[Evaluation of a fully automated microbiology system, RAISUS to determine antimicrobial susceptibility for the isolates of Haemophilus influenzae].

A fully automated microbiology system, RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated to determine antimicrobial susceptibility of the isolates of Haemophilus influenzae. The test procedures were fully automated from the inoculation of the test organism through the reading of growth-endpoint on the test plates. Also, Haemophilus Test Medium (HTM) broth supplemented with oxidation-reduction color dye, Redox, assures us of enough bacterial growth and of accurate interpretation of bacterial growth. In the evaluation, the RAISUS provided 99.2% (1,012 of 1,020 testings) agreement in MIC determinations, and 96.4% (983 of 1,020 testings) agreement in category interpretations when compared to the results obtained by the NCCLS M7-A6 and M100-S14 standards. Also, ampicillin (ABPC)-resistance in H. influenzae was characterized by the RAISUS through the addition of clavulanic acid (CVA) into the test broth. Sixteen beta-lactamase-positive isolates resulted in conversion to susceptible interpretation by addition of CVA, whereas the remaining 11 isolates did not. These unaffected isolates were presumptively identified as being beta-lactamase positive, ABPC/CVA-resistant (BLPACR). Of fifty-four beta-lactamase-negative isolates, twelve were regarded as being beta-lactamase negative, ampicillin-resistant (BLNAR) due to 4 microg/ml or greater MICs against ABPC and unaffected MICs by CVA. With the results obtained, it is concluded that the RAISUS can provide comparable determinations for the isolates of H. influenzae, in both MIC determinations and category interpretations, to the NCCLS standards, and is applicable to the routine testing in clinical laboratories.

[Antifungal susceptibility among the isolates of yeast from the University Hospital of the Ryukyus].

During the period from 1999 to 2003, A total of 2,255 isolates of yeast from the patients of the University Hospital of the Ryukyus were determined for their antifungal susceptibilities by the ASTY (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo) colorimetric microdilution testing. The isolates included 1,576 strains of Candida albicans, 409 of C. glabrata, 69 of C. tropicalis, 60 of C. parapsilosis and 141 of other Candida species. A high and uniform antifungal activity of amphotericin B against all the members of Candida was demonstrated with the most minimum inhibitory concentrations (MICs) ranging between 0.25 to 2.0 microg/ml. The MICs obtained from a nearly half of the isolates included against flucytosine distributed < or = 0.125 microg/ml, but 65 (2.9%) and 43 (1.9%) isolates were interpreted as being resistant and intermediate, respectively. The activity of fluconazole markedly varied by species. The resistant ratios of C. albicans and C. parapsilosis were 0.9% and 1.7%, whereas those of C. tropicalis and C. glabrata were estimated as being 40.6% and 13.7%, respectively. The activities of miconazole and micafungin were the most potent with MICs50, 0.125 and 0.06 microg/ml. However, the MICs of C. parapsilosis against miconazole and those of C. glabrata against micafungin were significantly higher when compared to those of C. albicans. By this study, it became apparent that the activities of the presently available antifungal agents were mostly similar for the time and place, but the susceptibilities of the agents among Candida species were markedly different, particularly against azole derivatives. With this, it is necessary for clinical microbiology laboratories to determine antifungal susceptibility for the isolates of yeast and to continuously monitor the effectiveness of each antifungal agent.

[Laboratory-based evaluation of a fully automated microbiology system, RAISUS for species-identification and antimicrobial susceptibility tests].

A fully automated microbiology system, RAISUS newly developed (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated for gram-positive and gram-negative clinical isolates. In the species-identification, the RAISUS comparably identified 200 of 202 isolates (99.0%) consisting of 10 species of gram-positive cocci and of 16 species of gram-negative bacilli. When compared to the MicroScan Walk/Away, which resulted in six misidentifications, the RAISUS showed higher accuracy in species-identification. In the evaluation of antimicrobial susceptibility test, the RAISUS gave comparable minimum inhibitory concentrations (MICs) and category interpretations. Of 693 testings for gram-positive and of 721 testings for gram-negative isolates, 655 (94.5%) and 688 (95.4%) testings gave comparable interpretations to the reference microdilution test, respectively. Most identification results were reported within 5-hour incubation by RAISUS, and susceptibility test results were 4 to 7 hour-incubations. With these results obtained through laboratory-based evaluation, it became apparent that the RAISUS has enough accuracy and rapidity to determine species-identification and antimicrobial susceptibility, and is applicable to the routine testing in clinical laboratories.

[Detection of vancomycin-resistant enterococci by a fully automated microbiology system, RAISUS].

