First outbreak of multidrug-resistant Klebsiella pneumoniae producing both SHV-12-type extended-spectrum beta-lactamase and DHA-1-type AmpC beta-lactamase at a Korean hospital.

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital.
MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE).
RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microg/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested.
CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.

INTRODUCTION

β-Lactamase production is the predominant mechanism for resistance to β-lactams in Enterobacteriaceae. Klebsiella pneumoniae isolates are often multidrug-resistant and are the major hosts for extended-spectrum β-lactamases (ESBLs).1 In particular, K. pneumoniae strains have also acquired plasmid-mediated AmpC enzymes. Unlike ESBLs, AmpCs are poorly inhibited by β-lactamase inhibitors and are less active against cefepime and cefpirome than ESBLs. AmpCs confer resistance to oxyimino- and 7-α-methoxy-cephalosporins.2 The prevalence of ESBLs and plasmid-mediated AmpC β-lactamases in K. pneumoniae has been rising in Korea. Of the ESBLs and the acquired AmpC enzymes, SHV-12 and DHA-1 are the most prevalent and widespread in Korea, respectively.3,4 Occurrence of ESBL and AmpC β-lactamase co-producing K. pneumoniae has been reported,5,6 but outbreaks of the isolates are extremely rare.

Over a three-month period in 2004, K. pneumoniae isolates resistant to cefotaxime, ceftazidime, cefepime, aztreonam, and cefoxitin were isolated from the patients hospitalized at a university hospital in Korea. The aim of our study was to analyze clinical and molecular epidemiology of the multidrug-resistant K. pneumoniae isolates.

MATERIALS AND METHODS

Patients and bacterial isolates

From May to July 2004, a total of 11 non-duplicated isolates of K. pneumoniae that were resistant to cefotaxime, ceftazidime, aztreonam, and cefoxitin, were identified by Clinical Laboratory and Standards Institute (CLSI) disk diffusion method.7 Ten of the 11 isolates were resistant to cefepime. All isolates were also resistant to aminoglycosides (amikacin, gentamicin, and tobramycin), ciprofloxacin, and trimethoprim-sulfamethoxazole. All isolates were suggested to produce ESBL based on the double disk test.8 Table 1 shows the clinical background of patients for each isolate and their respective treatment outcomes. The first isolate from each source of patient was included in the study.

Table 1

Clinical and PFGE Data of the Patients with SHV-12 and DHA-1 Co-Producing K. pneumoniae Isolates

MICU, medical intensive care unit; IM, internal medicine; RM, rehabilitation medicine; NS, neurosurgery; DM, diabetes mellitus; COPD, chronic obstructive pulmonary disease; SLE, systemic lupus erythematosus AMK, amikacin; CFT, cefotiam; CFS, cefoperazone-sulbactam; CIP, ciprofloxacin; CLI, clindamycin; CRO, ceftriaxone; CTX, cefotaxime; FEP, cefepime; FLM, flomoxef; IPM, imipenem; ISP, isepamicin; LEV, levofloxacin; MPM, meropenem; NET, netilmicin; PTZ, piperacillin-tazobactam; TEI, teicoplanin; VAN, vancomycin.

Susceptibility testing and β-lactamase phenotype

Minimal inhibitory concentrations (MICs) were determined by the broth microdilution method according to the CLSI guidelines.9 The antimicrobial agents used were piperacillin (Sigma, Steinheim, Germany), tazobactam (Yuhan, Seoul, Korea), cefoxitin (Sigma), ceftazidime (Glaxo SmithKline, Harlow, UK), aztreonam (Bristol-Myers Squibb, Prinston, NJ, USA), cefepime (Boryung, Seoul, Korea), and imipenem (Merk, West Point, PA, USA). ESBL and AmpC phenotypes were investigated by the CLSI ESBL confirmatory disk diffusion test7 and the AmpC disk test,10 respectively.

Isoelectric focusing (IEF)

Crude β-lactamase preparations were obtained from the isolates by sonication and subjected to analytical IEF as described previously.11 β-Lactamase activity was detected by overlaying the gels with 0.5 mM nitrocefin in 0.1 M phosphate buffer, pH 7.0.

PCR amplification and DNA sequencing

Plasmids from the isolates were extracted by a rapid alkaline lysis procedure.12 PCR with blaDHA-13,14 and blaSHV-specific primers and subsequent sequencing of the PCR products were performed. An 1,071-bp fragment of the blaSHV gene was amplified with the primers SHV-F (5'-CGC CGG GTT ATT CTT ATT TG-3') and SHV-R (5'-CCA CGT TTA TGG CGT TAC CT-3'). Sequence alignment and analysis were performed on-line using the BLAST program of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

Pulsed-field gel electrophoresis (PFGE)

PFGE was carried out with a CHEF-DRII System (Bio-Rad, Hercules, CA, USA). XbaI-digested genomic DNA was prepared according to the instruction of Bio-Rad, and fragments were separated for 20 h at 6 V/cm at 14℃, with initial and final pulse times of 5 and 40 s, respectively. DNA bands were visualized by staining the gel with ethidium bromide and photographed.

