Development of a simple screening method for analyzing cereulide toxin in fried rice using liquid chromatography-tandem mass spectrometry.

PURPOSE: The presence of cereulide, an emetic toxin produced by Bacillus cereus, in fried rice samples is critical evidence of food poisoning even in situations where B. cereus could not be detected. This study aims to develop a screening method for analyzing cereulide in fried rice using the QuEChERS procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Cereulide was identified and quantified in fried rice samples using the QuEChERS extraction method and LC-MS/MS. The accuracies of the methods were determined by analyzing fortified blank samples at two concentrations (10 and 50 µg/kg) conducted on three samples daily for five days. RESULTS: The QuEChERS procedure removed matrix compounds from fried rice. Characteristic MS/MS spectra enabled the identification of cereulide. As the matrix effects in seven fried rice samples were within ± 6%, an external solvent calibration curve could be used for quantification. This method exhibited good accuracy ranging from 88 to 89%. The relative standard deviations for both repeatability and intra-laboratory reproducibility were < 4%. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification was 2 µg/kg. The applicability of this method was confirmed using the analysis of cereulide in fried rice samples incubated with emetic Bacillus cereus. CONCLUSIONS: The QuEChERS extraction procedure described herein showed substantial promise as a reliable screening tool for cereulide in fried rice sample.

Multi-Locus Sequence Typing and Lipooligosaccharide Class Analysis of Campylobacter jejuni HS:19 Isolated in Japan.

Campylobacter jejuni is a major foodborne pathogen that causes enteritis in humans, and is also known to be an antecedent infectious factor for Guillain-Barré syndrome (GBS). The onset of GBS after C. jejuni infection results from molecular mimicry between human neuronal gangliosides and C. jejuni lipooligosaccharides (LOS). C. jejuni HS:19 has been previously isolated from GBS cases more frequently than other serotypes in Japan. Therefore, in this study, we performed molecular analysis of 88 HS:19 isolates from GBS cases, sporadic diarrhea patients, and poultry meat samples, using multi-locus sequence typing and LOS class analysis. As a result, 87 of the 88 HS:19 isolates were typed as ST22 / CC22 and LOS class A1, while one was typed as ST1947 / CC22 and LOS class A1. Furthermore, the analysis of another 331 isolates from sporadic enteritis cases showed that only 34 (10.3%) were classified as LOS class A, including HS:19 (25 isolates), HS:2 (8 isolates), and HS:4c (1 isolate). In conclusion, C. jejuni HS:19 had high clonality, regardless of its origin, compared to other capsule types in Japan.

A case of fulminant Clostridium perfringens infection: Role of macroscopic examination of the serum and peripheral blood smears.

A 69-year-old man was brought to our hospital by ambulance with a fever. The translucent pink color of the serum sample suggested severe hemolysis. His blood pressure dropped rapidly, and he later suffered a cardiopulmonary arrest and died approximately 30 h after arriving at our hospital. The day after the patient's death, Clostridium perfringens was detected in the blood culture taken at the time of hospital admission. When serum sample shows translucent pink to red color and bacilli from bacteria is identified in peripheral blood smear, Clostridium perfringens should be considered and appropriate medical treatment should be initiated immediately.

Comparison between patients with norovirus-related gastroenteritis and asymptomatic carriers with respect to distribution of antibody-complexed viral particles and intestinal flora.

Asymptomatic carriers have a major influence on the spreading of norovirus infections. The objective of this study was to examine the characteristics of patients and asymptomatic carriers affected by norovirus-related community gastroenteritis outbreaks. No significant difference between the two groups was observed in terms of the number of norovirus-antibody complexes with respect to total numbers. Principal coordinates analysis of the intestinal flora based on ß-diversity analysis, revealed a different bacterial composition between patients and asymptomatic carriers, particularly regarding the genera Pseudomonas, Bacteroides, and Erwinia, as well as the Ruminococcaceae family. Although the proportional changes between these intestinal microorganisms were not sufficient to explain gastroenteritis symptoms, they represent possible markers shared by asymptomatic norovirus carriers.

Adherence of Clostridium perfringens spores to human intestinal epithelial Caco-2 cells.

Clostridium perfringens is a gram-positive, spore-forming bacillus, and is a causative agent of foodborne infection, antibiotic-associated diarrhoea and sporadic diarrhoea in humans. In cases of antibiotic-associated and sporadic diarrhoea, C. perfringens colonises the intestine, proliferates and causes disease. However, bacterial colonisation of the intestine is not considered necessary in the pathogenesis of foodborne illness, because such pathogenesis can be explained by anchorage-independent production of diarrhoeic toxin by the bacterium in the intestine. In this study, we used an in vitro adherence assay to examine the adherence of C. perfringens spores to human intestinal Caco-2 cells. Adherence of spores from isolates of foodborne illness and nosocomial infection was observed within 15 min, and plateaued 60 min after inoculation. Electron microscopy revealed a tight association of spores with the surface of Caco-2 cells. The adherence of vegetative cells could not be confirmed by the same method, however. These results suggest that C. perfringens spores may adhere to intestinal epithelial cells in vivo, although its biological significance remains to be determined.

