We studied 101 strains of Enterohemorrhagic Escherichia coli (EHEC) O26 isolated from diarrhea patients in six prefectural institutes of public health in Japan during June 1996 and December 1997 and tried to establish an isolation medium for EHEC O26. None of the 101 EHEC O26 strains fermented rhamnose; Whereas all of the other EHEC including O157 and non-EHEC (166 strains) fermented rhamnose except 1 strain of non-EHEC. All of the randomly selected EHEC O26 (14 strains of O26: H11, 2 strains of O26: H-) showed a very high resistance to potassium tellurite (Minimal Inhibitory Concentration (MIC)≥50μg/ml), whereas all of the randomly selected non-EHEC (26 strains) but 1 showed a high sensitivity (MIC≤6.25μg/ml) to this compound.
On the basis of these results, we developed a Rhamnose MacConkey (RMAC) medium in which lactose in the MacConkey medium was replaced by rhamnose, and Cefixime-Potassium Tellurite-RMAC (CT-RMAC) medium in which Cefixime (0.05mg/l) and Potassium Tellurite (25mg/l) was added to RMAC for the isolation of EHEC O26 strains. We then evaluated the specifity of these selective media by growing a selected number of O26 (24 strains) and 9 selected strains of bacteria. All of the EHEC O26 strains generated rhamnose non-fermented colonies (white color) on both media. In contrast to the EHEC O26, the vast majority of E. coli strains (166/167=99.4%) other than EHEC O26 were theoretically assumed to generate red colonies on theRMAC medium because of their rhamnose fermenting character, and most of them were assumed not to grow on CT-RMAC medium because of their sensitivity to postassium tellurite. These findings and results indicate that EHEC O26 can be easily distinguished from other strains of E. coli including O157. Although EHEC O26 strains showed somewhat poor growth on CT-RMAC medium compared with that on RMAC medium, these O26 showed almost the same degree of growth on CT-RMAC as they showed on DHL media.
The results of the present study demonstrated that the use of RMAC and CT-MRAC media for the isolation of EHEC O26 is very reliable and efficient with RMAC having good sensitivity and CTRMAC having a better specificity for the isolation of this strain of EHEC.

View full abstract

A total of 341Shigella sonneistrains consisting of 94 domestic strains and 247 imported strains isolated during 1990-1997 in Tokyo, were examined regarding their colicine-type, drug-resistance and ornithine-utilization.
The colicine typing results showed that the domestic strains were classified into 7 types, and the imported strains were classified into 13 types. Among the colicine-types identified, 8-type, 0-type, 6-type and 12-type were predominant in the domestic strains, whereas 6-type, 0-type, 8-type, 9A-type and 12-type were predominant in the imported strains.
The drug-resistance test using 9 drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM and NFLX) showed that 89.4% of the domestic strains and 85.4% of the imported strains were resistant to some of the drugs except FOM and NFLX. Drugs with a high resistant rate were SM, TC and ST for both groups. Drug-resistance patterns of the resistant strains varied in 22 types. Among those, a triple drug-resistance type with TC·ESM·EST was found in the most frequent pattern in both groups.
The results of the ornithine-utilization test revealed that 28.7% of the domestic strains and 8.1% of the imported strains were negative. The ornithine-negative strains in the same source had a similar plasmid-profile, but generally there was no correlation between the different sources.

View full abstract

We have evaluated urine specimens of presumptive cases of legionnaires' disease (110 cases, 173 sample), collected in the past eight years (April, 1990-August, 1998) with the Binax EIA kit which detects the soluble antigen of Legionella pneumophila serogroup (SG) 1, and the Biotest EIA kit which detects Legionella species.
Seven cases (19 specimens) were positive for the Binax EIA kit, and nine cases (22 specimens) were positive for the Biotest EIA kit. The sensitivity for culture, PCR, IFA method were 100%, 100%, and 50%, the specificity for these method were 93%, 97.1%, and 90% respectively. Overall agreements for these method were 93.5%, 97.4%, 86.8%, these results suggested that the urinary antigen detection test had high sensitivity and specificity. Our study indicated that concentrated urine samples increase sensitivity.
We also evaluated the capabilities of both EIAs to detect soluble antigens were extracted from bacterial suspension of 18 strains of 5 Legionella species by heating. Both assays detected L. pneumophila serogroups 1 to 14, L. bozemanii. The Binax EIA proved to be useful as the Biotest EIA for diagnosis of legionellosis caused by Legionella species and serogroups other than L. pneumophila serogroup 1. Some cases have been shown to excrete antigen for prolonged period of times despite recovery from infection, so that the patient's history should be sought.
The urine antigen detection EIA methods proved to be rapid and easy to use, detect antigen in the early stage of the desease with high sensitivity and specificity. Its use for the definition of legionellosis should be considered in Japan.

