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 . 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 ) . 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 ) . analysis by pulsenet ( www.cdc.gov/pulsenet/protocols.htm ) standardized xbai pulsed - field gel electrophoresis ( pfge ) showed an indistinguishable profile , xtym-1 , associated with the multidrug - resistant s. enterica serotype typhimurium dt104 clone ( 10,11 ) ( figure a1 ) . multilocus variable number tandem repeat analysis ( mlva ) typing ( 12 ) showed an unique type 314721311 ( loci sttr956103 , respectively ) in 16/17 isolates and a single - locus variant ( differing by 1 repetition ) for the remaining isolate , which was an environmental isolate collected 1 month after the control measures were implemented ( slaughter of laying hens ) ( figure a1 ) . the tiramisu isolate 09ceb3100 was also characterized by multilocus sequence typing ( mlst ) for the flic and fljb genes encoding the flagellar 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 identified the flic gene encoding the i antigen and the fljb gene encoding for the 1,2 antigen , confirming that this salmonella spp . the investigation and molecular data concluded that a nonmotile variant strain of serotype typhimurium , genetically distinct from the emerging monophasic variant described worldwide , has been circulating in laying hens , whose contaminated eggs had likely caused food poisoning . during 20002009 , a total of 108,362 serotyped salmonella spp . isolates from humans were registered at the fnrc - salm , of which 374 ( 0.3% ) were nonmotile . such 1,4,,12:: strains have been rarely reported in food , environment or animals with 166 registered isolates collected by the french food safety agency salmonella network during 20012009 ( compared with 21,214 serotype typhimurium and 157,885 salmonella spp . was circulating , we studied 43 additional s. enterica serotype 1,4,,12:: strains : 22 human and 21 nonhuman strains ( 5 from laying hens , 6 from cattle , 2 from sheep , 2 from partridges , 1 from chickens , 3 from the environment , and 3 from foodstuffs ) isolated during 20012009 . these strains were selected on the basis of their diversity ( geographic area , year of isolation , and source ) . all the strains were characterized by antimicrobial drug susceptibility testing , pfge , mlva , and by flic and fljb genes sequencing . the 60 s. enterica serotype 1,4,,12:: isolates studied ( 17 isolates linked to the food poisoning and 43 additional isolates ) displayed 22 different pfge profiles . only 10 profiles , from 49 isolates ( 81.6% ) , matched with profiles from the fnrc - salm pfge typhimurium database ( which includes 207 profiles generated in routine surveillance on 632 strains isolated during 19812010 ) . all 49 strains were confirmed as nonmotile variants of typhimurium by the identification of the flic gene encoding the i antigen and the fljb gene encoding for the 1,2 antigen . the 11 strains whose pfge pattern did not match any typhimurium pfge profile in our database belonged to serotypes typhimurium ( corresponding to 4 new pfge profiles ) , paratyphi b ( n = 3 ) , agona , derby , indiana , and saintpaul ( 1 each ) . among the 49 strains with a xtym profile , 37 ( 75% ) belonged to xtym-1 ( figure a1 ) . 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 profile ( 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 flic gene ( asp251 encoding an asparagine residue ) compared with those available in public database , including serotype typhimurium reference strain lt2 . xtym-1 profiles exhibited 7 pfge profiles , suggesting the loss of motility was acquired independently by 7 distinct serotype typhimurium populations . the mlva type 3147 - 21311 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 ( 20052007 ) clustered at the internal extremity of the same branch . two laying - hen strains isolated in 2009 ( also from the same producer ) were grouped in the interconnecting node . this finding , combined with antimicrobial drug susceptibility testing , pfge , and flic sequencing data , suggested that the 2009 strain causing the food poisoning is a derivative of the 2005 strain ; both strains were isolated from the same egg producer during a 4-year interval . 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 notification 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 modified 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 .