[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.