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Antimicrobial Agents and Chemotherapy, April 2009, p. 1642-1644, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Two Clinical Strains of Klebsiella pneumoniae Carrying Plasmid-Borne blaIMP-4, blaSHV-12, and armA Isolated at a Pediatric Center in Shanghai, China{triangledown}

Ying Liu,1 Bei Zhang,2 Qing Cao,2 Weichun Huang,2 Lisong Shen,1 and Xuan Qin3,4*

Clinical Laboratory Diagnostic Center, Xin Hua Hospital, Shanghai, China,1 Infectious Disease Department, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, 1678 Dong Fang Road, Shanghai 200127, China,2 Microbiology Laboratory, Seattle Children's Hospital, Seattle, Washington,3 Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington4

Received 2 October 2008/ Returned for modification 6 November 2008/ Accepted 10 January 2009


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ABSTRACT
 
Two cases of pulmonary infection due to strains of multidrug-resistant Klebsiella pneumoniae were investigated. Beta-lactamase determinants, such as blaIMP-4 and blaSHV-12, and the 16S rRNA methyltransferase-encoding gene armA were detected in these plasmid-bearing organisms. The integron-borne blaIMP-4 and armA contained intervening sequences highly related to those of a Vibrio cholerae O139 plasmid found in Hangzhou, China.


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INTRODUCTION
 
Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases (ESBLs), particularly with the determinant blaSHV-12, have been extensively reported in China (15, 16). The use of carbapenems as the treatment of last resort for ESBL producers has contributed to the emergence of carbapenemase-producing organisms, such as Pseudomonas aeruginosa, Acinetobacter spp., and members of the Enterobacteriaceae (11). The plasmid-carried and integron-borne class A enzyme determinant blaKPC, and class B metallo-β-lactamase determinants blaIMP and blaVIM, as well as the aminoglycoside resistance gene armA, have been characterized (3, 11, 14). For this report, two multidrug-resistant K. pneumoniae clinical isolates containing multiple plasmids and multiple resistance gene determinants were analyzed.

Two 6-month-old infants with congenital heart disease were admitted to Shanghai Children's Medical Center for treatment of pulmonary infections in November 2005 and January 2006. Their hospital stays did not overlap. Prior to initial microbiological evaluation, these patients were treated with oxacillin, cefuroxime, cefotaxime (CTX), ceftazidime (CAZ), cefoperazone-sulbactam, and meropenem (MEM). Multidrug-resistant isolates of K. pneumoniae (one from each patient, designated KpSCMC1 and KpSCMC2) were recovered from tracheal aspirates. Antibiotic susceptibilities were determined initially with the Vitek system (bioMérieux), and multidrug resistance patterns were further evaluated by the disk diffusion and Etest (Biomérieux) MIC method using standard interpretive criteria (5). Both isolates showed similar susceptibility results, which included resistance to all cephalosporins and aminoglycosides (Table 1). It was of interest that the zones of inhibition to imipenem (IMP) measured 21 mm for both isolates but the zones of inhibition to MEM measured 13 mm and 14 mm. Given the criterion of ≥16 mm for "susceptible" to MEM and IMP by disk diffusion, both isolates were further tested by Etest. KpSCMC1 and KpSCMC2 produced MICs to IMP of 3 mg/liter and 2 mg/liter and to MEM of 8 mg/liter and 4 mg/liter, respectively, bordering precariously on the breakpoint of ≤4 mg/liter for susceptible. Both isolates were sensitive to ciprofloxacin, although KpSCMC2 was resistant to nalidixic acid (NAL). Their susceptibilities to trimethoprim-sulfamethoxazole appeared to be chiefly due to susceptibility to trimethoprim alone, but not to sulfonamides.


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  		TABLE 1. Relevant phenotypic and genotypic characteristics of the K. pneumoniae clinical isolates in this study

Potential gene determinants associated with these resistance phenotypes were further investigated by PCR and nucleotide sequencing using previously published primers (1, 8, 11). PCR amplification from plasmid DNA preparations of KpSCMC1 and KpSCMC2 yielded corresponding products for int1, blaIMP, blaSHV, blaTEM, armA, and sul1, but not for blaCTX-M, blaCMY, or qnr. Sequencing of the amplified products revealed the presence of blaIMP-4, blaSHV-12, blaTEM-1, and armA in both isolates.

