The trend of epidemiological study against MRSA stains which were isolated in 1992 and in 1993 was investigated. Number of stains tested yearly consisted of 30 isolates that were considered to play pathogenic roles for inpatients in clinical departments at our institute. In comparing with biological studies on MRSA stains and the epidemilogical surveillance of the background of the isolation, the data summerizes as followings;
1) No.of MRSA stains which were producible for TSST increased from 24/30, 80% up to 30/30, 100%.
2) No.of enterotoxin type harbouring bitype of B/C increased 0/30, 0% up to 12/30, 40%.
3) No.of type of plasmid DNA profile increased in varying from 3 types (A, B, C) to 8 types (A-H).
4) The in vitro activity of antimicrobials, as such MINO, GM, IPM, CMZ was less potent than that of the prior year, and even for VCM, ABK, the activity proved less potent in 1-2 tubes in MIC₉₀.
5) No.significant hospital acquired infection was detected between the inpatients, with MRSA infection and isolates from plasmid DNA profiles.
6) Since the ratio of the coincidence of plasmid DNA profiles of MRSA was only in 4 patients out of 27, 14.9 &, nosocomial infections with MRSA brought to patients have not only been considered by medical, paramedical staff, but that the infection may be caused by broad contamination at the institute.

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To study the cytotoxic effect of Helicobacter pylori on the gastric mucosa, gastric glands harvestedfrom guinea pigs were incubated with clinical isolates of H. pylori. H. pylon alone (H group), urea alone (U group), H. pylori plus urea (HU group), and H. pylori plus urea and the urease inhibitor acetohydroxamic acid (HUA group) were incubated with isolated gastric glands. The controls were incubated without additives. Incubation was for 30, 60 and 180 min at 37°C in a microaerophilic atmosphere. The HU group showed an increase in the ammonia concentration and pH of the culture supernatant; release of lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT) into the supernatant owing to cell disruption was also increased. In the HUA group, since urease activity was inhibited, the ammonia concentration and pH were also significantly lower (p<0.001), and LDH and GOT release into the supernatant was significantly reduced (p<0.001-0.01). Observation by light and electron microscopy showed clear intracellular vacuolization of the gastric glands and adherence of H. pylori to the cell surfaces. These results suggest that ammonia, a metabolite of urea released by H. pylori urease, is one of the important factors in cytotoxicity in isolated gastric glands.

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Staphylococcus spp. were isolated from the ward environment and antibiotic susceptibility tests were performed. Twenty-nine strains out of 274 isolates were S. aureus, and 41.4% of the S. aureusstrains were methicillin resistant (MRSA). All 12 strains of MRSA were also resistant to oxacillin, ceftizoxime, ampicillin and clindamycin. Among the coagulase-negative staphyloccocci (CNS), methicillin-resistant (MR) strains of S. epidermidis, S. capitis, S. warneri, S. haemolyticus, S. hominis, S. auricularis, S. saprophyticus and S. cohnii were isolated. Eight of the 10 S. haemolyticus strains were methicillin resistant. The femA gene was detected in S. aureus (MSSA and MRSA), but not in CNS by polymerase chain reaction (PCR) analysis and Southern blot analysis. The mecA gene was found in all the MRSA and MR-S. epidermidis strains tested, and one of the two MR-S. hominis strains, but not in MSSA, MS-S. epidermidis, MS-S. hominis, or MS-S. haemolyticus. DNA from one strain of MR-S. hominis and 2 strains of MR-S. haemolyticus was not amplified by PCR using the mecA gene primer, or hybridized by Southern blotting. The ambiguity that mecA was detected in some MR-CNS strains, but not in others is discussed.

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We studied the antibacterial effect of Hinokitiol against Staphylococcus aureus. As reference, we studied also the antibacterial effects of the oil and water from Thujopsis dolablata var. hondai Makino which contains Hinokitiol an important element of its antibacterial effect. Interestingly, Hinokitiol showed the double zone phenomenon, i.e., minimum inhibitory concentration of hinokitiol against MSSA was 15-125 μg/ml and against MSSA was 125μg/ml. But when the concentration of Hinokitiol was diluted to 1.87-0.94 μg/ml, then the growth of MSSA was inhibited (Tabel 1 and Fig. 1). This phenomenon was observed the study on antibacterial effect using disk containing Hinokitiol (Fig/ 3). Similar phenomenon were observed also in the study using water from Thujopsis dolablata (Table 2 & Fig. 2). We deduce that Hinokitiol has a strong antibacterial effect against S. aureus. But this effect shows double zone effect. The antibactrial effect of Hinokitiol is more prominent against MRSA than MSSA.

