Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

AIMS: Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. METHODS AND RESULTS: We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1 CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). CONCLUSIONS: Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

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.

Foodborne Outbreak of Group G Streptococcal Pharyngitis in a School Dormitory in Osaka, Japan.

In September 2016, 140 patients with primary symptoms of sore throat and fever were identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory investigations determined that these symptomatic conditions were from a foodborne outbreak of group G streptococcus (GGS), with GGS being isolated from samples from patients, cooks, and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp. equisimilis of two emm types (stG652.0 and stC36.0). The causative food, a broccoli salad, was contaminated with the two types of S. dysgalactiae subsp. equisimilis, totaling 1.3 × 104 CFU/g. Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks, and foods produced similar band patterns among samples with the same emm type. This result suggested the possibility of exposure from the contaminated food. The average onset time was 44.9 h and the prevalence rate was 62%. This is the first report to identify the causative food of a foodborne outbreak by Streptococcus dysgalactiae subsp. equisimilis.

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.

Prevalence, antibiotic resistance, and extended-spectrum and AmpC ß-lactamase productivity of Salmonella isolates from raw meat and seafood samples in Ho Chi Minh City, Vietnam.

Salmonellosis is a type of foodborne disease caused by Salmonella enterica and is a frequent cause of childhood diarrhea in Vietnam. Of particular concern is the dissemination of multidrug-resistant Salmonella, as extended-spectrum ß-lactamase (ESBL)-positive isolates were recently detected in children in Vietnam. In the present study, the prevalence and antibiotic resistance of Salmonella isolates obtained from 409 raw meat and seafood samples collected between October 2012 and March 2015 from slaughterhouses, wholesale fish market, and retail markets in Ho Chi Minh City, Vietnam were examined. A high rate of Salmonella contamination was detected in the pork (69.7%), poultry (65.3%), beef (58.3%), shrimp (49.1%), and farmed freshwater fish samples (36.6%). A total of 53 Salmonella serovars were found, of which S. Rissen, S. Weltevreden, S. London, S. Anatum, S. Typhimurium, and S. Corvallis were the most prevalent. In addition, 4 monophasic S. Typhimurium strains were identified using a PCR method for the detection of a specific IS200 fragment within the fliB-fliA intergenic region. The Salmonella isolates had a high prevalence (62.2%) of resistance to antimicrobial agents, particularly tetracycline (53.3%), ampicillin (43.8%), chloramphenicol (37.5%), and trimethoprim/sulfamethoxazole (31.3%). Isolates with resistance to three or more classes of antimicrobials were found (41.1%). Especially, isolates such as S. monophasic Typhimurium, S. Schwarzengrund, S. Indiana, S. Newport, S. Saintpaul and S. Bovismorbificans exhibited resistance to 6 classes of antimicrobials (3.3%). All 7 S. Indiana strains were resistant to between 4 and 6 classes of antimicrobials, including ciprofloxacin, which is commonly used for the treatment of human Salmonella infections. Two fish isolates were confirmed to be CTX-M-55 ESBL-producing Salmonella serovars Bovismorbificans and Newport, and five CMY-2 AmpC-producing Salmonella isolates of serovars Braenderup (4) and Typhimurium (1) were detected in poultry samples. The findings from this study, which is the first report of ESBL- and AmpC-producing Salmonella isolates from food in Vietnam, indicate that multidrug-resistant Salmonella are widely disseminated not only in meats, but also in seafood, within the food distribution system of Vietnam. The presence of these multidrug-resistant strains is a public health concern and suggests that the use of antimicrobial agents in both humans and animals in Vietnam should be tightly controlled.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB.

Clostridium perfringens causes food-borne gastroenteritis following the consumption of contaminated food by producing C. perfringens enterotoxin (CPE) in the intestines. Recently, we reported a novel enterotoxin, binary enterotoxin of C. perfringens (BEC) in C. perfringens isolates, which caused two disease outbreaks in Japan. Consequently, in the event of food poisoning outbreaks caused by C. perfringens, it is now necessary to screen for both the cpe and becAB genes by diagnostic PCR. Here, we present a simple multiplex PCR method for simultaneous detection of cpe, becAB and a C. perfringens control locus, phospholipase C (plc). Applying this method, we investigated the prevalence of cpe- or becAB-carrying C. perfringens strains in human stool and bovine rectum swab samples. Using a total of 169 isolates, we found that the percentage of becAB-carrying strains was very small (0.59%), one-tenth that of cpe-carrying strains. The simple method presented in this study with high specificity and sensitivity to C. perfringens will be a useful tool to survey the global prevalence of becAB-carrying C. perfringens strains.

Dissemination of Extended-Spectrum ß-Lactamase- and AmpC ß-Lactamase-Producing Escherichia coli within the Food Distribution System of Ho Chi Minh City, Vietnam.

To investigate the dissemination of ESBL/pAmpC-producing E. coli within the food distribution system of Ho Chi Minh City (HCMC), Vietnam, the prevalence of ESBL/pAmpC-producing E. coli strains in chicken meat, pork, beef, and fish/shrimp samples obtained from slaughterhouses, a wholesale market, and supermarkets was examined. Among the total of 330 collected food samples, ESBL/pAmpC-producing E. coli was detected in 150 samples (45.5%). The highest prevalence of these isolates was in chicken meat (76/82, 92.7%), followed by pork (32/92, 34.8%), beef (18/74, 34.3%), and fish/shrimp (24/82, 29.3%). A total of 342 strains of ESBL/pAmpC-producing E. coli were isolated from 150 positive food samples. The most prevalent genes responsible for ESBL or pAmpC activity belonged to the CTX-M-9 (110/342, 31.2%), CTX-M-1 (102/342, 29.8%), and CIT (118/342, 34.5%) groups. To our knowledge, this is the first report of the high occurrence of pAmpC (37.1%) in animal-based food in Vietnam. Among the 342 total ESBL/pAmpC-producing E. coli isolates, 276 (80.7%) were resistant to at least 6 antibiotic agents. Notably, high percentages of resistance to ciprofloxacin and fosfomycin were found in isolates from chicken (80.5% and 50.8%, resp.). These findings demonstrate that animal-based food products in HCMC represent a major reservoir of ESBL/pAmpC-producing E. coli.

