Shiga toxin (Stx)-producing
Escherichia coli
(STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.
A set of 8 multiplex real-time SYBR Green PCR (SG-PCR) assays including 3 target primers and an internal amplification control (IAC) primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit), offered detection of greater than 103-104 foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm) curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.
Campylobacter jejuni is one of the leading causes of gastrointestinal illness worldwide and is mainly transmitted from chicken through the food chain. Previous studies have provided increasing evidence that this pathogen can colonize and replicate in broiler chicken during its breeding; however, its temporal kinetics in laying hen are poorly understood. Considering the possible interaction between C. jejuni and gut microbiota, the current study was conducted to address the temporal dynamics of C. jejuni in the cecum of laying hen over 40 weeks, with possible alteration of the gut microbiota and fatty acid (FA) components. Following oral infection with C. jejuni 81-176, inocula were stably recovered from ceca for up to 8 weeks post-infection (p.i.). From 16 weeks p.i., most birds became negative for C. jejuni and remained negative up to 40 weeks p.i. 16S rRNA gene sequencing analyses revealed that most of the altered relative rRNA gene abundances occurred in the order Clostridiales, in which increased relative rRNA gene abundances were observed at >16 weeks p.i. in the families Clostridiaceae, Ruminococcaceae, Lachnospiraceae, and Peptococcaceae. Lipidome analyses revealed increased levels of sterols associated with bile acid metabolisms in the cecum at 16 and/or 24 weeks p.i. compared with those detected at 8 weeks p.i., suggesting that altered microbiota and bile acid metabolism might underlie the decreased colonization fitness of C. jejuni in the gut of laying hens.
SUMMARY:Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10 (ng DNA)/reaction, significantly lower (10-to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89z and 100z, respectively, for Salmonella spp. and 100z and 83z, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.
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