Mechanisms of Resistance to Imipenem and Ampicillin in Enterococcus faecalis

We found ampicillin- and imipenem-resistant isolates of vanA-possessing Enterococcus faecalis with MICs of 8 to 16 μg/ml and 4 to 32 μg/ml, respectively. There have been few reports about penicillin- and imipenem-resistant E. faecalis. Two mechanisms of beta-lactam resistance in E. faecalis, the production of beta-lactamase and the overproduction of penicillin-binding proteins (PBPs), have been reported. The resistant isolates in the current study did not produce any beta-lactamases and analysis of the PBPs showed no overproduction. However, the affinities of PBP4 for beta-lactams in the resistant strains were lower than those of susceptible strains but the affinities of other PBPs for beta-lactams did not change. Accordingly, whole pbp4 fragments from these resistant isolates were sequenced. Two amino acid substitutions at positions 520 and 605 were observed in the highly resistant strains compared to the susceptible ones, Pro520Ser and Tyr605His, and a single Tyr605His amino acid substitution was found in the low-resistance strains. These two point mutations exist in the region between the active-site-defining motifs SDN and KTG of the penicillin-binding domain, the main target of beta-lactams. A strong correlation was seen between these substitutions and decreasing affinities of PBP4 to beta-lactams. In E. faecalis, resistance due to mutations in PBPs has not been reported, though it has in Enterococcus faecium. Our results suggest that development of high-level resistance to penicillins and imipenem depends on point mutations of PBP4 at positions 520 and 605

Nasopharyngeal Haemophilus influenzae Carriage in Japanese Children Attending Day-Care Centers▿

We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Haemophilus influenzae carriage in healthy children in day-care centers (DCCs). A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 184) and the following winter (n = 179). We obtained 172 H. influenzae isolates and analyzed them by antimicrobial susceptibility testing, PCR for blaTEM-1 and the penicillin-binding protein (PBP) gene, and pulsed-field gel electrophoresis (PFGE). The overall carriage rate was 47.4% (172/363), and 37.2% of the isolates (64/172) were ampicillin (AMP) resistant. All the resistant isolates had a PBP mutation(s), while only three isolates had TEM-1. The carriage rate was significantly higher in the winter than in the summer (56.4% and 38.6%, respectively), owing to the increase in the numbers of AMP-susceptible H. influenzae isolates in the winter. Children aged ≤3 years showed a higher rate of carriage of H. influenzae isolates with an AMP resistance gene(s) than those aged ≥4 years (21.9% and 12.6%, respectively). Forty-two strains with different PFGE patterns were obtained from among the 172 isolates. Only five strains were observed in both seasons. None of the strains isolated in the summer was isolated from the same carrier in the winter. Twenty-seven strains (64.3%) were isolated from two or more children, and 25 of these were each isolated from children belonging to the same DCC. These results indicate the spread of H. influenzae, particularly those with a PBP mutation(s), and the highly vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan

Molecular epidemiology and clinical features of extended-spectrum beta-lactamase- or carbapenemase-producing Escherichia coli bacteremia in Japan.

OBJECTIVES:To identify risk factors and clinical outcomes in patients with bacteremia due to extended-spectrum beta-lactamase (ESBL) or carbapenemase-producing Escherichia coli, as well as to determine the prevalence and genetic background of such isolates. METHODS:Case control study was performed with patients with E. coli bacteremia between January 2008 and May 2013 (n = 115) at a tertiary university hospital in Japan. Cases had ESBL-producing E. coli (ESBL-EC) whereas controls had non-ESBL-producing E. coli (non-ESBL-EC) isolates. A retrospective chart review was performed to identify risk factors and clinical outcomes. Isolates were characterized by antimicrobial susceptibility testing, polymerase chain reaction analysis for beta-lactamase genes, and multi-locus sequence typing. RESULTS:Of 115 unique cases of E. coli bacteremia, 30 (26.1%) were due to ESBL-EC and three (2.6%) were due to carbapenemase-producing E. coli. All three carbapenemase-producing E. coli isolates were IMP-6 and concurrently produced ESBL (ESBL/IMP-6-EC). ESBL-EC isolates showed multidrug resistance. Of the ESBL-EC isolates, CTX-M-27 was the most prevalent (33.3%), followed by CTX-M-14 (30%). Multi-locus sequence typing revealed that 19 (63.3%) isolates were ST131. The multivariate analysis identified nursing home-associated infections and antibiotic administration in the preceding 30 days as risk factors for ESBL-EC bacteremia. The 14-day mortality non-ESBL-EC, ESBL-EC, and ESBL/IMP-6-EC was 4.7% (4/85), 20% (6/30), and 66.7% (2/3), respectively. CONCLUSIONS:CTX-M-27, CTX-M-14, and ST131 were the most prevalent ESBL-EC isolates from bacteremic patients in a Japanese hospital. Further studies with larger sample sizes are warranted to investigate the clinical significance of ESBL-EC and ESBL/IMP-6-EC

Induction of plasmid-mediated AmpC β-lactamase DHA-1 by piperacillin/tazobactam and other β-lactams in Enterobacteriaceae.

