Antimicrobial susceptibility studies
Plasmid-mediated, inducible AmpC β-lactamase (DHA-1)-producing Enterobacteriaceae at a Korean hospital: wide dissemination in Klebsiella pneumoniae and Klebsiella oxytoca and emergence in Proteus mirabilis

https://doi.org/10.1016/j.diagmicrobio.2005.03.008Get rights and content

Abstract

The aim of the study was to investigate the phenotypic and genetic characteristics of recently emerging cefoxitin-resistant and induction-positive isolates of Escherichia coli, Klebsiella species, and Proteus mirabilis. Strains of Enterobacteriaceae were isolated at a Korean tertiary care hospital between June and December 2002. Induction was tested using cefoxitin and aztreonam disks, the blaDHA allele was detected by PCR, and pulsed-field gel electrophoresis (PFGE) patterns were also analyzed. Among the cefoxitin-resistant isolates, 2.7% of E. coli, 21.1% of Klebsiella pneumoniae, 32.0% of Klebsiella oxytoca, and 8.3% of P. mirabilis isolates showed induction, and were blaDHA-1 allele positive. To the best of our knowledge, this is the first report of blaDHA-1 in P. mirabilis. The MICs of ceftazidime, cefotaxime, and aztreonam increased significantly by higher inoculum, suggesting that their clinical usefulness is limited. Presence of multiple PFGE patterns and identical patterns in some isolates suggest that the widely disseminated blaDHA-1 in Klebsiella species was because of both horizontal and clonal spread.

Introduction

β-Lactamase production is the predominant mechanism for resistance to β-lactam antibiotics in Gram-negative bacilli. Resistance due to extended-spectrum β-lactamase (ESBL) appeared in Klebsiella pneumoniae and in other enterobacterial species, but they remained susceptible to cephamycins (Philippon et al., 2002).

Plasmid-mediated AmpC β-lactamases were first detected in 1988 (Bauernfeind et al., 1989, Bauernfeind et al., 1998, Papanicolaou et al., 1990), and subsequently various AmpC enzymes have been found, particularly in Klebsiella spp., Escherichia coli, and Proteus mirabilis (Paterson et al., 2003, Philippon et al., 2002). The majority of plasmid-mediated AmpC enzymes are expressed constitutively, with the exceptions of DHA, ACT-1, and CFE-1. DHA-1 was first reported in 1992 in Salmonella enteritidis in Saudi Arabia and France (Gaillot et al., 1997), DHA-2 in K. pneumoniae in France (Fortineau et al., 2001), ACT-1 in K. pneumoniae in the United States (Reisbig and Hanson, 2002), and CFE-1 in Citrobacter freundii in Japan (Nakano et al., 2004).

In Korea, cefoxitin-resistant E. coli and K. pneumoniae has been increasingly noted. A recent investigation detected 15 isolates with undetermined blaDHA subtype (Song et al., 2002). Recently, we have increasingly noted presence of induction-positive isolates of K. pneumoniae at a tertiary care hospital laboratory.

Some of the plasmid-mediated AmpC enzyme-producing isolates are susceptible to third-generation cephalosporins and aztreonam in vitro (Philippon et al., 2002), but their clinical utility is uncertain. As for DHA-producing organisms, even in vitro susceptibility data are limited, because only a few studies have been reported (Alvarez et al., 2004, Yan et al., 2002). In addition to the treatment problem, accurate differentiating methods for plasmid-mediated AmpC β-lactamase-producing isolates are required to improve surveillance and infection control (Perez-Perez and Hanson, 2002), but the current guidelines from the National Committee for Clinical Laboratory Standards (NCCLS, 2004) do not include any recommendation. Recently, Perez-Perez and Hanson (2002) recommended the use of multiplex PCR to identify family-specific AmpC β-lactamase genes.

The aim of this study was to determine the phenotypic and genetic characteristics of induction-positive strains of E. coli, Klebsiella spp., and P. mirabilis isolated at a Korean hospital. We also investigated the susceptibility, inoculum effect on the minimum inhibitory concentrations of antimicrobial agents, and the molecular epidemiological features of blaDHA-1 allele-positive strains.

Section snippets

Screening of inducible AmpC β-lactamase– and ESBL-producing isolates

Bacterial strains were isolated from patients at a Korean tertiary care hospital between June and December 2002. Species were identified by conventional methods or by using the Vitek GNI system (bioMérieux, Marcy l'Etoile, France). Cefoxitin-resistant E. coli, Klebsiella spp., and P. mirabilis isolates, detected by the disk diffusion method (NCCLS, 2002) using commercial disks and Mueller-Hinton agar (Becton Dickinson, Cockeysville, MD), were tested for induction by applying cefoxitin and

Detection of DHA-producing isolates

Of the 3,109 unduplicated isolates of E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis tested, 15.0%, 29.0%, 16.8%, and 9.7%, respectively, were resistant to cefoxitin (Table 1). Of the cefoxitin-resistant isolates, induction was observed in 8 of 291 (2.7%) E. coli, 55 of 261 (21.1%) K. pneumoniae, 8 of 25 (32.0%) K. oxytoca, and 1 of 12 (8.3%) P. mirabilis isolates.

blaDHA alleles were detected by PCR in all of the 72 induction-positive isolates. All of these isolates showed positive PCR

Discussion

Plasmid-mediated AmpC β-lactamase-producing gram-negative bacilli are being found increasingly in many parts of the world (Philippon et al., 2002), but reports on DHA-producing organisms are relatively rare. CMY-1-producing K. pneumoniae and E. coli have been prevalent and single amino acid-substituted CMY-1b (CMY-10) has emerged in Korea (Bauernfeind et al., 1998). Therefore, most of the plasmid-mediated AmpC β-lactamases in Korea was presumed to be these enzymes until other types were

Acknowledgment

This study was supported by the Yonsei University Research Fund (2003-35).

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