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and ß/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and ß/γ-actin, but its sensitivity towards ß/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates ß/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and ß/γ-actin.

[Two Outbreaks of Yersinia enterocolitica O:8 Infections in Tokyo and the Characterization of Isolates].

Although the number of outbreaks caused by Yersinia enterocolitica has been very small in Japan, 4 outbreaks were occurred during the 2 years between 2012 and 2013. We describe herein 2 outbreaks which were examined in Tokyo in the present study. Outbreak 1: A total of 39 people (37 high school students and 2 staff) stayed at a hotel in mountain area in Japan had experienced abdominal pain, diarrhea and fever in August, 2012. The Y. enterocolitica serogroup O:8 was isolated from 18 (64.3%) out of 28 fecal specimens of 28 patients. The infection roots could not be revealed because Y. enterocolitica was not detected from any meals at the hotel or its environment. Outbreak 2: A total of 52 students at a dormitory had diarrhea and fever in April, 2013. The results of the bacteriological and virological examinations of fecal specimens of patients showed that the Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff. We performed bacteriological and virological examination of the stored and preserved foods at the kitchen of the dormitory to reveal the suspect food. For the detection of Y. enterocolitica, food samples. together with phosphate buffered saline (PBS) were incubated at 4 degrees C for 21 days. Then, a screening test for Y. enterocolitica using realtime-PCR targeting the ail gene was performed against the PBS culture. One sample (fresh vegetable salad) tested was positive on realtime-PCR. No Y. enterocolitica was isolated on CIN agar from the PBS culture because many bacteria colonies other than Y. enterocolitica appeared on the CIN agar. After the alkaline-treatments of the culture broth or the immunomagnetic beads concentration method using anti-Y. enterocolitica O:8 antibodies, Y. enterocolitica O:8 which was the same serogroup as the patients' isolates was successfully isolated from the PBS culture. The fresh vegetable salad was confirmed as the incrimination food of this outbreak.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period.

BACKGROUND: Although Clostridium perfringens (C. perfringens) is well known as the causative agent of several forms of enteric disease, precise epidemiological and pathobiological aspects are still unknown. METHODS: We retrospectively reviewed the culture results of samples collected in our hospital from 2001 through 2013. In addition, for the detection and toxinogenic typing of C. perfringens, polymerase-chain-reaction amplification (PCR)-based rapid analysis was performed in 6 cases using DNA extracted from paraffin-embedded tissues. RESULTS: A total of 35 samples from 33 cases were positive for C. perfringens, representing an incidence of 0.017% (35/205, 114). Among 33 patients, 21 patients manifested sepsis and 7 patients had bacteremia. One of the septic cases was complicated by fatal intravascular hemolysis and thus, the prevalence was estimated at 3.0% among C. perfringens infections (1/33). The direct causative disease or state for C. perfringens infection was identified in 18 patients: surgery or intervention for cancers, 8 patients; chemotherapy for cancer, 2 patients; surgery or intervention for non-neoplastic disease, 6 patients; liver cirrhosis, 3 patients, etc. PCR-based toxinogenic typing of C. perfringens detected the alpha-toxin gene only in tissue from a patient who died of massive hemolysis; none of the toxin genes could be amplified in the other 5 cases examined. CONCLUSIONS: The prevalence of overt C. perfringens infection is low, but upon detection, infected patients should be carefully monitored for fatal acute hemolysis caused by type A C. perfringens. Furthermore, PCR-based rapid detection of C. perfringens and toxinogenic typing by archival pathological material is applicable as a diagnostic tool.

Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.

The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan.

Staphylococcal food poisoning (SFP), one of the commonest food-borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin-encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

Bacteriological and epidemiological characteristics of enterotoxigenic Escherichia coli isolated in Tokyo, Japan, between 1966 and 2009.

Enterotoxigenic Escherichia coli (ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that E. coli of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.

[Colony-sweep PCR efficacy in food-borne outbreak associated with six types of enterotoxigenic Escherichia coli].

A box-lunch-associated food-borne outbreak occurred in Tokyo and Chiba Prefecture in June 2003 involved six types of enterotoxigenic Escherichia coli (ETEC). Fecal specimens from patients were screened for ETEC using colony-sweep polymerase chain reaction (PCR). Of the 84 fecal specimens examined, 56 (66.7%) were PCR-positive, i.e. 35 (41.7%) LT-gene-positive, 21 (25.0%) STp-gene-positive and 11 (13.1%) STh-gene-positive. Both of toxin-genes, i.e. LT and STp, LT and STh, STh and STp were positive in 11 patients. ETEC was isolated in confirmation testing from 48 (57.1%) fecal specimens. A single type of ETEC was isolated from 43 fecal samples. Serotype and toxin type of the isolates were O25:NM (LT) (21 samples), O27:H20 (STp) (12 samples), O148:H28 (STh) (8 samples), O25:NM (STh) (1 sample), and O27:7 (STp) (1 sample). Two types of ETEC were isolated from 5 fecal samples, i.e. O25:NM (LT) and O27:H20 (STp) (3 samples), O27:H20 (STp) and O148:H28 (STh) (1 sample), and O25:NM (LT) and O78:NM (STh) (1 sample).

