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We examined a total of 151 Salmonella enterica serovar Enteritidis strains isolated in Gifu Prefecture during the period from April 2000 to March 2003 by using bacteriophage typing and pulsed-field gel electrophoresis (PFGE). Bacteriophage typing classified them into twelve phage types (PT) and RDNC (reacted but did not conform). The predominant phage type was PT47 (34.4%) followed by PT1 (21.9%), PT4 (16.6%) and RDNC (11.3%). XbaI- and BlnI-digested PFGE analyses identified 17 and 44 PFGE patterns, respectively, indicating that PFGE with BlnI had more discriminating power than that with XbaI. Combination of the phage types and PFGE types of BlnI could make 53 subtypes. Some isolates with the same phage type were subdivided into different PFGE types, but those with PT47 were not. PT47 isolates were derived from sporadic patients with gastroenteritis, food poisoning outbreaks and healthy carriers through the years. This suggests that PT47 is highly clonal and disseminates over our prefecture.

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.

We examined drug-resistance patterns, coagulase types, and MRSA-phage types of 125 MRSA strains isolated from clinical specimens during the period of January 1990 and December 1994. No vancomycin-resistant strain was isolated. Twenty one antibiotics were divided into three classes, low-intermediate- and high-isolation-frequency class, based on isolation frequencies of resistant strains. Minocycline, chloramphenicol, streptomycin, and imipenem were found to be included in low-isolation-frequency class (16.8-40%). In intermediate-isolation-frequency class (45.6-62.9%), cefmetazole, amikacin, gentamicin, and tetracycline were included. Oxacillin, ampicillin, piperacillin, ceftizoxime, cefoperazone, cefazolin, erythromycin, oleandomycin, kitasamycin, clindamycin, kanamycin, tobramycin, and ofloxacin belonged to high-isolation-frequency class (97.6-100%). MIC90s of vancomycin and minocycline (1.56 and 25 micrograms/ml) were lower than that of other 13 drugs. Comparing medical ward with dental ward, imipenem-, gentamicin-, and minocycline-resistant strains at medical ward, chloramphenicol- and streptomycin-resistant strains at dental ward were isolated dominantly on each ward, MRSA isolates were classified to 39 types by drug-resistance patterns. The isolation frequencies of coagulase type II and type IV strains were 65.6% and 29.6%, respectively. At dental ward, the isolation frequency of coagulase type IV strains was higher than that of coagulase type II strains during 1990-1992. However, coagulase type II strains were isolated considerably more than type IV strains during 1993-1994. By MRSA-phage typing, MRSA isolates were grouped into 18 MRSA-phage types. One hundred and twenty five MRSA isolates were divided into 56 types by using drug-resistance patterns, coagulase typing, and MRSA-phage typing. It was considered that such classification in combination of three methods is useful to make decision of epidemic by the same MRSA strain.

Bullous Impetigo caused mainly by S. aureus is seen in high incidence in children. Three decades ago CEMs were much more effective for bullous impetigo than today. Recently, the causative S. aureus obtained resistance against not only PCs but multiple antibacterial drugs except a few agents. Therefore the therapy for the disease has become difficult, especially the first choice effective antibacterial drugs for general use. Sensitivity of antibacterial drugs against S. auresu isolated from skin lesions, including some MRSA was studied in 113 patients with bullous impetigo. The agents with high sensitivity against all strains were VCM (100%), MINO (100%), CLDM (96.5%), FMOX (96.3%) and against 39 MRSA strains were VCM (100%), MINO (100%), OFLX (92.3%), CLDM (87.2%) and FOM (82.1%). Clinically available antibacterial drugs for children are MINO granules and FOM drysyrup. Phage group and coagulase type of the S. aureus were simultaneously examined to elucidate which strains were causative or secondary infectious agents of the bullous impetigo.

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.

