogenic E. coli O-serogroups representing a major public health concern, including O26, O103, O111, O145, and O157, was potentially around 2.6% in minced meat and 4.8% in raw milk. The MPN values showed an overall contamination ranging from 1 to 2 MPN cells from highly pathogenic serogroups/kg. This survey would indicate that the human pathogenic potential of STEC present in these samples probably remains limited. No conclusion can be drawn at the moment concerning a potential risk for consumers. This rapid screening approach for evaluating the potential presence of highly pathogenic serogroups of STEC in food raw materials should help to improve risk assessment of food poisoning outbreaks.

Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples.

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.

A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter.

In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter (C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C. upsaliensis (CCUG19559). The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10CFU/ml carcass-rinse. The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8% in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method. The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermo-tolerant Campylobacter strains.

Detection by 5'-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most frequent clinical cases.

This paper describes 5'-nuclease PCR assays for detecting eight O-serogroups, H7 flagellar antigen and stx genes from the Shiga toxin-producing Escherichia coli (STEC) associated with the world's most frequent clinical cases. A single set of primers was used to detect the genes stx1 and stx2 in the same reaction by 5'-nuclease PCR. Serotyping by 5'-nuclease PCR of STEC was based on the selection of primers and probes targeting the O-antigen gene clusters of E. coli O26, O55, O91, O111, O113, O157, the eae gene of E. coli O103, the O-island 29 of E. coli O145, and the flagellar H7 antigen gene. Results obtained on a collection of 190 strains indicate that the 5'-nuclease PCR assays used here could serve as a basis for rapid specific stx, O and H7 typing of these major pathogenic serogroups of E. coli. This work provides sensitive and specific tests for the rapid, reliable detection of the main pathogenic E. coli O-serogroups of major public health concern.

Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria.

In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France.

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.