Isolation and plasmid characterisation of Salmonella enterica serovar Albany harbouring mcr-5 from retail chicken meat in Japan.

The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016. We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates. The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368). The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5. Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.

Prevalence and Antimicrobial Susceptibility of Bacterial Pathogens in Ready-to-Eat Foods Retailed in Osaka Prefecture, Japan.

The potential human health risk of Japanese ready-to-eat (RTE) foods was investigated by determining the contamination by foodborne bacterial pathogens, the prevalence of opportunistic and nosocomial pathogens, and the antibiotic susceptibility of the isolates recovered from 96 samples of lightly pickled vegetables, 88 samples of Western-style desserts, and 98 samples of RTE fish and seafood products sold at retail in Osaka, Japan. Staphylococcus aureus, including isolates producing staphylococcal enterotoxin (SE), were isolated from six lightly pickled vegetable products, seven Western-style dessert products, and three RTE fish and seafood products. Of these isolates, one SEC-producing isolate from a cake was identified as community-acquired methicillin-resistant S. aureus, which was multilocus sequence type 8 and staphylococcal cassette chromosome mec type IV. Enterobacteriaceae species, including Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens, Citrobacter freundii-Citrobacter braakii, and/or the Enterobacter cloacae complex, were isolated from 92 (95.8%) of the lightly pickled vegetable products, 39 (44.3%) of the Western-style dessert products, and 74 (75.5%) of the RTE fish and seafood products. On the basis of the antimicrobial susceptibility profiles of the opportunistic and nosocomial Enterobacteriaceae pathogens, the third-generation cephalosporin, fosfomycin, and quinolone resistance determinants were investigated. We detected AmpC products of the CIT group and a qnrB9 product in 5 and 1 C. freundii-C. braakii isolates, respectively, and fosA gene products in 15 E. cloacae complex isolates. Because RTE foods are consumed without a heating process, the spread of bacterial pathogens from contaminated food to human consumers is possible. RTE foods must be handled using hygienic procedures from the processing steps to the table to reduce the prevalence of potentially pathogenic or pathogenic bacteria and to prevent bacterial growth.

Prevalence of Listeria monocytogenes in Retail Lightly Pickled Vegetables and Its Successful Control at Processing Plants.

Incidences of food poisoning traced to nonanimal food products have been increasingly reported. One of these was a recent large outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 infection from the consumption of lightly pickled vegetables, indicating the necessity of imposing hygienic controls during manufacturing. However, little is known about the bacterial contamination levels in these minimally processed vegetables. Here we examined the prevalence of STEC, Salmonella spp., and Listeria monocytogenes in 100 lightly pickled vegetable products manufactured at 55 processing factories. Simultaneously, we also performed quantitative measurements of representative indicator bacteria (total viable counts, coliform counts, and ß-glucuronidase-producing E. coli counts). STEC and Salmonella spp. were not detected in any of the samples; L. monocytogenes was detected in 12 samples manufactured at five of the factories. Microbiological surveillance at two factories (two surveys at factory A and three surveys at factory B) between June 2014 and January 2015 determined that the areas predominantly contaminated with L. monocytogenes included the refrigerators and packaging rooms. Genotyping provided further evidence that the contaminants found in these areas were linked to those found in the final products. Taken together, we demonstrated the prevalence of L. monocytogenes in lightly pickled vegetables sold at the retail level. Microbiological surveillance at the manufacturing factories further clarified the sources of the contamination in the retail products. These data indicate the necessity of implementing adequate monitoring programs to minimize health risks attributable to the consumption of these minimally processed vegetables.

Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures.

Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

Characterization of specific alleles in InlA and PrfA of Listeria monocytogenes isolated from foods in Osaka, Japan and their ability to invade Caco-2 cells.

Listeria monocytogenes expresses the surface protein internalin A (InlA), enabling the invasion of human intestinal epithelial cells to cause severe food-borne diseases. Full-length sequence analysis of inlA of 114 food isolates resulted in the detection of 29 isolates with a premature stop codon (PMSC) mutation and 6 isolates with 3-codon deletion mutations (aa 738 to 740) in inlA. The isolates with inlA PMSCs demonstrated a significantly lower level of invasion than the other food isolates in a Caco-2 cell invasion assay (P<0.01), but the isolates with the 3-codon deletion exhibited invasion comparable to the isolates with non-truncated InlA (P>0.05). According to analysis of the positive regulatory factor A (PrfA) sequences of 114 L. monocytogenes isolates, 7 isolates of serotype 1/2a from chicken samples contained a PrfA protein with a 5-nucleotide deletion from 712 to 716, including a stop codon. Although the isolates with a 5-nucleotide deletion in prfA demonstrated invasion comparable to the isolates with non-truncated InlA and PrfA after growth at 30 °C (P>0.05), they exhibited a significantly higher level of invasion than the other isolates after growth at 20 °C (P<0.01). To the authors' knowledge, this is the first report of L. monocytogenes isolates with the stop-codon deletion of PrfA.

