[Influence of the mixed inoculum on the species-identification and antimicrobial susceptibility test results by the automated microbial systems].

The influence of the mixed inoculum on the species-identification and antimicrobial susceptibility test results was evaluated for the three automated microbial systems, WalkAway-40 (Dade MicroScan, West Sacramento, CA, USA), VITEK 2 Compact (bioMérieux, Marcy l'Etoile, France) and RAISUS (Nissui Pharmaceutical, Tokyo). In the evaluation, two different species or two different strains were mixed in serial ratios and adjusted to the inoculum cell suspension for the respective systems, and then tested for the species-identification and antimicrobial susceptibility. For the species-identification, all the three automated systems experienced incorrect identifications others from the species inoculated with higher likelihoods (> 90%), e.g. Enterobacter cloacae plus Klebsiella pneumoniae resulted in K. ornithinolytica or E. aerogenes with 93% to 97% likelihoods at the mixing ratio 9 to 1. Whereas, the mixings extended-spectrum beta-lactamase (ESBL)-producing and non-producing Escherichia coli, methicillin-resistant (MRSA) and methicillin-susceptible Staphylococcus aureus, and vancomycin-resistant (VR) and vancomycin-susceptible (VS) Enterococcus faecalis, always resulted in correct detection of a small portion of resistant cells. However, minimum ratios of resistant cells for the correct detection varied by the systems, that is, RAISUS required 70% of MRSA, VITEK 2 Compact was 8%, and the WalkAway-40 was 1%. Also, when the cell suspension of VS E. faecalis spiked with Proteus mirabilis was tested, the WalkAway-40 reported as being very rare biotype, but both VITEK 2 Compact and RAISUS reported as the test inoculum being VR E. faecalis. With these results, it can be concluded that: First, incorrect species-identifications others from the inoculated species easily occur when the inoculum contains different species even at the ratio visibly indiscernible on the primary isolation agar plate. Secondly, the automated microbial systems always intend to detect antimicrobial resistant cells in the inoculum rather than to detect major susceptible cells to prevent us from reporting very major error interpretation.

[Epidemiological search for vancomycin-resistant enterococci in mainland of the Ryukyus].

During the period from January through December 2007, a total of 1,814 stool specimens from the inpatients of nine regional hospitals in mainland of the Ryukyus, were tested to identify vancomycin-resistant enterococci (VRE). All the stool specimens were primarily cultured onto the VRE selective agar plates, and a total of 195 specimens yielded positive enterococcal growth. Of 195 isolates, VRE screening agar tests identified 106 phenotypic VRE isolates, consisting 24 isolates of Enterococcus casseliflavus, 12 of E. faecalis, 4 of E. faecium, and 66 of E. gallinarum. Then, the 106 VRE isolates were tested for vanA and vanB genes by polymerase chain reaction, the results indicating none of the isolates were positive for vanA or vanB genes. With these results, it can be concluded that, at present, mainland of the Ryukyus is VRE-free area, and it is necessary to continue careful search for incoming and spreading of VRE positive for vanA and vanB genes in Okinawa.

[Multicenter-evaluation of optimal cell density to determine antimicrobial susceptibility for Haemophilus influenzae by the automated RAISUS system when the early-harvested bacterial cells were used].

The fully automated microbial system, RAISUS (Nissui Pharmaceutical, Tokyo, Japan) can provide antimicrobial susceptibility test results for the isolates of Haemophilus influenzae. It is known that viable cell concentrations (colony forming unit/ml) of H. influenzae significantly vary depending on the incubation period. For the rapid reporting of antimicrobial susceptibility test results, we evaluated optimal cell density when we prepared the cell suspension using the early-harvested (6 hour incubation) cells for RAISUS. A total of 180 clinical isolates, comprising of 33 ampicillin-susceptible isolates, 114 beta-lactamase negative but ampicillin-resistant isolates and 33 beta-lactamase positive and amoxicillin/clavulanic acid susceptible or -resistant isolates, were included. All the isolates were genetically defined according to the detection of TEM gene and specific mutation (s) in fts I gene. The isolates were incubated on chocolate agar plates for 6 hours, and then the cell suspensions were prepared and adjusted to 0.5, 0.25 and 0.125 McFarland standards through serially dilutions. The respective cell suspensions were tested by the RAISUS AST panels. The % agreements between RAISUS and Clinical and Laboratory Standards Institute standard microdilutions in ampicillin category interpretations were 66.7%(McFarland 0.5), 77.8% (McFarland 0.25) and 83.9%(McFarland 0.125). When the McFarland 0.125 cell suspensions were inoculated, the majority of discrep ant interpretations were minor errors (15.0%) and the occurrence of major error was 3.4%. There was no very major error throughout the study. Essential agreement in MIC determinations (with or within +/- 1 doubling dilution) for 11 beta-lactam antimicrobial agents tested improved to 95.2% by McFarland 0.125 when compared to 77.4% by McFarland 0.5. It was also demonstrated that the viable cell concentrations prepared from 6 hour incubation cultures were 2.5 to 6.5 times higher than those from 22 hour-incubations. With these results, it can be concluded that the early harvested cell suspension of H. influenzae is applicable to RAISUS antimicrobial susceptibility test with lower cell density (McFarland 0.125). With this adjustment, the antimicrobial susceptibility test for H. influenzae will be completed by RAISUS within 26 hours after primary isolation.

[Species-identification and antimicrobial susceptibility tests by the fully automated RAISUS using an early-harvested cell suspension].

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.

[Interpretive compatibility of antimycobacterial susceptibility for Mycobacterium tuberculosis determined by proportion test method on egg-based Ogawa media and broth microdilution test, BrothMIC MTB].

The antimycobacterial susceptibility test method newly proposed by the Japanese Society for Tuberculosis, a proportion method on egg-based Ogawa media, was evaluated in comparison with microdilution test for Mycobacterium tuberculosis complex, BrothMIC MTB-1 (Kyokuto Pharmaceutical Inc., Tokyo). In the evaluation, five antimicrobial agents, streptomycin, ethambutol, kanamycin, isoniazid and rifampicin were included. Through repeated testings of the three reference strains against five antimicrobial agents, both test methods were found to be highly precise. All the minimum inhibitory concentrations (MICs) determined by BrothMIC MTB fell within 3 log2 dilutions, however a total of 11 MICs resulted in indeterminate(I) interpretations. Whereas, all the test results by a proportion method on Ogawa media were comparable to the expected interpretations. However, three of 48 testings resulted in undeterminable interpretations due to insufficient growth on the growth control media. A total of 127 clinical isolates of M. tuberculosis complex were tested by both methods, and 89 to 90% of the test results were comparable with each other in category interpretations. However, 7.1 to 9.4% of MICs determined by BrothMIC MTB resulted in indeterminate(I), and 0.8 to 3.1% of discrepant interpretations were observed. In conclusion, both test methods were highly precise and comparable in determining antimycobacterial susceptibility for M. tuberculosis complex. Several advantages and disadvantages in each test method were discussed.