Infantile botulism caused by Clostridium butyricum type E toxin.

The case of a 9-month-old boy with infantile botulism caused by Clostridium butyricum type E toxin is reported. Because infantile botulism is rare in Japan, it was difficult to diagnose it at an early stage. Electrophysiologic findings were useful for the diagnosis, and were characterized by incremental responses (waxing) to short intervals and rapid repetitive nerve stimulation. A bioassay for botulism in mice indicated that the patient had botulism due to type E or F botulinum toxin. C. butyricum type E was isolated from his feces, confirming the diagnosis. This is the first known case of infantile botulism due to C. butyricum type E toxin in Japan.

[Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks].

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.

[An outbreak of diarrheal disease caused by enterotoxigenic Clostridium perfringens following exposure to a contaminated environment in a nursing home].

We herein report an outbreak of non-food-borne diarrhea which occurred in a nursing home due to enterotoxigenic Clostridium perfringens. The regional public health center in Gifu, Japan, recognized 7 patients with diarrhea in a nursing home, suspecting a food-borne illness. Bacteriological and epidemiological studies indicated that enterotoxigenic C. perfringens was the causative agent. However, suspected foods, the kitchen and the cooks carried no enteropathogenic bacteria, indicating that this outbreak was a non-food-borne diarrhea. The swab specimens obtained from the residential area of the nursing home were found to have enterotoxigenic C. perfringens. Isolates from the stool specimens of patients and environment were all serotype TW47, showing susceptibilities to ampicillin, levofloxacin, and clindamycin very similar to each other, and had banding patterns identical to each other by pulsed-field gel electrophoresis. These results strongly supported the existence of monoclonal spread of an enterotoxigenic C. perfringens among the environment of the nursing home and the residents. During 3 weeks 14 residents were involved in this outbreak. The extensive effort of keeping the residential area clean led to a prompt cease of this outbreak.

Usefulness of a combination of pulsed-field gel electrophoresis and enrichment culture in laboratory investigation of a foodborne outbreak due to Clostridium perfringens.

Clostridium perfringens is ubiquitous in nature and normally detectable in human stools. Therefore, it is difficult to perform specific microbiologic diagnosis in foodborne outbreaks, particularly when only a few cultures are detected from fecal specimens. Usually, it has been necessary to detect over 10(6) spores/g of fecal sample as a diagnostic criterion of diarrhea due to C. perfringens. A relatively large foodborne outbreak occurred in Osaka City, Japan in October 2001. Although C. perfringens was suspected as the causal agent, four to seven days had passed after the onset of symptoms before fecal specimens were brought into our laboratory. The positive rate obtained by direct plating was quite low (13/83). We attempted to detect the organisms using enrichment culture after 75 degrees C 20 min heat-treatment, and C. perfringens enterotoxin gene (cpe)-positive strains were isolated from 53 of 81 samples. Pulsed-field gel electrophoresis (PFGE) and serotyping showed that 36 (67.9%) of these 53 strains had indistinguishable PFGE patterns and the same serotype, TW69. Our experience indicates that the enrichment culture could be useful for laboratory confirmation of a C. perfringens foodborne outbreak if it is used with adequate molecular epidemiologic methods.

A botulism case of a 12-year-old girl caused by intestinal colonization of Clostridium botulinum type Ab.

We encountered a 12-year-old girl, who had contracted food-borne botulism, and subsequently suffered from obstinate constipation for more than half a year. Even on hospital day 122, Clostridium botulinum and its toxin were detected in her stool specimens. The potency of the toxin of the blood serum sampled before treatment was 20 mouse minimum lethal dose per ml. The toxin in the blood had a molecular size equivalent to that of type A botulinum neurotoxin. On hospital day 250, the patient's serum detoxified type A neurotoxin. We confirmed that the patient had food-borne botulism caused by C. botulinum type Ab, followed by intestinal colonization-type botulism.

[A case of invasive Clostridium perfringens infection complicated intravascular hemolysis].

We experience a case of a 83-year-old male who was admitted complaining of chills, cramp, high fever and respiratory distress. His blood revealed marked hemolysis. Gram positive Rods was observed in the hemoliesed blood taken on admission. About 2 hours after admission, he suddenly fell into a critical condition. He died about 6 hours after admission in spite of resuscitation. Clostridium perfringens was detected from the blood and liver obtained by autopsy. We suspected that he died of acute intravascular hemolysis caused by alpha-toxin produced by C. perfringens. In conclusion, for a patient who has a high fever with strong hemolysis such as our case, C. perfringens infection should be considered.