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan.

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored blaCMY-2, a plasmid-mediated AmpC ß-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring blaCMY-2.

A foodborne outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli serotype O169:H41 in Osaka, Japan.

We describe our laboratory investigation of a massive foodborne outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 that occurred during a 2-day traditional festival held in September 2012 in Osaka Prefecture, Japan. Of 126 customers who patronized a particular Japanese restaurant during the event, 102 developed symptoms of gastrointestinal disease. We isolated strains of ETEC serotype O169:H41 from 1 food sample and from fecal samples collected from 19 of 34 patients and 2 of 4 food handlers. Pulsed-field gel electrophoresis analysis of these isolates suggested that the foodborne pathogen that caused the diarrheal outbreak was a specific clone of ETEC serotype O169:H41. Based on these findings and our interviews with the restaurant owner and employees, we concluded that a likely cause of the outbreak was an overwhelmed capacity of the restaurant kitchen in terms of preservation of sanitary procedures during the festival and the inability of the restaurant staff to handle the relatively large quantity of food to ensure a lack of contamination with ETEC. Thus, we reconfirm that ETEC strains of serotype O169:H41 remain important causes of domestic foodborne outbreaks in developed countries, including Japan.

Evaluation of the culture method NIHSJ-02 alternative to ISO 10272-1:2006 for the detection of campylobacter jejuni and campylobacter coli in chicken: collaborative study.

For the surveillance of the prevalence of Campylobacter jejuni and Campylobacter coli in raw chicken products in Japan, a qualitative method, National Institute of Health Sciences Japan (NIHSJ)-02, was developed as an alternative to International Organization for Standardization (ISO) 10272-1:2006. In the NIHSJ-02 culture method, the enrichment step is carried out in a reduced volume of Preston broth at 42 +/- 1 degrees C to reduce cost and space, and to prevent the overgrowth of background bacteria. To evaluate the performance of NIHSJ-02, a collaborative study was conducted, and the results obtained by NIHSJ-02 were compared with those obtained using the reference method, ISO 10272-1:2006. Fifteen laboratories participated; each examined 48 minced chicken samples consisting of test samples uninoculated, inoculated with C. jejuni at a low or high level, and inoculated with C. coli at a low level. The average probabilities of detection by NIHSJ-02 across laboratories were 0.033, 0.222, 0.678, and 0.267 in samples uninoculated, inoculated with C. jejuni at a low and high level, and with C. coli at a low level, respectively. Those by ISO 10272-1:2006 were 0.051, 0.128, 0.551, and 0.090. Significantly higher probabilities of detection were determined by NIHSJ-02 compared to ISO 10272-1:2006, except for uninoculated samples. On the other hand, significantly lower frequency of occurrence of background bacteria was observed by NIHSJ-02 (43.1%) compared with ISO 10272-1:2006 (92.6%). NIHSJ-02 showed better performance than ISO 10272-1:2006 with regard to the selective detection of C. jejuni and C. coli in chicken.

Detection of chromosomal blaCTX-M-15 in Escherichia coli O25b-B2-ST131 isolates from the Kinki region of Japan.