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Fifty-three granulocytopenic patients were studied in a randomized trial comparing trimethoprim-sulfamethoxaxole (ST) alone with ST+ciprofloxacin (CPFX) for prevention of bacterial infections.Seventeen febrile episodes occurrred in 24 patients receiving ST alone, and 9 febrile episodes occurred in 29 patients receiving ST+CPFX. ST+CPFX was significantly effective than ST alone (p<0.005).Although ST alone was effective to prevent infections in moderately granulocytopenic patients, it could not prevent infections in severely granulocytopenic patients whose minimal granulocyte count was less than 250/μl during prophylactic treatment.In contrast, ST+CPFX was effective in severely as well as in moderately granulocytopenic patients.Clinically significant adverse reactions were not observed in both regimens.
These results suggest that combination with ST and CPFX is more efficacious than ST alone for the prevention of bacterial infections in granulocytopenic patients.

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Female acute uncomplicated cystitis responds relatively well to antimicrobial chemotherapy. In particular, new quinolones are suited for use as antimicrobial agents in single-dose therapy of female acute uncomplicated cystitis since they have a long serum half-life and express potent antimicrobial activity against the causative microbes of this infection. Lomefloxacin (LFLX) is one such new quinolone which shows a long serum half-life, expresses potent antimicrobial activity against Escherichia coli (E. coli) and maintains an effetive urinary drug concentration for approximately three days after a single administration. The authors carried out a comparative investigation of the clinical efficacy of single doses of 100mg and 300mg of LFLX in the treatment of female acute uncomplicated cystitis. The clinical efficacy rates with these doses, evaluated on the 3rd day after administration, were 98.2%(56/57 cases) for the 100mg-LFLX dose and 100%(62/62 cases) for the 300-mg LFLX dose. When the evaluation was performed on the 7th day after administration, the clinical efficacy rates were 91.3%(42/46 cases) for the 100-mg LFLX dose and 95.8%(46/48 cases) for the 300-mg LFLX dose. In addition, the microbial eradication rates were 73.7%(42/57 cases) for the 100-mg LFLX group and 75.8%(47/62 cases) for the 300-mg LFLX group on the 3rd day after administration, and 71.7%(33/46 cases) for the 100-mg LFLX group and 83.3%(40/48 cases) for the 300-mg LFLX group on the 7th day after administration. Although there were no statistically significant differences between the two LFLX dosage groups for these parameters at either of the evaluation times, the rates for the 300-mg LFLX dose were slightly superior. The investigators judged the efficacy of the LFLX treatment as having been insufficient in 12 patients, and urological examinations performed on six of those cases determined that there were mild underlying diseases in four cases, such as stenosis of the urethral meatus. On the basis of the findings described above, it is clear that a single 100-mg dose of LFLX provided sufficient clinical efficacy in the treatment of female acute uncomplicated cystitis, but the efficacy of the 300-mg dose of LFLX was even better. In addition, it was surmised that performance of detailed urological examinations provides an opportunity to detect mild underlying diseases that may be the cause of the intractability in female acute uncomplicated cystitis cases showing an insufficient response to treatment with antimicrobial agents such as LFLX.

