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.

Phenotypic and genetic analyses of Campylobacter jejuni Lior serotype 76 isolated from chicken meat and clinical specimens.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

A foodborne outbreak of sapovirus linked to catered box lunches in Japan.

Sapovirus (SaV) is a common cause of acute viral gastroenteritis worldwide, and SaV outbreaks have become more frequent in recent years. In January 2010, an outbreak of acute gastroenteritis due to SaV occurred in Aichi, Gifu and Mie Prefectures, Japan. The illness was strongly associated with eating a delivered box lunch prepared by one catering company. In total, 655 (17.1 %) of 3827 individuals developed gastroenteritic symptoms. SaV was detected in seven of the nine people who became ill and in seven of the 52 food handlers at the catering company, but all the tested samples were negative for norovirus and enteropathogenic bacteria. Sequence analysis of RT-PCR products indicated that the nucleotide sequences of SaV strains from the people who became ill and the food handlers were identical. The detected SaV strains were genogrouped as SaV genotype I.2. This was the largest foodborne outbreak of sapovirus in Japan.

An enrichment medium for increasing a very small number of vibrio parahaemolyticus cells to the detection limit of the loop-mediated isothermal amplification (LAMP) assay.

We developed an enrichment medium for use with the loop-mediated isothermal amplification (LAMP) assay (enrichment media + LAMP assay) to quickly increase a very small number of Vibrio parahaemolyticus cells to the detection limit of the assay. Thirty-nine different enrichment media were prepared based on evaluating 12 potential ingredients. From our assessment of the 39 media, enrichment medium #36, which contained 2% sodium chloride, 1% proteose peptone no. 2, 0.1% trehalose, 0.5% α-ketoglutaric acid, 0.25% pyruvic acid, and 0.5% yeast extract (pH 8.6), was found to be most effective at enhancing the proliferation of V. parahaemolyticus during incubation for 3 h at 40ºC. We compared the detection limits of the LAMP assay, the enrichment medium #36 + LAMP assay, and the cultivation method using bacterial cell and spiked shrimp sample tests. The detection limits of the LAMP assay, the medium #36 + LAMP assay, and the cultivation method were 10(3), 10(0)-10(-1), and 10(-1) CFU ml(-1), respectively. Enrichment medium #36 promoted a 10(3)- to 10(4)-fold increase in the bacterial population, and the detection limit of the enrichment media + LAMP assay was the same as that of the cultivation method.

Antimicrobial ointments and methicillin-resistant Staphylococcus aureus USA300.

We tested 259 methicillin-resistant Staphylococcus aureus isolates and 2 USA300 ATCC type strains for susceptibility to bacitracin and neomycin contained in over-the-counter antibacterial ointments. Resistance to both bacitracin and neomycin was found only in USA300. The use of over-the counter antimicrobial drugs may select for the USA300 clone.

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype.

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.

Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: a collaborative study.

In order to establish a rapid and sensitive method for the detection of Verotoxigenic Escherichia coli O157 and O26, a collaborative study was conducted focusing on a comparison of the efficiency of loop-mediated amplification (LAMP) assay targeting the Verocytotoxin (also called Shiga toxin) gene, utilizing a direct plating method and a plating method with immunomagnetic separation (IMS-plating method) using various agar media. In combination with enrichment with the modified EC supplemented with novobiocin, E. coli O157 was detected in most samples of ground beef and alfalfa sprouts by LAMP assay, the direct plating method and the IMS-plating method. E. coli O26 was detected in approximately 100% of the food samples by LAMP assay. However, the IMS-plating and direct plating methods recovered 80 and 50% in ground beef samples, respectively. As a result, it was demonstrated the LAMP assay is superior to the IMS-plating method. Based on these results, it appears LAMP assay is effective as a screening assay to detect E. coli O157 and O26 from positive samples.

Ability of Shiga toxin-producing Escherichia coli and Salmonella spp. to survive in a desiccation model system and in dry foods.

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35 degrees C for 24 h in paper disks. At an inoculum level of 10(7) CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10(3) to 10(4) CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10(2) CFU/disk). After 22 to 24 months of subsequent storage at 4 degrees C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10(3) to 10(4) CFU/disk). In contrast to the case for storage at 4 degrees C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25 degrees C and 35 degrees C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70 degrees C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25 degrees C.

[Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001].

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.

[Drug resistance of enterohemorrhagic Escherichia coli O157 and a possible relation of plasmids to the drug-resistance].

Antimicrobial susceptibility was examined using 89 enterohemorrhagic Escherichia coli O157 isolates obtained from diarrhea patients in Aichi Prefecture, Japan between June 1996 and June 1997. Among the 89 isolates, 15 (16.9%) were found to be resistant to 6 of 9 antibiotics examined. These 6 antibiotics were ampicillin (ABPC), cefaloridine (CER), chloramphenicol (CP), kanamycin (KM), streptomycin (SM), and tetracycline (TC). Among the 15 drug-resistant isolates, 7 were resistant to 4 drugs (ABPC, CER, SM, TC), 3 were resistant to 3 (ABPC and 2 of CER, SM, TC), 2 were resistant to 2 (SM, TC), one each to KM or SM. Another isolate showed resistance to 5 drugs (ABPC, CP, KM, SM, TC). Selected 13 drug-sensitive and selected 12 multi-drug resistant isolates were tested for the presence of plasmids. All of the drug-sensitive isolates had 54 MDa plasmid and the majority (8/13) had 2.0 MDa plasmids, whereas; all of the drug-resistant isolates except one (1/12) had 54 MDa plasmid and the majority had 8.0 MDa (9/12) and 4.2 MDa (11/12) plasmids. The first transformation test revealed that plasmids of 8.0 MDa (3/4) and 46 MDa (1/4) were transferred to a donor cell with ABPC resistance. 54 MDa plasmid was transferred to a donor cell with both of ABPC and TC resistance. In the second transformation test, only the 8.0 MDa plasmid was confirmed to be transferred to a donor cell with ABPC resistance. Accordingly, it was indicated that the ABPC resistant gene was carried on 8.0 MDa plasmid, and it was suggested that resistant genes for ABPC and TC, and ABPC were carried on 54 MDa, and on 46 MDa plasmids, respectively.

Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains.

We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H-) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC > or = 50 microg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC < or = 1.56 microg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.