Escherichia coli O25b-B2-ST131 isolates harbouring bla(CTX-M-15) are distributed worldwide. The bla(CTX-M-15) transposition unit has often been found on plasmids and the genetic contexts have been examined; however, less is known about the frequency and contexts of the bla(CTX-M-15) transposition unit on the chromosome. This study was performed to determine the chromosomal location of the bla(CTX-M-15) transposition unit and to analyse the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit in E. coli O25b-B2-ST131 isolates. Twenty-two E. coli O25b-B2-ST131 strains harbouring bla(CTX-M-15) that had been isolated from university hospital patients and nursing home residents in the Kinki region of Japan were examined. Inverse PCR (iPCR) targeting bla(CTX-M-15) was performed to classify the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit. The isolates were classified into nine types (types A-I) considering the iPCR results; type A was the most prevalent type (13/22 isolates). Sequences of the iPCR-amplified DNA fragments showed that the bla(CTX-M-15) transposition unit consisted of ISEcp1, bla(CTX-M-15) and orf477Δ. A homology search of the obtained sequences showed that the bla(CTX-M-15) transposition unit was inserted into different chromosomal regions in eight of the nine classified types. Although 21 of the 22 E. coli isolates possessed chromosomally located bla(CTX-M-15) transposition units, clonal spread was not evident on pulsed-field gel electrophoresis (PFGE) analysis. Taken together, these data indicate that certain E. coli O25b-B2-ST131 strains harbouring chromosomal bla(CTX-M-15) have emerged and spread in the Kinki region of Japan.

Laboratory investigation of an Escherichia coli O157:H7 strain possessing a vtx2c gene with an IS1203 variant insertion sequence isolated from an asymptomatic food handler in Japan.

We isolated an Escherichia coli O157:H7 strain that was negative for verocytotoxin production, but positive for the vtx2 gene using commercial kits, from an asymptomatic food handler. The laboratory investigations revealed that a 1310-bp insertion sequence, IS1203 variant, was present in the B subunit-coding region of the vtx2c gene.

Isolation and characterization of vanA genotype vancomycin-resistant Enterococcus cecorum from retail poultry in Japan.

The isolation rate of high-level vancomycin-resistant enterococci (VRE) from poultry samples in Japan has increased in recent years. As this raises concerns for the potential spread of genes encoding vancomycin resistance, poultry is routinely screened for VRE. Here, we report the isolation and characterization of a vanA genotype vancomycin-resistant Enterococcus cecorum strain (E. cecorum IPHa84) from retail domestic poultry in September 2009. The species identification was performed by biochemical testing and sequencing of the 16S rRNA and manganese-dependent superoxide dismutase genes. The vancomycin and teicoplanin susceptibility tests showed that E. cecorum IPHa84 was resistant to vancomycin and susceptible to teicoplanin, demonstrating that this isolate was VanB phenotype-vanA genotype VRE. Moreover, a vanA gene cluster was found in a chromosomally encoded Tn1546-related element, which exhibited the characteristic structure of the prototype Tn1546 element, but contained eight point mutations. The vanS sequence of E. cecorum IPHa84 contained three point mutations and was 100% identical to those of VRE isolated from different broiler droppings in Japan prior to the banning of avoparcin, indicating that the Tn1546-related element may be stable in poultry production environments, even in the absence of selective pressure. The isolation of a novel enterococcal species harboring the vanA gene reconfirms that poultry can serve as a reservoir of VanA-type VRE or vancomycin resistance genes, and suggests that the transmission of these risk factors from poultry to humans through the food chain remains a potential threat in Japan.

Molecular epidemiological investigation of a diffuse outbreak caused by Salmonella enterica serotype Montevideo isolates in Osaka Prefecture, Japan.

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.

Whole-genome analysis of Salmonella enterica serovar Typhimurium T000240 reveals the acquisition of a genomic island involved in multidrug resistance via IS1 derivatives on the chromosome.

Salmonella enterica serovar Typhimurium is frequently associated with life-threatening systemic infections, and the recent global emergence of multidrug resistance in S. enterica isolates from agricultural and clinical settings has raised concerns. In this study, we determined the whole-genome sequence of fluoroquinolone-resistant S. enterica serovar Typhimurium T000240 strain (DT12) isolated from human gastroenteritis in 2000. Comparative genome analysis revealed that T000240 displays high sequence similarity to strain LT2, which was originally isolated in 1940, indicating that progeny of LT2 might be reemerging. T000240 possesses a unique 82-kb genomic island, designated as GI-DT12, which is composed of multidrug resistance determinants, including a Tn2670-like composite transposon (class 1 integron [intI1, bla(oxa-30), aadA1, qacEΔ1, and sul1], mercury resistance proteins, and chloramphenicol acetyltransferase), a Tn10-like tetracycline resistance protein (tetA), the aerobactin iron-acquisition siderophore system (lutA and lucABC), and an iron transporter (sitABCD). Since GI-DT12 is flanked by IS1 derivatives, IS1-mediated recombination likely played a role in the acquisition of this genomic island through horizontal gene transfer. The aminoglycoside-(3)-N-acetyltransferase (aac(3)) gene and a class 1 integron harboring the dfrA1 gene cassette responsible for gentamicin and trimethoprim resistance, respectively, were identified on plasmid pSTMDT12_L and appeared to have been acquired through homologous recombination with IS26. This study represents the first characterization of the unique genomic island GI-DT12 that appears to be associated with possible IS1-mediated recombination in S. enterica serovar Typhimurium. It is expected that future whole-genome studies will aid in the characterization of the horizontal gene transfer events for the emerging S. enterica serovar Typhimurium strains.