RESULTS

Bacterial strains and clinical characteristics

K. pneumoniae isolates were first found to show resistance to cefotaxime, ceftazidime, cefepime, aztreonam, and cefoxitin, evidenced by disk diffusion test. During a particular time period, a total of 11 isolates showing very similar antibiogram were isolated. These results strongly suggested that there is coexistence of ESBL and AmpC in these isolates. The isolates were obtained from 11 inpatients at a hospital; 43 to 87 years old, 9 males and 2 females, from May to July 2004. Of 11 isolates, 6 isolates were recovered from sputum, 4 isolates from urine, and 1 isolate from a wound. Isepamicin and cefoperazone-sulbactam had most frequently been administered in 10 and 9 patients, respectively. Two of these 11 patients died, and the infection in the remaining 9 patients improved with therapy (Table 1).

Antimicrobial susceptibility and β-lactamase phenotype

These 11 isolates showed very similar susceptibility profiles, characterized by elevated MICs (≥ 128 µ g/mL) of piperacillin, piperacillin-tazobactam, cefoxitin, ceftazidime, and aztreonam, while MICs of imipenem for all of them were ≤ 2 µ g/mL. The MICs of cefepime varied among the strains isolated (Table 2). All the isolates yielded positive to AmpC disk tests for AmpCs, double disk tests and CLSI ESBL confirmatory disk for ESBLs.

Table 2

MIC Distributions of the 11 SHV-12 and DHA-1 Co-Producing K. pneumoniae Isolates

β-Lactamase genotype and PFGE profiles

These isolates had β-lactamase bands at isoelectric points of 7.6 and 7.8. The blaSHV and blaDHA alleles were detected by PCR, and the sequences were identical to those of blaSHV-12 and blaDHA-1. The PFGE analysis identified one major profile with four subtypes (Ia, Ib, Ic, and Id) (Table 1, Fig. 1).

Fig. 1

PFGE dendrograms of 11 SHV-12 and DHA-1 co-producing K. pneumoniae isolates. The scale indicates the percent of genetic similarity. The molecular size marker includes yeast chromosomes, Saccharomyces cerevisiae (Bio-Rad Laboratories, Hercules, CA, USA).

DISCUSSION

Expanded-spectrum cephalosporin-resistant organisms due to ESBL production have been reported in many countries.15 ESBL production among K. pneumoniae isolates in Korea was 28.4% during 2002 - 2003,16 which is significantly higher than the United States, Taiwan, or Hong Kong.1,6 SHV-12, SHV-2a, and TEM-52 were common in the late 1990s in Korea.17 In 2002, an SHV-12-like enzyme was prevalent, but SHV-2a and TEM-52 were found in a few isolates in Korea.18 DHA-1, an inducible plasmid-mediated AmpC that was first identified in Salmonella enteritidis isolated in Saudi Arabia in 1992,19 has been reported in several other countries.5,20 DHA-1 enzyme is the most frequently identified among the AmpC β-lactamase-producing K. pneumoniae isolates in Korea.4 Genes encoding both ESBLs and plasmid-mediated AmpC β-lactamases are usually located on large multidrug resistance plasmids. Therefore, both types of enzymes are typically associated with resistance to multiple antibiotics, thus leaving few therapeutic options.21

In our study, the isolates that co-produced SHV-12 and DHA-1 enzymes showed high MICs of ceftazidime, cefepime, aztreonam, and cefoxitin. Four (8.7%) of 46 plasmid-mediated AmpC-producing K. pneumoniae isolates also had an ESBL at a Korean hospital.14 Furthermore, the outbreak of K. pneumoniae isolates co-producing SHV-2a ESBL and DHA-1 AmpC has been reported in Korea.22 The nosocomial infections caused by DHA-1 and SHV-12 β-lactamase producers occurred in Japan.23 However, this is the first report of its occurrence in Korea.

In this study, K. pneumoniae strains were recovered from patients with a mean age of 58 years. These data agree with a previous study showing usually older with ESBL.24 However, the mean age (55 years old) of the other 164 patients who were infected with broad-spectrum cephalosporins-susceptible K. pneumoniae was older in age (data not shown). The extended-spectrum cephalosporins had preferentially been used as first-line drugs at the hospital. Among 11 patients, all of them had received the extended-spectrum cephalosporins (9 had received cefoperazone-sulbactam). The emergence of multi-resistant organisms, characterized by a heavy use of antimicrobial agents, may provide nosocomial transmission.25 In our present study, the emergence of K. pneumoniae isolates co-producing SHV-12 and DHA-1 could be correlated with heavy use of extended-spectrum cephalosporins. PFGE of XbaI-digested genomic DNA revealed that 10 out of the 11 isolates in this study, likely corresponded to a single clone, whereas one isolate (WJ34) was not clonally related.

In conclusion, the first outbreak caused by K. pneumoniae isolates co-producing SHV-12 ESBL and DHA-1 AmpC β-lactamase occurred at a university hospital in Korea. The spreads was caused by an endemic clone of the resistance gene.