An arbitrarily primed polymerase chain reaction (AP-PCR) DNA profile was applied to epidemiological analysis of Salmonella enterica subsp. enterica serovar Enteritidis. A total 21 strains of S. Enteritidis isolated from 21 cases (10 cases of healthy persons, 7 cases of food poisoning outbreaks and 4 sporadic diarrhea cases), during the period between December 1991 and August 1996 in Wakayama City, were used. A total of 60 arbitrary primers (DNA oligomer (12) set, Wako) were screened with 4 S. Enteritidis strains of different cases. A-11, B-32, C-42 and C-45 primers were chosen. Plasmid DNA profiles, antimicrobial susceptibility patterns and phage types were also examined. The combination of these three methods resolved the collection into five groups (A to E). And type C strains were found in 17 cases (81%) out of 21 cases. However, according to AP-PCR DNA profile, all 21 strains were classified into six groups (I to VI), and 17 type C strains were classified into three groups (III, IV and V). Type IV was predominant in Wakayama City, and type C.IV was found in 15 cases (71%). In conclusion, we considered that AP-PCR DNA profile using appropriate primers was an effective epidemiological marker.

A total of 263 Salmonella strains isolated in Metro Manila, the Philippines from 1992 to 1994, were examined for their serovar-distribution and drug-resistance. Salmonella serovar Typhi (S. Typhi) and S. Enteritidis phage type was also tested. The serological typing results showed that the strains were classified into 10 O-groups and 32 serovars. S. Weltevreden, S. Enteritidis, S. Typhi, S. Typhimurium, and S. Derby were predominant in the serovars identified. The drug-resistance test using nine drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM, and NFLX) showed that 95 strains (36.1%) were resistant to any of the seven drugs except FOM and NFLX. The serovars of a high resistant rate were S. Ruiru (100%), S. Typhimurium (87.0%), S. Senftenberg (80.0%), S. Derby (66.7%), and S. Agona (50.0%). Drug-resistance patterns of the resistant strains varied to 21 types, and 80 of them were multidrug-resistant. Phage types of 37 S. Typhi strains were B1 (27 strains), E2 (6 strains), and others (4 strains). Phage types of 38 S. Enteritidis strains were 4 (32 strains), 7 (3 strains), and others (3 strains).

A total of 603 strains of Salmonella serovar Enteritidis (SE) isolated from patients with sporadic diarrhea during the period from April 1985 to December 1995 in Yamanashi Prefecture, were examined for their yearly and monthly frequency of SE isolates, drug susceptibility, phage typing and plasmid profile. The results can be summarized as follows: 1) The rate of sporadic diarrhea due to SE to all the Salmonella isolates were 4.4% during the period from 1985 to 1988. However it suddenly increased to 47.1% in 1989. After that, the rates decreased to half every year as compared to 1989. And yet, in the last 3 years it became as high as 69.2%, 68.9% and 73.5% in 1993, 1994, and 1995, respectively. 2) Monthly frequency of isolates was 20.1% in September, 19.4% in August, 15.3% in October, 10.9% in July. 3) The predominant ages of isolates were 2 years of age (7.3%), 1 year of age (6.1%), 4 years of age (5.8%), 3 years of age (5.6%). 4) The rates of isolates from males was higher than females (Male: 59.5%, Female: 40.5%). 5) The rate of resistant isolates was 85.4% in the period. The predominant resistance pattern was SM single (417 strains: 69.2%), SA.SM.TC resistance (76 strains: 12.7%). 6) The phage type (PT) of isolates were classified into 15 different patterns. After 1990, the most prevalent PT was PT4. And recently, new types of PT appeared in isolation of PT22 in 1994, PT5 in 1995. 7) The predominant plasmid profile (PP) of SE isolates was 60 kb plasmid. Almost all SE strains of PT4 carried 60 kb plasmid. SE strains of PT1 possessed 2 patterns of PP. One pattern showed that SM single resistance strains carried only 60 kb plasmid, on the other hand SA. SM.TC resistance strains carried 60, 50, 7.5 kb plasmids.

Since 1989, outbreaks of Salmonella ser. Enteritidis food poisoning has dramatically increased in Tokyo, and a total of 95 outbreaks has been reported between 1989 and 1993. As the results of examination of phage type by the method of Ward, et al., strains isolated from 31 (32.6%) of 95 outbreaks were phage type 34 which is a very rare type in Europe and America. To characterize these unique strains of phage type (PT) 34, 302 isolates obtained from patients and vehicle foods of the outbreaks, eggs and environments in Tokyo were examined for plasmid DNA profiles and antimicrobial resistance patterns. Plasmid DNA was extracted by the Kado's method, and analysed by agalose gel electrophoresis. Antimicrobial susceptibility was tested for CP, TC, SM, KM, ABPC, NA, SXT, FOM and NFLX by the K-B disc method. The strains of PT 34 were further subdivided to 4 types by plasmid profile and antibiogram. The prevalent pattern of PT 34 strains was type plasmid profile which carried 2 different plasmids (> 250 and 60 kb) and was streptomysin-resistant. A total of 28 (90.3%) of 31 outbreaks were found to be caused by this specific type of Salmonella ser. Enteritidis. Several kinds of egg-related foods were suspected for the vehicles of transmission among 12 outbreaks. Especially, in 5 outbreaks, this specific type (type B plasmid profile and streptomysin resistant) of Salmonella ser. Enteritidis strains were isolated both from patients and incriminated food which were cooked with eggs. The phase type of Salmonella ser. Enteritidis strains isolated from raw egg and poultry or environmental specimens from poultry farm showed that the prevailing phage types were almost the same as those of foods poisoning outbreaks. This strongly suggests that these foods may be considered as a source of infection in Salmonella ser. Enteritidis outbreaks.