The resistance gene organization and environments were further resolved by overlapping PCR techniques. The blaIMP-4-containing plasmids identified in both isolates appeared identical in their complex integron structures as carriers of antimicrobial resistance genes. The gene determinants were organized in the order of int1-blaIMP-4-orfII-orfIII-qacE{Delta}1-sul1-orf5-orf6-orf513 (or ISCR)-tnp-Acp1-armA (GenBank FJ384365). A segment of DNA encoding both OrfII (a putative reverse transcriptase) and OrfIII (a conserved hypothetical protein) was found to contain sequence similarity to a cognate segment (GenBank EU116440) described in a plasmid (pMR150) isolated from a Vibrio cholerae O139 strain in Hangzhou, China (9). This similarity in corresponding regions was found both at the DNA level (88% nucleotide identity), over a 1.5-kb segment, and at the deduced amino acid level (93% identity over 392 OrfII residues). Based on a GenBank search, only one other integron-borne orfII region showed a close match at the DNA level (85% nucleotide identity), over a 1.2-kb fragment (GenBank AY204504) in two Salmonella enterica serovar Typhimurium strains (DT208) isolated from retail meat in the United States (4).

It was of note that the parent strain KpSCMC2 gave rise to a fully carbapenem-sensitive segregant after unintentional in vitro passages, which was thus designated KpSCMC2c (carbapenemase-cured phenotype). Strain KpSCMC2c exhibited a characteristic ESBL phenotype with much reduced MICs to IMP (0.064 mg/liter) and MEM (0.25 mg/liter) and was fully susceptible to all aminoglycosides (Table 1). Strain KpSCMC2c was PCR positive only for blaTEM-1 and blaSHV-12 and was negative for int1, blaIMP, orf513, and armA, suggesting that blaSHV-12 was not located on the same plasmid with the int1 cluster and thus did not cosegregate with the int1 integron-borne resistance genes. Strain KpSCMC2c evidently retained its parental NAL resistance phenotype, likely due to a chromosomal mutation(s) which has been commonly documented in the quinolone resistance-determining regions of gyrA and/or parC (2). By genetic fingerprinting using pulsed-field gel electrophoresis, KpSCMC1 was found to be unrelated to KpSCMC2 and KpSCMC2c, while the latter two were indistinguishable (Fig. 1).


Figure 1
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  		FIG. 1. Pulsed-field gel electrophoresis patterns for the K. pneumoniae strains. Restriction enzyme XbaI was used for genomic DNA digestion, and K. pneumoniae ATCC 13883 was an unrelated type strain used as a control. (Pulsed-field gel electrophoresis was performed by Molecular Epidemiology, Inc., Lake Forest Park, WA.)

Similar to results in previous reports (6, 7), the two K. pneumoniae isolates in this study showed decreased susceptibilities to MEM but troublesome initial "susceptible" results to IMP by both disk diffusion and Etest. Given the MIC susceptibility breakpoint of ≤4 mg/liter for both MEM and IMP, routine susceptibility testing using automated instrumentation may fail to detect such resistance. As with the false-negative results using Etest MBL strips to characterize metallo-β-lactamase production, we agree with others that Enterobacteriaceae isolates showing an IMP MIC of ≥2 mg/liter should be checked for carbapenem resistance (7, 10, 13, 17).

In summary, this study investigated two K. pneumoniae clinical isolates that showed multidrug resistance to β-lactam and aminoglycoside antimicrobial drug classes, together with the emergence of quinolone resistance in the second isolate. Integron-borne gene cassettes featuring int1 and/or ISCR are remarkably versatile in trapping multidrug-resistance genes, which has resulted in some untreatable "superbugs" (11, 12). The identification of a putative reverse transcriptase (OrfII) coding sequence that is highly related to those reported in a V. cholerae O139 strain from a nearby province, together with the coexistence of blaIMP-4-armA, raises the possibility that active dissemination of resistance genes may proceed by means of capturing recombinant elements of all kinds. Moreover, the finding of blaSHV-12 on a separate plasmid, together with what is likely a chromosomal quinolone resistance-determining region mutation(s) in strain KpSCMC2, illustrates the magnitude of the genetic capacity in these organisms when encountering antimicrobial pressure in compromised human hosts. The emergence of multidrug resistance in an otherwise commensal enteric colonizer is an operational conundrum for a pediatric surgery center where the patient population is especially vulnerable. This also represents a serious diagnostic challenge for microbiology laboratories, as well as challenges for infection control and antimicrobial drug management.


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Nucleotide sequence accession number.
 
The sequence of the blaIMP-4-containing plasmid was submitted to GenBank under accession no. FJ384365.


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ACKNOWLEDGMENTS
 
We thank Justin Kwong and Treva Tsosie for their technical assistance. We thank Emmanouil Galanakis for his review of these cases and Michael A. McNutt for his critical review of the manuscript.

This study was supported in part by a Project HOPE (Millwood, VA) training grant for physicians specializing in clinical microbiology and infectious diseases.


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology Laboratory, A6901, Seattle Children's Hospital, Seattle, Washington 98105. Phone: (206) 987-2586. Fax: (206) 987-3840. E-mail: xuan.qin{at}seattlechildrens.org Back

{triangledown} Published ahead of print on 21 January 2009. Back


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Antimicrobial Agents and Chemotherapy, April 2009, p. 1642-1644, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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