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Female acute uncomplicated cystitis responds relatively well to antimicrobial chemotherapy, but this is also a disease which shows a high frequency of recurrence. However, there have been no published reports regarding long-term monitoring of the course of this disease after therapy has been administered. Accordingly, using primarily a questionnaire, the authors carried out long-term monitoring (for a mean of 242 days) of the natural course of cases of female acute uncomplicated cystitis after single-dose therapy with lomefloxacin (LFLX), a new quinolone antimicrobial agent. The subjects of this study were female patients diagnosed as having acute uncomplicated cystitis with pain upon urination, pyuria ( & ge;10 WBCs/hpf) and bacteriuria ( & ge;10⁴cfu/ml). LFLX was orally administered as a single dose of 100 mg or 300 mg, and the therapeutic efficacy was evaluated on the 3rd and 7th days thereafter. In principle, the evaluation of cure was performed on the 7th day after LFLX administration, and monitoring was conducted to detect early recurrence during the next 7 days (i.e., through the 14th day after treatment). Then the subjects were monitored for late recurrence during a mean follow-up period of 242 days by means of a questionnaire. Confirmation of recurrence was carried out to the greatest extent possible.
It was possible to carry out long-term monitoring of the natural course of 101 cases of female acute uncomplicated cystitis in which the clinical efficacy on the 3rd day after LFLX treatment had been evaluated as good or excellent. Analysis of the pattern of change in the recurrence rate by application of the Kaplan-Meier method revealed that, for patients monitored for 270 days or more, the recurrence rate was 23.4% for 47 cases in the LFLX 100-mg group and 11.1% for 54 cases in the LFLX 300-mg group. The overall recurrence rate was 16.8%. These data indicate that recurrence was less likely to occur in the LFLX 300-mg group, but the difference between the two dosage groups was not statistically significant. According, it was surmised that recurrence is the natural course in approximately 17% of cases of female acute uncomplicated cystitis during the roughly nine-months period after single-dose antimicrobial therapy.

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We measured the chemiluminescent activity (CL-index) in both the whole blood and isolated neutrophils from 12 patients with acute respiratory infection (7 cases; pneumonia, 4 cases; in the exacerbated phase, chronic lower tract infection, and one; acute bronchitis) two times per each case: before and after chemotherapy. Before the initiation of chemotherapy, neutrophil and whole blood CL was high but whole blood CL was higher. After the completion of chemotherapy, whole blood CL was decreased more siginicantly than neutrophil CL. There was no corelation between neutrophil CL and whole blood CL. However the neutrophil CL-index·N (neutrophil numbers×neutrophil CL-index) was correlated with the whole blood CL. Thus, we think the number of neutrophils is a critical factor for phagocytic function of neutrophils as determined by CL.

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We studied the protective effect of human macrophage colony-stimulating factor (hM-CSF) on fungal infection due to systemic candidiasis in vivo and the activities of macrophages in vitro, in order to demonstrate the usefulness of M-CSF on fungal infection.
The effect of hM-CSF on systemic candidiasis was examined by using normal and immunosuppressed mice. In addition, the effects of hM-CSF on the activity of reticuloendothelial system (RES) organ and on the phagocytic activity and NBT reduction activity of mouse macrophage were also examined in vitro.
HM-CSF improved the survival rate of systemic candidiasis in both normal and immunosuppressed mice. Combination therapy with hM-CSF and fluconazole showed higher survival rate more than in the therapy with either hM-CSF or fluconazole alone. Furthermore, hM-CSF enhanced the activity of RES organ, phagocytosis by macrophages and NBT reduction by macrophages, significantly.
These results indicate that hM-CSF enhances the phagocytic cactivity and candicidal activity by macrophages in vivo, thereby preventing dissemination of fungal infection.

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We investigated the role of respiratory syncytial virus (RSV) in respiratory tract infections of up-to-3-months old infants. This prospective study was carried out from April 1993 to March 1994. Detection of RSV antigen in nasopharyngeal specimens was done by enzyme immunoassay using TESTPACK RSV^® at our out patient clinic.
During this study period, 65 young infants with respiratory tract infection visited our clinic.
Seventeen patients (26%) were diagnosed as having RSV infection. Fifteen out of the 17 infants with RSV infection were observed in spring and winter. Fourteen out of the 17 infants with RSV infection had acute bronchiolitis or pneumonia. In contrast to RSV infection, only three patients out of the 48 infants with non-RSV infection had lower respiratory tract infection. In conclusion, the majority of young infants infected with RSV suffered from bronchiolitis or pneumonia.