A fully automated microbiology system, RAISUS recently developed (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated for identification of enterococci and for detection of vancomycin-resistant enterococci (VRE). When a total of 124 enterococcal isolates were tested, RAISUS correctly identified 122 (98.4%) isolates. Two isolates resulted in species-identifications disagreed with the reference but agreed as belonging to the genus of Enterococcus. When a total of fifty-seven VRE isolates confirmed to be positive for vanA and/or vanB genes were tested against vancomycin, the current RAISUS susceptibility program version 1.76 could detect 41 (71.9%) isolates of VRE as having > or = 32 microg/ml MIC for vancomycin, but one was intermediate (MIC, 8.0 microg/ml) and the remaining 15 vanB-type isolates were incorrectly interpreted as vancomycin-susceptible (MIC, < or = 4.0 microg/ml). The test program based on the algorism to determine bacterial growth in the presence of vancomycin was developed and evaluated. With this test program, all the VRE isolates positive for vanA and/or vanB genes were identified as being vancomycin-resistant or intermediate interpretation. However, eight of 19 clinical isolates of E. casseliflavus and E. gallinarum intrinsically possessing vanC gene were determined as being < or = 4.0 microg/ml MIC for vancomycin. With the influence of program revision, RAISUS became to incubate the test plate longer than with the current program, but 50% of enterococcal isolates including vancomycin-resistant and vancomycin-susceptible isolates were determined within 5 hour-incubations and 90% were within 9 to 10 hour-incubations. With these results, we can conclude that the revised test program for enterococcal isolates could rapidly and correctly identify vancomycin-resistance, and will be applicable to the routine susceptibility test in clinical laboratories.

[Multicenter evaluation of a newly developed microdilution test, brothMIC NTM to determine minimum inhibitory concentrations of antimicrobial agents for nontuberculous mycobacteria].

We developed a new broth microdilution susceptibility test for nontuberculous mycobacteria to determine minimum inhibitory concentrations(MICs). The test method utilized air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The nine agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, clarithromycin, ethionamide and amikacin. The test plates were reconstituted by inoculation of 0.1 ml of cell suspensions prepared in distilled water by a 1:100 dilution of the 0.5 McFarland suspension. After inoculation, the isolates resulted in incubation at 36 degrees C with clarithromycin at pH 7.4, and with the remaining agents at pH 6.6. The growth endpoints were visually read after 7-day and 10-day incubations for slowly growing nontuberculous mycobacteria, and after 3-day and 5-day incubations for rapidly growing mycobacteria, respectively. The reproducibility was evaluated with the five ATCC reference strains of nontuberculous mycobacteria. Of the 1,287 repeated tests of the four ATCC slowly growing strains(M. avium, M. intracellulare, M. kansasii and M. gordonae), 1,200(93.2%) of the MICs read after 7-day incubation fell within 3 log 2 dilutions. Also, a strain of rapidly growing mycobacteria, M. fortuitum, was repeatedly tested, and the reproducibility was estimated to be 93.3% after 3-day incubation. A total of 728 clinical isolates of nontuberculous mycobacteria comprising 14 species were tested against nine agents. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of rifampicin, levofloxacin, clarithromycin were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative means to determine MICs for nontuberculous mycobacteria in clinical laboratories.

[Antimicrobial susceptibility and prevalence of beta-lactamase producing clinical isolates in southern Kyushu. The results of collaborative study from 1999 to 2000].

The positivity of beta-lactamase and antimicrobial susceptibility were determined in a total of 1,358 clinical isolates at 15 hospitals and clinics in four prefectures in southern Kyushu (Okinawa, Miyazaki, Kagoshima and Kumamoto) during the period from December 1999 to February 2000. The isolates collected comprised of 176 strains of S. aureus, 203 of H. influenzae, 102 of M. catarrhalis, 206 of E. coli, 153 of K. pneumoniae, 99 of E. cloacae, 95 of S. marcescens, 201 of P. aeruginosa, 79 of E. faecalis, and 44 of E. faecium. The frequency of CPDX resistance among E. coli in particular varied geographically, and was found to be higher in Kumamoto and Kagoshima. The strains of K. pneumoniae and E. cloacae resistant to common antimicrobial agents were particularly found in Kagoshima, and one strain of IPM-resistant E. cloacae was isolated in Miyazaki. Also, the geographical difference in the frequency of LVFX resistance among the isolates of E. cloacae was noted, the results indicating the higher prevalence in Okinawa and Kagoshima. Resistant isolates of P. aeruginosa were less common in Kagoshima, and four isolates of P. aeruginosa from Miyazaki were found to be resistant to CAZ and IPM. None of the isolates of S. aureus and Enterococcus spp. was resistant to VCM or TEIC at all. The isolates of E. faecalis resistant at high-level GM (500 micrograms/ml) and SM (1,000 micrograms/ml) were found in 27.8% and 22.8%, and those of E. faecium were 6.8% and 38.6%, respectively. Overall, the ratio of MRSA among S. aureus was 67.6%, and three isolates were resistant to ABK with no less than 8 micrograms/ml of MIC. The frequency of BLNAR (beta-lactamase-negative, ampicillin resistant) among H. influenzae isolated in Okinawa was markedly higher (isolation ratio, 37.9%) when compared with other prefectures, and the isolates of BLPACR (beta-lactamase-positive, AMPC/CVA resistant) were found only in Okinawa with a ratio of 41.6%. A total of 18 strains of ESBL defined by the NCCLS criteria (M100-S11) were isolated, eight strains of K. pneumoniae and 10 strains of E. coli. Of 18 isolates of ESBL, 13 were from Kagoshima and the remaining five were from Kumamoto.