Molecular Characteristics of Extended-Spectrum Beta-Lactamases and qnr Determinants in Enterobacter Species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing blaCTX-M, blaSHV, or blaTEM were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan

Distribution of antimicrobial resistance genes among ESBL-producing <i>Enterobacter</i> spp.

*<p>All <i>qnrA</i> were <i>qnrA1</i>.</p

Dendrogram and PFGE of <i>Xba</i>I-digested genomic DNAs from ESBL-producing <i>Enterobacter</i> species.

<p>EC: <i>Enterobacter cloacae</i>, EA: <i>Enterobacter aerogenes</i>. Strain No. and PFGE type correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037967#pone-0037967-t002" target="_blank">Table 2</a>.</p

Profile of <i>Enterobacter</i> spp. carrying ESBL genes and results of the boronic acid disk test.

<p>CTX: cefotaxime, CAZ: ceftazidime, FOX: cefoxitin, FEP: cefepime, MEM: meropenem, LVX: levofloxacin, AMK: amikacin, BA: 3-aminophenylboronic acid. EC: <i>Enterobacter cloacae</i>, EA: <i>Enterobacter aerogenes.</i></p