Rinsho Biseibutsu Jinsoku Shindan Kenkyukai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology最新文献

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

For the rapid and sensitive detection of influenza A and B viruses, a latex turbidimetric immunoassay (LTIA) was developed using latex reagents prepared by the sensitization of anti-influenza A or B monoclonal antibodies on latex particles. We measured the immunoreactivity of these latex reagents to influenza A and B viral antigens. The sensitivity and specificity of LTIA and reverse transcription-polymerase chain reaction (RT-PCR) for the detection of these viruses in clinical specimens (96 nasal swabs) were compared. The absorbance change in the latex agglutination reaction increased for each latex reagent with increasing concentration of the viral antigens. Reaction curves were obtained with each concentration of viral antigens for 5 min. The effective concentration ranges were 0-10 microg/ml for influenza A and 0-20 microg/ml for influenza B. The LTIA using clinical specimens revealed 8 positive and 73 negative results for influenza A and 15 positive and 52 negative results for influenza B. The sensitivities and specificities were 89% (8/9) and 84% (73/87), respectively, for influenza A and 100% (15/15) and 64% (52/81), respectively, for influenza B. The corresponding positive predictive values (PPV) were 36% (8/22) for influenza A and 34% (15/44) for influenza B. The negative predictive values (NPV) were approximately 99% (73/74) for influenza A and 100% (52/52) for influenza B. The LTIA is a rapid and sensitive method for detection of the influenza virus; It can be used for high throughput assay by automatic measurement and can potentially be used during influenza pandemics.

The TOX A/B QUIK CHEK "NISSUI" which detects both toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile in stool specimens through immunochromatography was first approved to be released in Japan, and we evaluated its accuracy. In the evaluation, the TOX A/B QUIK CHEK "NISSUI" could correctly detect TcdA and TcdB in solution and in stool specimens spiked with culture broth of TcdA and/or TcdB-producing isolates of C. difficile. The minimum detectable concentrations for TcdA and TcdB were determined to be < or =0.32 ng/ml and < or =0.63 ng/ml, respectively. The TOX A/B QUIK CHEK "NISSUI" gave the consistent results with the colon-endoscopic diagnosis, that is, all the 10 stool specimens from the patients with pseudomembranous colitis were read as being positive, but negative for five patients without any C. difficile-associated disease (CDAD). Of 10 positive stool specimens, one was read as being negative by the commercially available test reagents that can detect only TcdA. In clinical evaluation, a total of 240 stool specimens were tested. Of these, the TOX A/B QUIK CHEK "NISSUI" gave 19 positive results, and TcdA and/or TcdB-producing strains of C. difficile were successfully isolated from all the positive stool specimens, except one. Whereas, of 221 negative stool specimens, 28 isolates of C. difficile were recovered and 11 isolates were identified as TcdA and/or TcdB-producing strains. With these results, it can be concluded that the TOX A/B QUIK CHEK "NISSUI" can correctly detect both TcdA and TcdB of C. difficile, and should be promptly applied to clinical microbiology laboratory to make a definite diagnosis of CDAD, particularly for the CDAD caused by the TcdA-negative but TcdB-positive mutant strains.

Anoxomat Mart II (Mart Microbiology BV, Lichtenvooorde, Netherlands, Central Scientific Commerce Inc., Tokyo, Japan) is an anaerobic jar apparatus which uses a vacuum pump in combination with catalyst as gas replacement procedure to remove all traces of oxygen. As we had a chance to use Anoxomat Mart II, we compared it with other two anaerobic culture methods; namely AnaeroPack anaero (Mitsubishi Gas Chemical Co., Tokyo, Japan) which employs anaerobic jar method, and Concept400 (RUSKINN TECHNOLOGY LTD, England; Central Scientific Commerce INc., Tokyo, Japan) which uses anaerobic chamber method. We used 10 different species of anaerobic bacteria obtained from ATCC. One strain each of 10 species was cultured and examined for measurement of the sensitibity of an anaerobic indicator, th number of bacteria after 48 hour culture, the diameter of colonies, and MIC value. As a result, the time to reach the anaerobic condition was around 30 minutes by the Mart II against around 60 minutes by the AnaeroPack anaero. There was no difference concerning the number of bacteria after 48 hour culture among three methods. But anaerobic bacteria cultured by Mart II tended to make bigger colonies compared to other two methods in the 5 strains out of 9, except for one strain in which the diameter of colonies could not be measured. On the other hand, the comparison of MIC value showed good correlation in 11 antibiotics out of 12 among three methods. The MIC value of 11 antibiotics fitted within 1-fold difference, and 2-fold difference was observed in only one antibiotic. Mart II is so small that it does cheep consumables. From these reasons, we concluded that Mart II can be one of the useful anerobic culture methods.

beta-D-Glucan (beta-G) measurement in blood is useful for prompt diagnosis, selection of treatment and therapeutic evaluation for deep fungal infection. A patient with high beta-G blood was not improved by strong anti-fungal treatment in our hospital, and the beta-G levels in the blood fell immediately after discontinuing ingestion of the agaricus mushroom extracted element (sennseiro). To verify the elevation of beta-G concentration in the blood after intake of an agaricus mushroom extraction element, beta-G concentration in the blood was measured 4 days after 9 volunteers drank 2 g of agaricus mushroom extraction element, "SSG plus 35" twice a day for 3 days. A low value of beta-G in blood was detected in one person (11.1%), which demonstrated that beta-G concentration in the blood increased by oral ingestion of the agaricus element although the reason was unclear why it was detected in only one person. Taken together, when high beta-D-glucan is identified by uncertain cause or in spite of enough anti-fungal medication, it is necessary to confirm whether a patient has ingested health food containing an agaricus mushroom element or other fungus elements to avoid needless treatment with anti-fungal medicine.