Activities of antimicrobial agents were examined against Staphylococcus aureus strains isolated in 1990. Strains resistant to benzylpenicillin were isolated at the highest frequency (93.6%), and followed by those resistant to kanamycin (51.5%), erythromycin (49.0%), gentamicin (45.1%), fluoroquinolone (33.4%) and minocycline (12.3%). Most of these drug resistant strains of S. aureus were also resistant to methicillin. It should be noted that the MRSA strains were also resistant to other multiple drugs. Annual changes of MRSA in clinical isolates have been increasing since 1981. The isolation frequencies of MRSA strains were 18.9%, 44.8% in 1981 and 1990, respectively. Among methicillin susceptible S. aureus (MSSA), however, drug resistance to other antibiotics have been decreasing year by year. Many strains among both MRSA (73%) and MSSA (86%) were sensitive to typing phages of 100 x RTD. 76% of the MRSA strains were phage typed into groups III or mixed, and 60% of the MSSA strains were typed into these two groups, also.

Phage types were determined for 102 strains of motile Aeromonas isolated from patients with diarrhea at four metropolitan hospitals in Tokyo. The following results were obtained. 1) Of the 102 strains examined, 52 (51.0%) were divided into 28 phage types. This rate was considerably higher, compared to our previous results, namely, 21.7% for strains isolated from natural environments and 25% for those isolated from meats. By bacterial species, phage types could be determined for 33 (52.4%) of 63 strains of A. hydrophila, for 16 (45.7%) of 35 strains of A. sobria, 2 (50.0%) of 4 strains of A. caviae and 1 (100%) strain of Aeromonas spp. 2) Of the 52 strains for which the phage types could be determined, the greatest number (16 strains, 30.8%) were identified as belonging to Type I group. These are followed by 5 strains (9.6%) which were identified as Type I/III group and 2 strains (3.4%) each identified as Type I/II, I/II/V, IV, V and VI groups. The remaining 21 strains were identified as belonging to one of the other phage type groups. Thirty-five (67.3%) of the strains for which the phage types were identified, were found to belong either to Type I group or to combinations with Type I. This demonstrated that 34.0% of the isolates from patients with diarrhea were related to Type I.

Many kinds of staphylococcal typing systems have been developed and tested for epidemiology of Staphylococcus aureus. Coagulase typing and phage typing are more useful and important for this epidemiology. The methods of coagulase typing and phage typing were discussed in this paper. A large number of toxins and proteins of Staphylococcus aureus have been released in the field of infectious environment. Some of these toxins and proteins, working as causative agents of staphylococcal infection, are of interest and discussed here. MRSA shows the same mechanisms of infection as methicillin sensitive Staphylococcus aureus.

An epidemiologic investigation was carried out in Ogaki Municipal Hospital to clarify the status of nosocomial MRSA Infection between 1989 and 1991. In 1989, coagulase type IV, enterotoxin A-producing, and phage group I strains, which were highly resistant to multiple antibiotics and isolated in the internal wards, accounted for 43.4% of all MRSA strains clinically isolated in the entire hospital. In 1990, coagulase type II strains that were sensitive to GM but resistant to FMOX and IPM increased. There were significant differences in the frequency of detection of various strains among wards, suggesting an inter-ward variation in MRSA strains. Changes in environmental strains reflected those in clinical strains. The findings suggest the necessity of measures not only for long-hospitalized MRSA carriers themselves but also for the environment of patients, medical staff, and those taking care of patients.