A collaborative study on a method to detect Salmonella in food.

Fourteen laboratories with expertise in Salmonella detection in food joined in a collaborative study. The laboratories performed qualitative analyses of ground pork samples using the proposed detection method. Salmonella Typhimurium (hydrogen sulfide-producing strain) and Salmonella Senftenberg (hydrogen sulfide-nonproducing strain) were used for inoculation. Three levels of Salmonella contamination were used for the study (0, 1-10, and 11-100 cfu/25 g). We evaluated the presence of Salmonella in each sample and the serological O group. Unmarked samples delivered to the laboratories were accurately judged to be inoculated or not inoculated with Salmonella at a 99.8% (419/420) detection rate in this collaborative study. The proposed method is suitable as a standard method to detect Salmonella in food.

Development of loop-mediated isothermal amplification and PCR assays for rapid and simple detection of Campylobacter fetus subsp. venerealis.

Campylobacter fetus is divided into CFV and CFF. Because CFV causes bovine genital campylobacteriosis, differentiation of the two subspecies is essential to the implementation of efficient CFV control and eradication programs. We have developed LAMP and duplex PCR assays for rapid and simple detection of CFV. The LAMP assay correctly detected 7 CFV strains and did not detect 53 CFF, 35 non-fetus Campylobacter and 25 non-Campylobacter strains. The PCR assay successfully differentiated the two subspecies. The LAMP and PCR assays were faster than conventional biochemical assays, requiring for detection less than 50 min and less than 4 hr, respectively, from the beginning of DNA extraction from a single colony on blood agar to final determination. Our LAMP and PCR assays are rapid and practical tools for detection of CFV.

Isolation of VanA-type vancomycin-resistant Enterococcus strains from domestic poultry products with enrichment by incubation in buffered peptone water at 42 degrees C.

Eight VanA-type enterococcal strains were isolated from 8 of 171 domestic poultry products by using enrichment by incubation in buffered peptone water at 35 degrees C and 42 degrees C. The pulsed-field gel electrophoresis patterns of all six VanA-type Enterococcus faecalis isolates were nearly indistinguishable, indicating the presence of a specific clone in Japan.

Plasmid-mediated quinolone resistance in Salmonella isolated from patients with overseas travelers' diarrhea in Japan.

Twenty-eight of the 302 Salmonella strains isolated from patients with overseas travelers' diarrhea who were examined at Kansai Airport Department Station during the 6-year period between 2001 and 2007 showed decreased susceptibility to both nalidixic acid (NA) and ciprofloxacin (CPFX) (MIC of NA, 16-64 microg/mL; MIC of CPFX, 0.064-2.0 microg/mL). These 28 strains revealed no variations in the quinolone resistance-determining region; however, 25 of the strains showed retention of the qnr gene. The qnr-retaining Salmonella belonged to 6 serotypes, and 21 and 4 of the 25 strains showed qnrS1 and qnrS2, respectively. The most common serovar was S. Corvallis (17 strains).

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Campylobacter fetus.

We developed a sensitive and rapid loop-mediated isothermal amplification (LAMP) assay for detection of Campylobacter fetus. This assay provides simpler and more rapid detection of C. fetus than conventional biochemical and PCR assays. The assay correctly detected 60 C. fetus strains but not 55 non-fetusCampylobacter and 30 non-Campylobacter strains. The sensitivity of the LAMP assay in pure cultures and in a spiked bovine liver specimen was 10-fold more sensitive than that of the conventional PCR assay. The sensitivity of the LAMP assay in a spiked bovine vaginal mucus specimen was similar to that of the conventional PCR assay. The assay was markedly faster, requiring less than 40min for detection of C. fetus in a single colony on blood agar and 80min in spiked bovine specimens from the beginning of DNA extraction to final determination. Our LAMP assay is a simple and practical tool for detection of C. fetus regardless of subspecies.