Prevalence and Antimicrobial Susceptibility of Enterobacteriaceae Isolated from Retail Pepper in Vietnam.

To investigate the microbial quality of retail pepper in Vietnam, the enumeration and detection of Enterobacteriaceae and the screening of cefotaxime (CTX)-resistant coliforms were performed by using 84 commercial samples. Although Enterobacteriaceae were isolated from 78 samples, the number of Enterobacteriaceae was lower than 1.0 log CFU/g in 46 samples. For the detection of Enterobacteriaceae with the International Organization for Standardization methods, Salmonella spp., Escherichia coli, Klebsiella pneumoniae, Cronobacter sakazakii, and Enterobacter cloacae complex were isolated from 5, 12, 36, 19, and 30 samples, respectively. During screening of CTX-resistant coliforms, K. pneumoniae, C. sakazakii, and E. cloacae complex were isolated from 8, 1, and 21 samples, respectively. Seven K. pneumoniae and seven E. cloacae complex isolates obtained in the screening of CTX-resistant coliforms were resistant to at least one of the three third-generation cephalosporins (CTX, ceftazidime, and cefpodoxime). Moreover, one E. cloacae complex cluster IV and all K. pneumoniae isolates were positive for extended-spectrum ß-lactamase genes or plasmid-mediated AmpC ß-lactamase genes or both. Additionally, two extended-spectrum ß-lactamase-producing K. pneumoniae isolates and one AmpC ß-lactamase-producing E. cloacae complex cluster IV isolate were positive for the plasmid-mediated quinolone resistance determinants and also had amino acid alterations in the quinolone resistance-determining regions of GyrA and ParC. Furthermore, 10 E. cloacae complex isolates were positive for the plasmid-mediated fosfomycin resistance gene fosA. As pepper is often consumed without a heating process, the possible spread to humans of foodborne, opportunistic, and nosocomial infection pathogens or resistance genes from foods prepared or seasoned with pepper cannot be excluded. Therefore, it is necessary to handle pepper by using hygienic conditions during the cultivation, harvesting and processing steps.

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.

A highly rapid and simple competitive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poisoning toxins in shellfish.

Using a streptavidin-coated well plate, a biotin-labelled anti-gonyautoxin 2/3 monoclonal antibody GT-13A, and a decarbamoyl saxitoxin-peroxidase conjugate, a direct competitive enzyme-linked immunosorbent assay (PSP-ELISA) was developed for monitoring paralytic shellfish poisoning (PSP) toxins in shellfish. This assay is simple to perform and can be completed in approximately 20 min. The PSP-ELISA was compared to the mouse bioassay (MBA) for the detection of PSP toxins in shellfish samples (n=83) collected from the coast of Osaka Prefecture, Japan. When positive and negative results were indicated based on the regulatory limit for PSP toxins (4 mouse unit(MU)/g of shellfish meat), the PSP-ELISA results showed a sensitivity of 100% (25 of 25) and a specificity of 89.7% (52 of 58 samples) compared to the MBA results. These results suggest that the PSP-ELISA could be used as a rapid and simple screening method prior to the MBA.

Simultaneous Immunomagnetic Separation Method for the Detection of Escherichia coli O26, O111, and O157 from Food Samples.

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef, and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain.

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.

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.

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.

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.

Simultaneous enrichment of shiga toxin-producing Escherichia coli O157 and O26 and Salmonella in food samples using universal preenrichment broth.

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin-producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42 degrees C recovered significantly more cells from inoculated beef than UPB at 35 degrees C and from radish sprout samples than UPB at 35 degrees C and mEC+n. With regard to Salmonella, UPB incubated at 42 degrees C was as effective as UPB at 35 degrees C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42 degrees C and UPB at 35 degrees C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42 degrees C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42 degrees C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42 degrees C.

Application of a modified loop-mediated isothermal amplification kit for detecting Norovirus genogroups I and II.

Norovirus is a major etiologic agent in worldwide outbreaks of gastroenteritis associated with food as well as person-to-person transmission. The ubiquitous nature of Norovirus necessitates simple and rapid detection methods with high accuracy and sensitivity. To this end, several investigators have evaluated the usefulness of commercial reverse-transcription loop-mediated isothermal amplification (RT-LAMP) kits for detecting Norovirus genogroups I (GI) and II (GII). In previous studies, the conventional Loopamp kit for Norovirus GII showed a relatively high detection rate, while that for Norovirus GI showed a relatively low detection rate. In the present study, clinical Norovirus specimens were used to compare the detection rate of a modified Loopamp kit for Norovirus GI with the rates of the conventional Loopamp kit for Norovirus GI and an "in-house" RT-LAMP GI primer set, methods which had a high detection rate. Results from the present study showed that the modified Loopamp kit for Norovirus GI had a higher detection rate for two viral genotypes (GI.3, GI.11). On comparison with an "in-house" GII primer set using genotype GII.4 viruses circulating recently, the detection rate by the Loopamp kit for Norovirus GII was found to be higher, with a 98% detection rate. These results indicate the applicability of the modified LAMP kit for GI and the conventional LAMP kit for GII for detection of Noroviruses in clinical samples.