Objective: To investigate the specificity of a newly developed Lactobacillus medium (LB medium) and its usefulness for the detection of Lactobacillus.

Method: Chlorophenol red was added to Rogosa SL Broth to make the LB medium whose pH was adjusted to 5.90. After incubation of Lactobacillus in LB medium for 24 hours, the correlation between the number of bacteria and the medium pH was examined. The change of pH was also examined when other microbes coexisted with Lactobacillus.

Results: Six species of Lactobacillus were tested. The medium pH decreased to 4 approximately 5 after incubation of any of these species for 24 hours. Though there were some differences in the acid-producing ability among species, a significant negative correlation (r = -0.93 approximately -0.99) was found between the initial number of bacteria and the medium pH with all species tested. The color of the medium did not change after incubation of other bacteria that might also be found in the normal vagina. The performance of the medium was not affected by the presence of large numbers of other microbes. In L. gasseri JCM 1131, the test microbe for color sample, the number of microbes whose color changed (red-->orange-->yellow) were approximately consistent with that of microbes reported in bacterial vaginosis (BV).

Conclusion: This method can be easily applied to detect Lactobacillus species as the number of Lactobacillus can be estimated based on the change in color of the medium for less than 24 hours after incubation.

We evaluated the usefulness of an early-harvested bacterial cell suspension to the fully automated RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) to provide the results of species-identification and antimicrobial susceptibility testings within a day after overnight-incubation of the primary cultures. A single, well-separated colony appeared on the primary culture plate was transferred onto a blood agar or chocolate agar plates, then incubated for 3 to 6 hours. The cell suspension to the RAISUS was properly prepared to the McFarland 0.5 turbidity from the early-harvested bacterial cells. When the five ATCC reference strains, consisting of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were repeatedly tested for the species-identification, all the identification results were acceptable. Antimicrobial susceptibility tests were evaluated with the above five strains and Haemophilus influenzae ATCC 49247. The results obtained indicated that the most susceptibility test results were comparable to those MICs obtained by the standard test procedure, but some strains, in particular, H. influenzae and P. aeruginosa gave significantly discrepant MICs for certain antimicrobial agents. The significant discrepancy in MIC determinations regarded the difference of viable cell concentrations in the cell suspension prepared respectively. Through the analysis of laboratory workflow, it became to apparent that 18S to 20S of the tests were completed by 5:00 p.m., and it required to wait until 3:00 a.m. to complete 90S of the tests. With these results, the early-harvested bacterial cell suspension is applicable to species-identification by RAISUS, but it is necessary to adjust viable cell concentrations to antimicrobial susceptibility test. Also, it is urgent to reconstitute a daily workflow to improve the rapidity of RAISUS test function.

Commonly isolated anaerobic gram-negative rods (4 genus 64 strains), some other important gram-negative anaerobic species (9 genus 45 strains), and cigar-shaped clostridia (11 strains) were studied on their susceptibility patterns to 6 agents on "Microring AN". Some modifications were made in the methods and interpretation of results. Susceptibility patterns to erythromycin, rifampicin, colistin, benzylpenicillin, kanamycin, and vancomycin were following (sensitive [S], intermediate [I], resistant [R], variable [V]): for Bacteroides fragilis group, V, S, R, R, R, R, respectively; for non-pigmented Prevotella, V, S, V, V, R, R, respectively; for pigmented Prevotella, S, S, SR, V, V, R, respectively: for Fusobacterium nucleatum/necrophorum, R(S), S(I), S(IR), S(R), S, R, respectively; and for F. varium, R, R, S/I, R(S), S, R, respectively. Some results were different from that in the data table in the instruction of "Microring AN", because of differences of methodology and changes of susceptibility of those species during years. As to the other groups, that are not included in the data table in the instruction, results were following: for Bilophila wadsworthia, R, R, S, R, S, R, respectively; Desulfovibrio, V, R(S), R, R, S, R, respectively; for cigar-shaped clostridia, V, S(R), R, R, S(R), S, respectively. "Microring AN" was useful for presumptive identification in genus, species, or group level, though morphological observation and some additional simple tests such as bile-sensitivity and catalase were essential.

In Aug, 2002 (heisei 14), a 74-year-old male inpatient had been complaining of fever and diarrhea. A feces culture test was done and a few colonies were discovered and were suspected to be Salmonella O7 and were later confirmed to be as such. A further investigation and interviewing of patients was undertaken. As a result we suspected an internal infection, and did feces culture tests with 7 out of 15 inpatients who had recently had a stomachache or diarrhea and were able to be tested, and 2 discharged patients. Salmonella O7 was detected by direct fulguration in 4 of them and by enrichment in 2, for a total of 6 out of 9 patients. We considered the source to be food poisoning originating in hospital meals and in adherence to the Food Hygiene Law, turned to the proper authorities, which was the Nagaoka Board of Health, for administrative guidance. To search for the cause of this infection, environmental inspections of the patients' rooms and hospital kitchen were undertaken. Further interviews with the patients were also done but they cleared neither the infection source nor the infection route. Even without a genic test, the biotypes were all the same as 53525040. This was discovered by an Autoscan 4 (DADE BEHRING) and the result gave a strong suspicion of an internal infection. The Nagaoka Board of Health was of the opinion that there was a low possibility of the hospital meals being a source of the infection. The reasons were because the diarrhea did not present at the same time among all the patients, there were a distinct few cases of diarrhea among the hospital meal fed patients, and there were no contamination factors found by their investigation of the staff or the system for preparing the meals. Three patients were given an antimicrobic and the measures for training and educating the staff, maintaining a clean environment and fighting infection were re-examined. The case was brought to its conclusion after about 40 days.