View full abstract

Aspiration of potentially pathogenic bacteria (PPB) colonized in the upper airway is a major cause of bacterial pneumonia. We hypothesized that PVP-I nasal inhalation is effective in removing PB from the upper airway. The aim of this study was to investigate the effectiveness and safety of PVP-I nasal inhalation. Methods: Patients with asymptomatic PPB (MRSA and/or aerobic GNB i. e. Pseudomonas aeruginosa, Enterobacteriaceae) colonization in the pharynx were enrolled in this study. These patients were divided randomly into two groups as follows: a PVP-I nasal inhalation group (N group) which was asked to inhale 1% PVP-I solution×2/day nasally by a jet nebulizer and gargling with PVP-I soultion×2/day, and a control group (C group), which was asked to gargle with PVP-I solution×2/day. The study period was 2 weeks in both groups. Results: Group N consisted of 16 cases, which included9 (56%) cases with chronic respiratory complications and group C consisted of 14 cases which included 6 (43%) cases with complications. In N and C group, PPB disappearance from the pharynx was observed in 44% and 14% of patients after the study period, respectively. In the patients of group N, without chronic respiratory complication, PPB disappeared in 86% ot the cases. There was no adverse effect correlated with PVP-I nasal inhalation. Conclusion: We conclude that PVP-I nasal inhalation is a safe procedure for removing PPB from the upper airway, and this method may contribute to preventing bacterial pneumonia.

View full abstract

Since the first outbreak in 1990, the incidence of Salmonella ser. Enteritidis (S. Enteritidis) phage type (PT) 1 food poisoining has gradually increased in Tokyo and has reached approximately 30% of the total S. Enteritidis outbreaks reported. To characterise these S. Enteritidis PT 1 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 (60kb), SM and TC resistance and non producibility from glycols), and type B (plasmid profile carrying two plasmids (60 and 20kb, ), 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, enviroments, and/or foodhandlers. 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 PT34 outbreaks was also suspected as a vehicle of transmission in two outbreaks.

View full abstract

To clarify the source of infection and route of transmission of Verocytotoxin-producing Escherichia coli (VTEC) in humans, we collected fresh feces from healthy dairy cattle reared in Hokkaido, Fukushima, Kanagawa and Okinawa prefectures between June 1996 and March 1997, and attempted to isolate VTEC. The results are described below.
1) VTEC was isolated from 68 (27.1%) of 251 fecal samples tested. VTEC was isolated from 14 (28.0%) of 50 in Hokkaido, 13 (26.0%) of 50 in Fukushima, 20 (39.2%) of 51 in Kanagawa and 21 (21.0%) of 100 in Okinawa. There were no difference in the prevalence among the prefectures.
2) Toxin type and serotype of 85 isolates were determined. Thirty-three isolaties (38.8%) were classified into VT1 toxin and VT2 toxin, respectively, and 19 isolates (22.4%) were classified as the strain that produces both VT1and VT2 toxins. The toxin types of these isolates were divided by serotypes. The VT1-producing isolates were the most frequent among O111: H-. The VT2-producing isolates included O2: H12, O2: H29, O2: H-, O82: H8, O82: HUT, O153: H19, O153: H42 and O153: H-. Among the isolates producing both VT1 and VT2 toxins, O153: H19 was relatively frequent.
Based on findings that many bacterial strains coinciding with toxintypes and serotypes of human-derived VTEC isolated from dairy cattle, it was suggested that dairy cattle are closely related to VTEC infection in human as a source of infection.