A total of 184 strains of S. Enteritidis isolated from patients with sporadic cases of diarrhea during the period from April 1985 to March 1991 in Yamanashi Prefecture, were examined for their drug susceptibility, phage typing and plasmid profile. The results can be summarized as follows: 1) In 1989, cases of sporadic diarrhea due to S. Enteritidis has drastically increased twenty-eight-fold from 4 to 112 cases. 2) The most predominant ages of S. Enteritidis isolates were that of 2 years age. 3) The predominant phage type (PT), accounting for almost 71% of the strains isolated, was PT 34 in 1989. 4) Eighty-two (73.2%) out of 112 strains of S. Enteritidis were resistant to SM single in 1989. In 1990, SM single resistant strains were 49 (90.7%) out of 54 strains of S. Enteritidis. 5) The predominant plasmid profile of S. Enteritidis isolates were 60, 55 Kbp plasmids. 6) In 1989, the characteristic of S. Enteritidis isolated from the cases of sporadic diarrhea were resistant to SM single, identified as belonging to PT 34 or PT 4. Almost S. Enteritidis strains of PT 34 carrying 60, 55 Kbp plasmids and many strains of PT 4 S. Enteritidis possessed only 60 Kbp plasmid.

We succeeded in isolating Aeromonas-susceptible phages from river water and mud after a few years (1983-1985) of painstaking effort. For the first time in Japan, we investigated phagetypes of the bacterial strains isolated. The results obtained are summarized as follows: 1) Aeromonas-susceptible phages were isolated from 82 (40.1%) of 195 samples of river water and from 23 (25.6%) of 90 samples of river mud. By the cross-matching test of the phages isolated from the 105 samples, these were classified into 13 type groups (Groups I-XIII). 2) When 594 Aeromonas strains isolated from river water, lake water, river mud and fresh-water fish were examined using the phage group patterns developed by us, 129 (21.7%) strains classified into these phagetypes. Phagetyping was possible for 11 (15.5%) of the 71 strains isolated from river and lake water, for 29 (35.4%) of the 82 strains from river mud and for 89 (20.2%) of the 441 strains from fresh-water fish. By bacterial species, phagetyping was possible for 53 (51.5%) of the 103 strains of A. hydrophila. 21 (7.2%) of the 292 strains of A. sobria, 13 (8.8%) of the 148 strains of A. caviae and 42 (82.4%) of the 51 strains of Aeromonas spp.. Especially the phages classified as Groups I, IV and VI amounted to the majority. Thus we succeeded in isolating Aeromonas-susceptible phages which could be classified into Groups I-XIII. The results suggested the possibility of utilizing this phagetyping for analysis of the ecological distribution of genus Aeromonas.

The characteristics of 167 isolates of S. aureus from 106 mice suffering dermatitis were examined. All 167 isolates coagulated both rabbit and human plasmas and 161 of them also coagulated bovine plasma. All the isolates produced heat-stable and heat-labile DNase, phosphatase and yellow pigment, reduced nitrate, hydrolysed egg yolk, Tween 80, and hippurate, and grew on crystal violet agar in colonies of the negative type C and on medium with 10% NaCl. The majority of them produced fibrinolysin, protease and acetoin. Fifty-three percent were gelatinase positive. In hemolysis tests, 25, 57 and 45 isolates showed alpha-, beta-, alpha beta-hemolysis, respectively. Forty isolates did not produce hemolysins in the rabbit and sheep blood agar. All of 75 isolates tested produced acid from fructose, galactose, glucose, glycerol and mannose, but did not from arabinose, dextrin, inulin, raffinose, salicin, sorbitol and xylose. Most of these isolates produced acid from lactose, mannitol, sucrose and trehalose. All of the 75 isolates were highly sensitive to penicillin, methylphenylisoxazolyl penicillin, erythromycin, spiramycin, lincomycin, chloramphenicol, tetracycline, kanamycin, gentamicin and cephaloridine, but were resistant to sulfisoxazole. With phages of human set, all 167 isolates were typable at 100 X RTD. All but one of the typable isolates belonged to mixed lytic groups. These were I + III (35 isolates), I + M (1), I + III + M (124) and I + II + III + M (6), with long phage patterns. When the 167 isolates were biotyped as described by Hájek and Marsálek [7, 8], 5 belonged to biotype A, 1 to biotype B and 60 to biotype C.(ABSTRACT TRUNCATED AT 250 WORDS)