Investigations on yearly changes in tebipenem susceptibility of bacterial isolates from pediatric patients -A post-marketing surveillance of tebipenem pivoxil granules for pediatric.

We conducted the post-marketing surveillance of tebipenem pivoxil (Orapeneme fine granules 10% for pediatric), an oral carbapenem antibacterial agent, to investigate changes in bacterial susceptibility against tebipenem (TBPM). Bacterial strains used in this surveillance were methicillin-susceptible Staphylococcus aureus (MSSA: 303 strains), Streptococcus pneumoniae (554 strains), other Streptococcus spp. (242 strains: including Streptococcus pyogenes 133 strains), Moraxella catarrhalis (306 strains) and Haemophilus influenzae (506 strains) isolated from pediatric patients in 15 medical facilities in Japan between April 2010 and March 2015. Investigation was conducted three times (April 2010-March 2011, April 2012-March 2013 and April 2014-March 2015), and in any of these investigation periods, there were a large number of isolates from infants in terms of the frequency of isolates by age. The MIC90s of TBPM against MSSA, S. pneumoniae, other Streptococcus spp., M. catarrhalis and H. influenzae in these investigations were 0.015-0.03, 0.06, 0.008-0.015 (0.002 for S. pyogenes), 0.03 and 0.5-1 µg/mL, respectively, which were less than 2-fold, and a remarkable increase in MIC90 was not shown. On the other hand, the MIC50s of carbapenems including TBPM and penicillins against S. pneumoniae decreased to 1/4-1/8 during the investigation periods, and decreased gPRSP*¹ (48.7% - 26.1%) and increased gPISP (2x)*² (24.1% -+ 46.8%) were suggested to be involved in these changes in susceptibility. In S. pneumoniae, a decrease of macrolides-resistant strains due to mefA*³ (38.5% - 18.8%) and an increase of macrolides-resistant strains due to ermB*4 (41.7% - 62.4%) were noted. In H. influenzae, the frequencies of gBLNAR*5 and ß-lactamase-producing strains were about 60-70% and 7-9%, respectively, and a remarkable change in susceptibility was not shown. As a result of investigations in the susceptibility of clinical isolates collected from pediatric patients as post-marketing surveillance, there was no decrease in TBPM susceptibility noted.

Horizontal gene transfer of ftsI, encoding penicillin-binding protein 3, in Haemophilus influenzae.

Horizontal gene transfer has been identified in only a small number of genes in Haemophilus influenzae, an organism which is naturally competent for transformation. This report provides evidence for the genetic transfer of the ftsI gene, which encodes penicillin-binding protein 3, in H. influenzae. Mosaic structures of the ftsI gene were found in several clinical isolates of H. influenzae. To identify the origin of the mosaic sequence, complete sequences of the corresponding gene from seven type strains of Haemophilus species were determined. Comparison of these sequences with mosaic regions identified a homologous recombination of the ftsI gene between H. influenzae and Haemophilus haemolyticus. Subsequently, ampicillin-resistant H. influenzae strains harboring identical ftsI sequences were genotyped by pulsed-field gel electrophoresis (PFGE). Divergent PFGE patterns among beta-lactamase-nonproducing ampicillin-resistant (BLNAR) strains from different hospitals indicated the potential for the genetic transfer of the mutated ftsI gene between these isolates. Moreover, transfer of the ftsI gene from BLNAR strains to beta-lactamase-nonproducing ampicillin-susceptible (BLNAS) H. influenzae strains was evaluated in vitro. Coincubation of a BLNAS strain (a rifampin-resistant mutant of strain Rd) and BLNAR strains resulted in the emergence of rifampin- and cefdinir-resistant clones at frequencies of 5.1 x 10(-7) to 1.5 x 10(-6). Characterization of these doubly resistant mutants by DNA sequencing of the ftsI gene, susceptibility testing, and genotyping by PFGE revealed that the ftsI genes of BLNAR strains had transferred to BLNAS strains during coincubation. In conclusion, horizontal transfer of the ftsI gene in H. influenzae can occur in an intraspecies and an interspecies manner.

Amino acid substitutions in mosaic penicillin-binding protein 2 associated with reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae.

The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

Molecular evolution of beta-lactam-resistant Haemophilus influenzae: 9-year surveillance of penicillin-binding protein 3 mutations in isolates from Japan.

A total of 621 clinical isolates of Haemophilus influenzae collected in Japan between 1995 and 2003 were studied for their susceptibilities to several antimicrobial agents, beta-lactamase production, and amino acid substitutions in penicillin-binding protein 3 (PBP 3). Over the four study periods (first period, 1995 to 1996; second period, 1997 to 1998; third period, 2000 to 2001; fourth period, 2002 to 2003), the susceptibilities to beta-lactam agents decreased and the incidence of isolates with substitutions at positions 377, 385, 389, 517, and/or 526 in PBP 3 increased from 28.8% to 52.0%. Five hundred seventy-one beta-lactamase-nonproducing isolates were grouped into 18 classes, based on the pattern of the five mutations in PBP 3. The Asp526Lys substitution led to 6.0-, 4.3-, 2.4-, and 5.4-fold increases in amoxicillin-clavulanic acid, cefdinir, cefditoren, and faropenem resistance, respectively. PBP 3 with multiple substitutions (Met377Ile, Ser385Thr, and/or Leu389Phe) together with Asp526Lys resulted in increased resistance compared to that for PBP 3 with the Asp526Lys substitution alone. These results indicate that mutations at these five positions increased resistance to most beta-lactams. Although a significant change in the prevalence of beta-lactamase-producing strains was not observed, the proportions of those possessing both PBP 3 alterations and beta-lactamase production have slightly increased (from 1.4% to 5.0%). The ROB-1 beta-lactamase was rare, but this is the first report of this beta-lactamase in Japan.