View full abstract

A21years-old pregnant woman developed diarrhea, fresh bloody stoolsand abdominal pain on April 6^th1997 at 32 weeks of gestation, and was admitted to the hospital on April ¹¹th. The stool culture on admission was positive for enterohemorrhagic Escherichia coli (EHEC) O157: H7 (Stxl and 2). Clinical laboratory data during admission showed only slight elevation of β-microglobulin and Nacetyl glucosaminidase in the urine, and no neurological or hemolytic symptoms were seen. After the antibiotic and lactobacillus administration, all her symptoms were relieved and no abnormal findings in pregnancy were observed. She delivered a baby girl normally on May 30 ^th. Serum (between 41 and 120days from the onset) and milk (between 4 and 64 days post partum) samples from the mother, and serum (64 days of age) from a baby and cord blood were obtainedto monitor the immune status against EHEC O157: H7and against Shiga toxins (Stx). Anti-E. coli O157LPS antibodies (IgA, G and M) were assayed by the ELISA method. Neutralizing anti-Stx antibodies were measured by using ACHN cell cytotoxicity assay.
In the colostrum and mature milk, high levels of IgA and IgM, and no IgG antibodies against EHEC O157 LPS were detected. In one of the control colostrum samplesobtained from4healthy mothers IgA antibody against EHEC O157 LPS was detected. To assess the potency of protection against EHEC O157: H7by the breast milk, we monitored it by the bactericidal activity for the organism under complement-coincubation experiment, and by the neutralization test for the Stx cytotoxicity. As a result, breast milk samples (both colostrum and mature milk) from a patient were demonstrated to kill the organisms. One of 4 healthy milk samples, showed bactericidal activity though it was negative in O157-LPS antibody. This bactericidal activity seen in one healthy colostrum is possibly due to a nonspecific reaction caused by non-0157E. coli infection. From these observations, it was suggested that the bactericidal activity was due to the IgM class antibody against EHEC O157: H7. However, the neutralizing antibody against Stxl and 2could not be detected in any sample.
EHEC infection at late gestation did not cause adverse effects to afetus, and breastfeeding may have advantage for the protection of a baby against EHEC infection.

View full abstract

“HITAZYME C. pneumoniae”(or “HITAZYME CPN”, for short) is a diagnostic reagent that has been recently developed by adopting an ELISA method for detection of anti-Chlamydia pneumoniae (C. pneumoniae) antibodies. When this reagent is used under a current diagnostic standard that has been set as a provisional standard, however, high antibody positive rates are often produced for both IgG and IgA even using the specimens of healthy persons. So, it is difficult to distinguish C. pneumoniae-infected patients from healthy persons. Therefore, this time, we tried to establish a new diagnostic standard by setting up of special cut-off values for a single serum and rise rates of antibody titers for paired sera to improve the accuracy for diagnosis of C. pneumoniae infection.
For a single serum testing, we set a special cut-off value at ID3.00for both IgG and IgA, so that most healthy persons fall within the range of the “negative” zone. This value was based on the calculation of “Mean+2SD” using measurement results(or IDs)of healthy persons. When this cut-off value was applied, the rate o f≥ID 3.00for either IgG or IgA was7.6% for healthy persons, and 64.9% for infected patients .(The rate reached 76.4% when the highest IDs of multiple specimens taken from each patient for this test were used in calculation) As a diagnostic standard for a single serum, therefore, it was defined that: “If ID is 3.00 or greater for IgG and/or IgA, it is highly likely that the case has an acute or a present infection.”
Using paired sera, we could confirm almost a linear relationship between the results by HITAZYME CPN and those by micro-IF method. Under micro-If method, if the antibody titer increases four times or greater using paired sera, acute infection is diagnosed. As it was found that the fourfold increase in antibody titer corresponds to the increase of 1.35 in ID for IgG and 1.00 for IgA, we defined a diagnostic standard for paired sera as follows: “If ID increases by 1.35 or greater for IgG, and/or if ID increases by 1.00 or greater for IgA, the case may be diagnosed as acute infection.”

