We investigated the susceptibility to antibacterial agents of 197 strains of Haemophilus influenzae isolated from pediatric patients at medical facilities in Gifu and Aichi prefectures between 2009 and 2010. Those strains were also examined for the mutations of ftsI coding for penicillin-binding protein 3, presence of bla TEM-1, serotype andμ-lactamase producing ability.

Among the 197 strains, the most prevalent serotype was non-typeable (89.8%), followed by serotype b (8.1%), e (1.5%) and f (0.5%).

Based on the susceptibility among the 197 strains to antibacterial agents,μ-lactamase non- producing ampicillin-susceptible H. influenzae (BLNAS) accounted for 27.4%,μ-lactamase non- producing ampicillin-resistant H. influenzae (BLNAR) for 62.4%,μ-lactamase producing ampicillin-resistant H. influenzae (BLPAR) for 6.1% andμ-lactamase producing amoxicillin/ clavulanic acid-resistant H. influenzae (BLPACR) for 4.1%.

According to PCR-based genotyping, the strains were classified into 6 categories: gBLNAS, gLow-BLNAR, gBLNAR, gBLPAR, gBLPACR-I and gBLPACR-II. The incidences of each resistant class were 17.3% for gBLNAS, 6.6% for gLow-BLNAR, 66.0% for gBLNAR, 5.6% for gBLPAR and 4.6% for gBLPACR-II. The combined incidence of gLow-BLNAR and gBLNAR was 72.6%, which was higher than that of BLNAR (62.4%).

The MIC₉₀s of antibacterial agents against the 197 strains were as follows; 0.0156μg/mL for tosufloxacin and garenoxacin, 0.0313μg/mL for levofloxacin and pazufloxacin, 0.0625μg/mL for norfloxacin, 0.25μg/mL for tazobactam/piperacillin (TAZ/PIPC) and ceftriaxone, 0.5μg/mL for TAZ/PIPC (1 : 8) and cefditoren, 1μg/mL for piperacillin, cefteram, cefotaxime, meropenem, tebipenem and minocycline, 2μg/mL for doripenem, 4μg/mL for cefcapene, imipenem and azithromycin, 8μg/mL for sulbactam/ampicillin, clavulanic acid/amoxicillin (1 : 2, CVA/AMPC) and cefdinir, 16μg/mL for CVA/AMPC (1 : 14), flomoxef and clarithromycin, 32μg/mL for ampicillin.

Although there was no rapid increase in the antibacterial resistance, the prevalence of BLNAR was still over 50%. In order to ensure the appropriate chemotherapy, it is important to continue the surveillance of susceptibility among H. influenzae.

Morphological changes in penicillin-resistant Streptococcus pneumoniae(PRSP) and β-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae(BLNAR) after exposure to oral antibacterial agents could be observed over time under a phase-contrast microscope. Morphological changes in BLNAR were also observed using a scanning electron microscope. The organisms used in this study were ME19F strain identified as genotypi (c g) gPRSP (serotype: 19F) and JPH002 strain identified as gBLNAR (serotype: b). The antibacterial agents used were amoxicillin (AMPC), cefditoren (CDTR), tebipenem (TBPM), and tosufloxacin (TFLX). The concentration of each antibacterial agent to which the bacteria were exposed was set at the blood level one hour after Cmax when administered to children at the usual dose.

Bacteriolysis of gPRSP cells started after exposure of only 20 minutes to TBPM, and 90% of the cells were lysed within 2 hours. A high bactericidal action of TBPM on gPRSP was supported by these findings.

When gBLNAR was exposed to AMPC and TBPM, lysis from spheroplasts and cells with vacuoles were sometimes observed. In contrast, after gBLNAR was exposed to CDTR, lysis occurred after marked filamentation in the cells, but after exposure to TFLX, cells deduced to be killed after mild filamentation without lysis.

Time-dependent morphological changes that reflect the differences in bactericidal activity and PBP affinity among β-lactams provide beneficial information to select antibacterial agents.

This study aimed to compare the susceptibilities of 5 reference strains and 28 isolates of Candida spp., to micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and miconazole, obtained by visually determined minimum inhibitory concentration (MIC) using the agitation method (V-A), as described in the Clinical and Laboratory Standards Institute M27-A3 document; visual determinations without agitation (V-NA); and spectrophotometric determinations for the presence or absence of agitation (SP-A and SP-NA, respectively). Our results indicate that when the V-NA, SP-A, and SP-NA—the 3 alternative microdilution procedures for MIC endpoint determinations—were compared with the V-A, excellent agreements were observed between the V-NA and V-A rather than with the spectrophotometric methods (between the SP-A or SP-NA, and V-A). Furthermore, many errors occurred while using the SP-A method in the presence of agitation and some isolates showed major errors. Three of 5 isolates that showed very major errors between the spectrophotometric SP-A or SP-NA, and the reference V-A method were trailing isolates. Therefore, it was suggested that the MICs of Candida spp. obtained by the V-NA method were more precise than those by the conventional SP-A method.