Chromosomal AmpC β-lactamase induction by several types of β-lactams has been reported, but not enough data are available on DHA-1 β-lactamase, a plasmid-mediated AmpC β-lactamase. Therefore, we evaluated the DHA-1 β-lactamase induction by various antibiotics including piperacillin/tazobactam (PIP/TZB) in this study. Six strains (Enterobacter cloacae 2 strains, Citrobacter freundii 1 strain, Serratia marcescens 2 strain, and Morganella morganii 1 strain) possessing chromosomal inducible AmpC β-lactamase were used as controls. Four strains (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain, and C. koseri 1 strain) possessing DHA-1 β-lactamase were used. The β-lactamase activities were determined by a spectrophotometer using nitrocefin. β-lactamase induction by PIP, PIP/TZB was not observed in any strains and β-lactamase induction by third- and fourth-generation cephems was not observed in most strains. The induction ratios of the chromosomal AmpC β-lactamase in the reference group by PIP/TZB were <1.51, and those of the DHA-1 β-lactamase were <1.36, except for K. pneumoniae Rkp2004 (2.22). The β-lactamase induction by first- and second-generation cephems, flomoxef, and carbapenem differed in each strain. Cefmetazole (CMZ) strongly induced β-lactamase. This study demonstrated that the induction of DHA-1 β-lactamase was similar to that of chromosomal AmpC using various Enterobacteriaceae, although the induction of β-lactamase in both groups by PIP/TZB was low. We also reported that the induction of PIP/TZB, a β-lactamase inhibitor combination antibiotic, against various AmpC-producing Enterobacteriaceae, including DHA-1 producers, was low

Genetic Profiles of Fluoroquinolone-Resistant Escherichia coli Isolates Obtained from Patients with Cystitis: Phylogeny, Virulence Factors, PAIusp Subtypes, and Mutation Patterns▿

The low virulence of quinolone- and fluoroquinolone-resistant Escherichia coli strains is known, although the reasons for this remain unclear. We surveyed the mutation patterns of quinolone resistance determining regions (QRDRs), phylogenetic distribution, prevalence of 18 urovirulence genes, and PAIusp subtypes in 89 fluoroquinolone-resistant E. coli (FQREC) isolates obtained from patients with cystitis and compared them with those of their fluoroquinolone-susceptible counterparts (FQSEC). Phylogenetic group B2 was significantly less prevalent in FQREC than in FQSEC (49% versus 78%; P = 0.0138), but it still dominated, followed by phylogroup D (35%), in FQREC. When the prevalences of virulence factor (VF) genes were compared between FQREC and FQSEC, sfa/foc, cnf1, hly, kpsMT, ompT, ibeA, usp, and iroN showed significantly lower prevalences in FQREC than in FQSEC (1.1% versus 24% [P < 0.0001], 0% versus 29% [P < 0.0001], 7.9% versus 33% [P < 0.0001], 74% versus 90% [P = 0.01], 71% versus 87% [P = 0.017], 5.6% versus 37% [P < 0.0001], 54% versus 82% [P < 0.0001], and 7.9% versus 32% [P = 0.0001], respectively), whereas aer, iha, and ETTT showed significantly higher prevalences in FQREC (85% versus 36% [P < 0.0001], 66% versus 29% [P < 0.0001], and 53% versus 16% [P < 0.0001], respectively). Furthermore, a similar difference in prevalences of uropathogenic VF genes was seen between FQREC and FQSEC in phylogroup B2. This indicated that the low virulence in FQREC was intimately correlated with a lesser distribution of VFs in phylogroup B2, which dominated in FQREC and FQSEC. It was interesting that the mutation pattern of Ser83Leu and Asp87Asn encoded in gyrA and Ser80Ile and Glu84Val encoded in parC was frequently found in FQREC isolates that belonged to phylogroup B2 and that most of these isolates showed PAIusp subtype 2a. PAIusp subtypes 1a, 1b, and 2b, which were frequently seen in FQSEC, were rarely found in FQREC. These results suggested that the acquisition of fluoroquinolone resistance, e.g., mutations in QRDRs, might be a specific event in limited strains, such as those that possess PAIusp subtype 2a in phylogroup B2

Outbreak of Cefozopran (Penicillin, Oral Cephems, and Aztreonam)-Resistant Neisseria gonorrhoeae in Japan

We have previously reported that the Neisseria gonorrhoeae isolates from clinical failure cases treated with cefdinir and aztreonam, β-lactams exhibited high MICs. These resistant isolates were clearly separated from the isolates exhibiting a low level of resistance to β-lactams as shown by the MIC distribution of cefozopran. Restriction fragment length polymorphism DNA typing revealed that the outbreak of cefozopran-resistant isolates in Kitakyushu, Japan, occurred as a result of clonal spread