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Since 1957 no case of rabies has been reported in Japan except for one imported rabies case diagnosed in 1970. There are many regions in the would, however, where animal rabies remains epizootic and human rabies cases also occur. Statistical examination was performed on the 29 subjects who visited our vaccine clinic to receive postexposure rabies vaccination because they had been bitten by suspected rabid animals abroad. The region where the most subjects were bitten was Thailand, the site where the most vaccinee were injured was in the lower extremity, and the animal which bit the most subjects were stray or domestic dogs. The number of subjects who received postexposure treatment at the local medical institutions were nearly equal to the subjects who did not receive the anti-rabies treatment. However, 76% of subjects who were bitten in Asia and Africa were vaccinated against rabies or given a dose of rabies immune globulin. It is reasonably expected that more Japanse people will be injured by possible rabid animals on abroad, and that imported rabies case will appear in Japan, because the number of Japanese going abroad is growing larger and larger. Therefore, the Japanese medical facilities for rabies postexposure treatment and imported rabies cases should be ameliorated and increased in number.

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We studied the polymerase chain reaction (PCR) method with 5 strains of Legionella pneumophila serogroup 4, 7 strains of L. pneumophila serogroup 5, 7 strains of L. pneumophila UT and 15 strains of Legionella like organisms (LLO). We used mip-primers reported by Mahbubani et al and LEG-primers reported by Starnbach et al.
Positive PCR results were obtained by all strains of L. pneumophila serogroup 4, L. pneumophila serogroup 5, L. pneumophila UT and 11 strains of LLO using mip-primers. Using LEG-primers, positive results were obtained by 2, 6, 5 and 8 strains respectively.
All strains that showed positive results using LEG-primer PCR, were containe by the strains that showed positive results using mip-primer PCR. Conversely, there were some strains that were positive in mip-primer PCR and negative in LEG-primer simultaneously. Therefore, mip-primers is recommended to detect L. pneumophila in clinical specimens by PCR method.

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We screened for the presence of Escherichia coli eae gene, one of the pathogenic factors of enteropathogenic E. coli (EPEC), by using the PCR method and compared the results with those from localized adherence to HeLa cells, presence of the EAF plasmid and the serotyping. One thousand one hundred and four E. coli strains isolated from diarrhea were examined, 144 (13.0%) possessed the eag gene. Of these eae positive strains, 93 (8.4%) exhibited localized adherence to HeLa cells and carried the EAF plasmid. And of these, 79 (7.2%) belonged to EPEC serotypes and 14 (1.3%) belonged to non-EPEC serotypes. Five (0.5%) with weak adherence, and 46 (4.2%) with no adherence were EAF plasmid negative.
Predominant serotypes of EPEC were 055: H-, 086: H34, 0111: H2, 0119: 116, 0128: H2 and main serotype of non-EPEC was 088: H25. Predominant serotypes of E. coli with no adherence to HeLa cells were 026: H-, 055: H-, 0101: H-.
EPEC and non-EPEC with localized adherence to HeLa cells were associated with diarrhea. Some of the eae positive strains with no adherence to HeLa cells, may have lost the EAF plasmid during storage. Though several variants of eae gene exist, it is necessary to confirm whether the strains possessing the eae. gene without localized adherence to HeLa cells can cause diarrhea.

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Bactericidal activities of an amphoteric surfactant (12w/v% Alkyl diaminoethylglycine hydrochloride, 3w/v% Alkyl dietylenetriaminoglycole hydrochloride, Nissan Anon #300^®, Inui Shouji Co., ADG) against Mycobacterium tuberculosis were examined independently or in cooperation with ethanol. The results obtained in the study are as follows:
1. In the treatment with the surfactant diluted with distilled water in one hundredth, the numbers of tubercle bacilli which survived were 500cfu/0.1ml after one minute-treatment, and 25cfu/0.1ml after ten minutes-treatment. On the other hand, the surfactant containing 23v/v% ethanol decreased the bacterial numbers to seven cfu/0.1ml after one minute and less than one cfu/0.1ml after ten minutes. The numbers of the survived bacilli treated with the surfactant containing 20v/v% ethanol were 500cfu/0.1 ml after one minute-treatment, and less than one cfu/0.1ml after ten minutes though 20v/v% ethanol alone was ineffective in ten minutestreatment.
2. The bactericidal activities of the surfactant against tubercle bacilli in sputum samples in which 1.65×10⁴cfu/0.1ml of the organism were contained were also investigated. The numbers of tubercle bacilli which survived were 25cfu/0.1ml after one minute-treatment with the surfactant in one fiftieth, two cfu/0.1ml after five minutes-treatmnet and one cfu/0.1ml after ten minutes-treatment. The numbers of the organism treated with the surfactant containing 23v/v% ethanol were less than one cfu/0.1ml after five minutes.
3. When the scanning electoron microscopies were performed, it was observed that the cell-surface of the organisms exposed to the surfactant in one fiftieth and the surfactant containing 23v/v% ethanol became to sticky and fusible. The morphological chang was greater in the former than in the later.
Those findings indicate that the bactericidal activity of the surfactant against M. tuberculosis is enhanced by cooparation with ethanol.

