Foodborne Outbreak and Nonmotile Salmonella enterica Variant, France

We report a food-related outbreak of salmonellosis in humans caused by a nonmotile variant of Salmonella enterica serotype Typhimurium in France in 2009. This nonmotile variant had been circulating in laying hens but was not considered as Typhimurium and consequently escaped European poultry flock regulations.

We report a food-related outbreak of salmonellosis in humans caused by a nonmotile variant of Salmonella enterica serotype Typhimurium in France in 2009. This nonmotile variant had been circulating in laying hens but was not considered as Typhimurium and consequently escaped European poultry fl ock regulations.

The Study
In May 2009, diarrhea and fever developed in 8 persons living in southwestern France 1 day after they ate a homemade tiramisu prepared with raw eggs. Fecal analysis was performed on samples from 5 of the 8 persons.
We also cultured a sample from the tiramisu. In medical laboratories, the isolation was performed by using standard procedures (i.e., use of conventional selective media). Isolation from the food sample was performed as required by the current International Organization for Standardization ISO 6579:2002 (i.e., by 2 selective enrichment media) (9). All cultures yielded S. enterica subsp. enterica 4,5,12:-:-.
An investigation at the suspected layer farm was conducted and showed the presence of 11 nonmotile Salmonella spp. isolates (with the same antigenic formula) in dust and feces collected from laying-hen houses. The layer farm, located in northwestern France, is a major farm that produces >32,000,000 eggs per year. All 17 isolates (5 from humans, 1 from the tiramisu, and 11 from the laying hens) were pan-susceptible to all antimicrobial drugs tested (10).
The tiramisu isolate 09CEB3100 was also characterized by multilocus sequence typing (MLST) and for the fl iC and fl jB genes encoding the fl agellar antigens (13). The isolate belonged to MLST sequence type 19, the main sequence type of serotype Typhimurium (http://mlst.ucc.ie/mlst/ dbs/Senterica). PCR and sequencing identifi ed the fl iC gene encoding the i antigen and the fl jB gene encoding for the 1,2 antigen, confi rming that this Salmonella spp. strain of the antigenic formula 4,5,12:-:-was a nonmotile variant of serotype Typhimurium. The investigation and molecular data concluded that a nonmotile variant strain Among the 49 strains with a XTYM profi le, 37 (75%) belonged to XTYM-1 (online Appendix Figure). The nonmotile XTYM-1 strains were divided into 2 groups regarding their susceptibility to antimicrobial agents: pan-susceptible for those (17) linked to the food poisoning plus 6 strains isolated before 2009 (3 human and 3 nonhuman isolates) and penta-resistant profi le (resistant to amoxicillin, streptomycin/spectinomycin, sulfonamide, chloramphenicol, and tetracycline) for the 14 remaining strains. Notably, all 23 nonmotile XTYM-1 pan-susceptible strains had a single amino acid substitution in the fl iC gene (Asp251 encoding an asparagine residue) compared with those available in public database, including serotype Typhimurium reference strain LT2. The search was made with BLASTN (www.ncbi.nlm.nih.gov). The 12 nonmotile strains that matched other non-XTYM-1 profi les exhibited 7 PFGE profi les, suggesting the loss of motility was acquired independently by 7 distinct serotype Typhimurium populations.
Thirty-eight MLVA types were found after testing the 60 isolates. The MLVA type 3-14-7-21-311 found in the food poisoning strains was not observed in the other strains tested. A minimum-spanning tree based on the MLVA types is shown in the Figure. The strains from the food poisoning clustered at the external extremity of a tree branch, whereas 3 human and 3 laying hen strains (from the same producer) isolated earlier (2005)(2006)(2007) clustered at the internal extremity of the same branch. The 2 MLVA types differed by only 1 repetition difference at 2 loci. Two laying-hen strains isolated in 2009 (also from the same producer) were grouped in the interconnecting node. This fi nding, combined with antimicrobial drug susceptibility testing, PFGE, and fl iC sequencing data, suggested that the 2009 strain causing the food poisoning is a derivative of the  strain; both strains were isolated from the same egg producer during a 4-year interval.

Conclusions
Regarding the European Directive and the Commission Regulation on the monitoring and reduction of zoonotic agents (14), the French Regulation has extended the target for reduction of prevalence of Salmonella spp. in poultry producers to include notifi cation of monophasic (because of the recent emergence in humans) and nonmotile (because of this food poisoning) variants of Typhimurium after January 2010 (15). This food poisoning outbreak also highlighted the need for a second selective enrichment media for Salmonella spp. detection not based on the motility in complement to the modifi ed semisolid Rappaport-Vassiladis medium recommended as a single medium by the European Directive.
We report a foodborne outbreak caused by a nonmotile S. enterica 4,5,12:-:-strain in France. This strain has been present in laying hens in France for the past decade. Despite continuous advances in food safety and disease surveillance, control, and prevention, atypical pathogenic Salmonella spp. strains that bypass existing procedures do emerge. Foodborne bacterial infections remain a major public health concern.