View full abstract

Toxin detection from stool specimens is prerequisite for Clostridium difficile-associated diarrhea and colitis. However, in Japan only one toxin detection kit is commercially available, which requires computerized VIDAS ^® fluorescence reader. In this study we evaluated ImmunoCard Toxin A ^®, which is an enzyme immunoassay with a format of individual cassetteand needs no special equipment to perform, by comparing with the VIDAS CDA ^® kit. Of 61 stoolspecimens 12 were positive and 39 were negative by both assays, 7 were VIDAS ^® positive-ImmunoCard ^® negative, 2 were VIDAS ^® invalid-ImmunoCard ^® negative, and 1 was invalid by both assays. Stool specimens which gave inconsistent results between two assays were subjected to cellculture assay to detect toxin B and culture for C. difficile followed by testing of toxin producibility of isolates. Of 7 VIDAS ^® positive-ImmunoCard ^® negative specimens 5 were negative for cell culture assay and toxigenic C. difficile culture and 2 were positive for cell culture assay. By omitting 3 VIDAS ^® invalid specimens and counting 5 specimens, which were cell culture negative but VIDAS ^® positive, as negative and 2, which were cell culture posotive and VIDAS ^® positive, as positive, 12 of the 14 VIDAS ^® positive specimens were ImmunoCard ^® positive (sensitivity, 85.7%) and all 44 VIDAS ^® negative were ImmunoCard ^® negative (specificity, 100%). These results indicate that although in comparison with VIDAS CDA ^®, Immuno-Card Toxin A ^® is slightly less sensitive, ImmunoCard ^® is a rapid, simple, and reliable C. difficile toxin A detection kit, which has the advantage of requiring no special equipment to perform and no centrifuge step, as small as 25μL, L of a specimen, and as short as approximately 15 min of time to complete the whole testing.

View full abstract

A case of post-operative abdominal lymphocyst infection caused by vanB type vancomycin resistant enterococcus (VRE) in reported. A27-year-old female was diagnosed as pregnancy with uterine cervical carcinoma and underwent Cesarean section and radical hysterectomy. After discharge, she developed a high fever which was diagnosed as a lymphocyst infection. Microbiological examination demonstrated the presence of vanB typeVRE in the cyst fluid. Cyst cleaning and minocyclin injection were effective. This is the first case of VRE infection in Japan.

View full abstract

We report a patient with systemic lupus erythematosus (SLE) complicated with nocardiosis. This case is very important that the complication of nocardiosis in SLE is very rare and the treatment to both SLE and nocardiosis is very difficult.
A twenty-one-year old female was adwmitted to our hospital because of thoracic empyema and active lupus nephritis. Her medical history revealed that the diagnose of SLE was made when she was 18 with lymphocytopenia, proteinuria, positive antinuclear antibodies, and high titer of antibodies to native DNA. She was treated with prednisolne60mg daily and became better. Proteinuria appeared again in September1995and she was admitted to the former hospital. Renal biopsy proved diffuse proliferative glomeluronephritis (WHO IVb). She was treatedwith lg per day of methylprednisolone for3days and succeeded with60mg day of prednisolone. In early November she developed left chest pain and fever and chest X-ray demonstrated left pleuraleffusion. Antibiotics, antituberculosis, and antifungal therapy failed to subside her pleuritis and it turned to empyema. Then she was transferred to our hospital for further treatment. Nocardia farcinica was detected from the aspirated pleural fluid obtained at the former hospital. Drainage and intrathoracic impenem injection were effective. While long usage of minocycline was continued for the nocardiosis, 500mg of cyclophosphamide pulse therapy to lupus nephritis was administrated. Two weeks later a new pulmonary lesion with left chest pain and liver abscess developed. Administration of trimethoprimsulfamethoxazole subsided the nocardiosis. She was discharged with1g per day of proteinuria the prescribed 13 mg per day of prednisolone and continuous TMP-SMZ intake for nocardial infection.
When immunosuppressive therapy must be given to the immunocompromised host, a more potent therapy must be added to avoid infection.

View full abstract

A57-year-old woman who had been operated on for colon cancer and given chemotherapy, presented in September 1995 with worsening cough and abnormalities on her chest X-ray film. Acid-fast bacilli were isolated from the sputum. The organism was classified as M.gordonae by biochemical tests and DNA/DNA hybridization. The patient was treated with rifampicin and clarithromycin. Subsequently, sputum cultures became negative and the chest x-ray film showed a decrease infiltration. The findings in the present case suggest that M. gordonae may cause pulmonary infection and should be considered as an opportunistic pathogen.

View full abstract