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Peripheral blood leukocyte (PBL) specimens and urine specimens of 18 renal transplant patients were examined weekly or biweekly for the presence of human cytomegalovirus (HCMV), using polymelase chain reaction (PCR) technique, shell vial procedure and culture. In 11 out of 18 patients, HCMV was detected from either or both of PBL and urine within one to three months after transplantation. HCMV DNA was detected by PCR for a longer period of time, in comparison to the shell vial procedure, showing that PCR was more sensitive. However, only 3 patients showed symptomatic HCMV infection with pyrexia leukocytopenia and liver dysfunction, whose PBL and urine specimens were positive for HCMV by both of the methods. In these 3 patients, HCMV was detected for a longer period of time, whereas in the other asymptomatic patients, it was detectable for only one or two weeks. Moreover, using DNA obtained from PBL in symptomatic phase, HCMV major immediate early (MIE) gene was detected by PCR after amplification for fewer cycles, whereas DNA obtained from these 3 patients in the asymptomatic period or at the end to active HCMV infection, and from other asymptomatic patients, it was detectable after 30 cycles of amplification.

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Isolated organisms from the respiratory tract have been studied in our hospital from 1986 to 1993. The total number of samples were 18, 345 and samples which showed 105 cfu/ml organisms were 8648 in our hospital for 8 years. Enterobacteriacae, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pneumoniae, and glucose nonfermenting gram-negative rods were major isolates in 8 years.
Haemophilus influenzae, which used to be the commonest isolate, decreased from 10.9% in 1993 while Enterobacteriacae increased from 8.9% in 1986 to 17.6% in 1993. S. pneumoniae and H. influenzae were major isolates from out-patients consisting of 50%, followed by Enterobacteriacae, P. aeruginosa and MSSA. Enterobacteriacae and P. aeruginosa were major isolates from in-patients, followed by MRSA and beta-Streptococcus. Streptococcus agalactiae, Serratia marcescens and Corynebacterium spp. prevailed especially in the geriatric ward. S. pneumoniae, H. influenzae and M. catarrhalis were major isolates from patients with pneumoconiosis, especially in winter.

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Confocal laser scanning microscope (CLSM) is a new type of microscope, which permits non-invasive optical sectioning of biomaterials by reducing out-of-focus haze.
A biofilm of Pseudomonas aeruginosa was developed on the silicon disks using a modified Robbins device and used to visualize hydrated living biofilm with CLSM.
In order to examine optimum staining agents and conditions, acridine orange, fluorescein isothiocyanate (FITC), FITC-conjugated concanavalin A (FITC-ConA), Evans blue, safranine and rhodamine-conjugated concanavalin A were used in three different medium (pH 4.5, pH 7.5, pH 9.5).
Only extracellular matrix was stained as a net-like structure for FITC-ConA and only bacteria for acridine orange and safranine. Although the staining patterns with FITC and Evans blue were affected markedly by the medium pH, those with other staining agents were not affected significantly. It was considered that the staining characteristics specific for each agent and changes of staining pattern by pH were probably due to relative differences among matrix, bacteria and staining agents in their electrostatic charges.
In addiition to sagittal sectioning images of P. aeruginosa biofilm, clear doubles staining images, which differentiates bacteria from matrix in the identical material, could be obtained with the combination of safranine and FITC-ConA.

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