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Campylobacter jejuni is a major foodborne pathogen causing enteritis in humans and is also known as an antecedent infectious factor for Guillain-Barre syndrome (GBS). The onset of GBS after C. jejuni infection results from molecular mimicry between human neuronal ganglioside and C. jejuni lipooligosaccharide (LOS). C. jejuni HS:19 has been previously reported to be 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 diarrheal patients, and poultry meats 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 other 331 isolates from sporadic enteritis cases shows that only 34 (10.3%) were typed 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, over other capsule types 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 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.

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.

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.

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.

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 β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/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 β/γ-actin.

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.

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.

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).

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.

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.

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.

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.

食肉中に残留する抗菌性物質を微生物学的方法により簡易, 迅速に系統別に推定する検査法の開発をした. すなわち, 厚生省法の分別同定法を改良した簡易な方法で抽出. 分画し, その分画液を試験液とし, これに対するB. subtilis, M. luteus及びB. cereusの3試験菌の感受性パターンから抗菌性物質を系統別に推定する方法, すなわち, 簡易系統別検査法を考案した. 本法によれば, MLsとSAsはクロロホルム層に抽出される分画液A, TCsとPCsはC18カラムで抽出される分画液B, AGsは COOH型カラムで抽出される分画液Cにそれぞれ分画された.分画液Aに対してM. luteus>B. subtilisの順に感受性を示す場合はMLs, B. subtilis (SAs用平板) のみが感受性を示す場合はSAsであった. 分画液Bに対してB. cereus>B. subtilisの順に感受性を示す場合はTCs, M. luteus>B. subtilisの順に感受性を示す場合はPCsであった. 分画液Cに対してB. subtilis>B. cereusの順に感受性を示す場合はAGsであった.本法の検出感度は, MLs, TCs及びAGsは分別同定法と同等であり, PCs及びSAsは残留抗菌性物質検査としては十分満足できる値であった.本法を屠畜場で注射痕を認めたウシ及びブタの筋肉及び腎臓に応用した結果, PCs, TCs及びSAsがそれぞれ単剤で, SAs及びTCsあるいはPCs及びAGsが複合でそれぞれ検出され, 分別同定法及び簡易検査法より高い確率で検出できた.

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.

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.

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.

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°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.

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.

Since the first outbreak in 1990, the incidence of Salmonella ser. Enteritidis (S. Enteritidis) phage type (PT) 1 food poisoning has gradually increased in Tokyo and has reached approximately 30% of the total S. Enteritidis outbreaks reported. To characterise these S. Enteritidis PT1 food poisoning, a total of 198 strains obtained from 44 outbreaks between 1990 and 1996 were examined for antimicrobial resistance, acid producibility from glycols (propylene and ethylene glycol) and plasmid DNA profiles. The 44 PT1 outbreaks analysed were further subdivided into 11 types by epidemiological markers. The most common patterns were type A (plasmid profile carrying only one plasmid (60 kb). SM and TC resistance and non producibility from glycols), and type B (plasmid profile carrying two plasmids (60 and 20 kb), SM resistance and no producibility from glycols) and were responsible for 21 (47.7%) and 15 (34.1%) outbreaks, respectively. In 11 of 44 outbreaks, strains carrying identical epidemiological markers were isolated both from patients and vehicle foods, environments, and/or food-handlers. Similar to PT4 and PT34 outbreaks reported in Japan, egg and egg-related foods were also suspected in 8 of these 11 outbreaks. Of interest, chicken which were not pointed out in PT4 and PT 34 outbreaks was also suspected as a vehicle of transmission in two outbreaks.

Since 1989, outbreaks of Salmonella ser. Enteritidis (S. Enteritidis) food poisoning have dramatically increased in Tokyo, and a total of 31 outbreaks has been reported in 1989. Twenty-one of these 31 outbreaks were caused by S. Enteritidis PT34, but 8 outbreaks were caused by S. Enteritidis PT4. After 1990 instead of SE PT34, food poisoning due to PT4, which was a very common phage type in the UK, has increased in Tokyo. Between 1989 and 1995, there were 144 food poisoning outbreaks caused by S. Enteritidis, and 64 of these outbreaks were by due to S. Enteritidis PT4, which was one of the main phage types in Tokyo. To characterize these strains of phage type (PT) 4,293 isolates from patients, and vehicle foods, eggs and environment in Tokyo were examined for plasmid DNA profiles, acid productivity from glycols (propylene and ethylene) and antimicrobial resistance patterns. Plasmid DNA was extracted by Kado's method, and analyzed by agarose gel electrophoresis. The acid productivity from propylene glycol or ethylene glycol were tested using Barsicow medium with 1% propylene glycol or ethylene glycol. Antimicrobial susceptibility to AM, CP, TC, SM, KM, NA, ST, FOM and NFLX was tested by the K-B disc method. The strains of PT4 were further subdivided into 9 types by those epidemiologic marker analysis. The prevalent pattern of PT4 strains was type A plasmid profile carrying only one plasmid (60 kb) and there were 2 kinds of antibiograms. One was SM resistant, while the other was susceptible. A total of 56 (87.5%) of 64 outbreaks was found to have been caused by these types of S. Enteritidis. Several kinds of egg-related foods were suspected as the vehicles of transmission among 24 outbreaks. Especially, in 5 outbreaks, S. Enteritidis strains were isolated both from patients and suspected food which were cooked with egg. This strongly suggests that these foods may be the potential source of infection in S. Enteritidis PT4 outbreaks.

A simplified method was developed for the detection of residual tetracyclines and sulfa drugs in honey by microbiological assay. A honey sample was homogenized in 0.01M di-Na EDTA McIlvaine buffer (pH 4.0) and centrifuged. The supernatant was mixed well with chloroform and centrifuged. The chloroform layer was evaporated to dryness and the residue was dissolved in phosphate buffer (pH 8.0) to prepare fraction A. The aqueous layer was passed through a Sep-pak C18 cartridge column. The Sep-pak C18 cartridge column was washed with water and absorbed antibacterial agents were eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in phosphate buffer (pH 4.5) to prepare fraction B.Fractions A and B contained sulfa drugs and tetracyclines, respectively. The pulp disk method with Bacillus subtilis ATCC 6633 and Bacillus cereus var. mycoides ATCC 11778 as test organisms was employed for the assay of sulfa drugs and tetracyclines. As little as 0.05μg/g (oxytetracycline), 0.01μg/g (chlortetracycline) or 0.1μg/g (sulfa drugs) was detectable.Thin layer chromatographic identification was performed for positive test solutions. The test solution A was spotted on an aluminum oxide plate (Merck 5550) and a silica gel plate (Merck 5553), then developed in chloroform-methanol (7:3) and ethyl ether-chloroform-n-butanol (5:1:1), respectively. The test solution B was spotted on a cellulose plate (Merck 5552), which was developed in n-butanol saturated with water. Rf values were determined by bioautography with B. subtilis and B. cereus as the test organisms. Microbioautography was suitable for separation and identification of sulfa drugs and tetracyclines in honey. We used this method to identify sulfa drugs and tetracyclines detected in 22 samples out of 297 commercial honeys. It was found that 8 specimens contained oxytetracycline, 12 specimens chlortetracycline, 1 specimen sulfadimethoxine, and 1 specimen sulfisomidine.