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Antimicrobial Agents and Chemotherapy, July 2008, p. 2680-2682, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.00158-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Presence of Plasmid-Mediated Quinolone Resistance in Klebsiella pneumoniae Isolates Possessing blaKPC in the United States{triangledown}

Andrea Endimiani,1* Lenore L. Carias,1 Andrea M. Hujer,1 Christopher R. Bethel,1 Kristine M. Hujer,1 Federico Perez,2 Rebecca A. Hutton,1 William R. Fox,3 Geraldine S. Hall,3 Michael R. Jacobs,4 David L. Paterson,5 Louis B. Rice,1 Stephen G. Jenkins,6 Fred C. Tenover,7 and Robert A. Bonomo1,8*

Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106,1 Division of Infectious Diseases and HIV Medicine, University Hospital Case Medical Center, Cleveland, Ohio 44106,2 Cleveland Clinic Foundation, Cleveland, Ohio 44195,3 Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,4 University of Pittsburgh Medical Center, Pittsburgh, PA 15212,5 Mount Sinai Medical Center, New York, New York 10029,6 Office of Antimicrobial Resistance, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,7 Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 441068

Received 4 February 2008/ Returned for modification 16 March 2008/ Accepted 12 April 2008


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ABSTRACT
 
The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA] was evaluated among 42 blaKPC-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the blaKPC-3 and qnrB19 genes on the same conjugative plasmid, whereas another carried the blaKPC-3 and qnrA1 genes on separate plasmids.


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TEXT
 
The rapid spread of Klebsiella pneumoniae carbapenemases (KPCs) among members of the family Enterobacteriaceae represents an escalating global threat (15). Since blaKPC genes confer resistance to all β-lactams, the only therapeutic options for treating infections due to organisms possessing these β-lactamases are quinolones, aminoglycosides, polymyxins, or combinations of agents for which there are few data on efficacy (15). Unfortunately, high-level resistance to ciprofloxacin, gentamicin, and amikacin is also frequently observed among blaKPC-containing K. pneumoniae isolates (2, 3).

Quinolone resistance among Enterobacteriaceae is usually mediated by chromosomal mutations in the genes encoding DNA gyrase and topoisomerase IV. Plasmid-mediated quinolone resistance (PMQR) can arise from the expression of proteins encoded by the qnrA, -B, and -S genes that are able to protect the DNA gyrase. In addition, an aminoglycoside acetyltransferase encoded by the aac(6')-Ib-cr gene also confers ciprofloxacin resistance (10, 12). The qnr and aac(6')-Ib-cr loci are frequently found among Enterobacteriaceae producing AmpC enzymes, extended-spectrum β-lactamases, or both (1, 7, 8, 13). A plasmid-mediated quinolone efflux pump (i.e., the qepA gene) also has been described in Escherichia coli (17).

Recently, Mendes et al. reported the detection of a single K. pneumoniae isolate in China possessing blaKPC-2 and qnrB4 on the same conjugative plasmid (6). However, the prevalence of PMQR determinants among K. pneumoniae isolates producing KPCs has not yet been examined. Therefore, we studied a set of K. pneumoniae isolates collected at five major health care institutions located in the eastern United States to estimate the frequency of PMQR among KPC producers.

Eighty-five nonreplicated K. pneumoniae clinical isolates showing reduced susceptibility (i.e., MIC, ≥0.5 mg/liter) to imipenem, meropenem, or ertapenem were studied by using PCR amplification and DNA sequencing to detect the presence of the blaKPC genes and to confirm their identity (forward primer, 5'-ATGTCACTGTATCGCCGTC-3'; reverse primer, 5'-TTACTGCCCGTTGACGCC-3'). The isolates were randomly collected from January 2006 to October 2007 from Mount Sinai Medical Center in New York (MSMC), the University of Pittsburgh Medical Center (UPMC), and three Cleveland institutions, including the University Hospital Case Medical Center (UHCMC), the Cleveland Clinic Foundation (CCF), and the Louis Stokes Cleveland Department of Veterans Affairs Medical Center (LSVAMC).

Overall, 42 K. pneumoniae clinical isolates possessed a blaKPC gene (MSMC, n = 24; UPMC, n = 4; UHCMC, n = 2; CCF, n = 9; LSVAMC, n = 3). In particular, 25 isolates amplified blaKPC-2, and the remaining 17 amplified blaKPC-3. PCR and DNA sequence analysis of the PMQR determinants, the blaTEM, blaSHV, and blaCTX-M genes, were performed as previously reported (5, 7, 13, 14, 16). Two of the 42 (4.8%) K. pneumoniae isolates containing blaKPC also encoded a PMQR determinant. For these two isolates, conjugation experiments were performed using E. coli J53 (rifampin resistant) as the recipient strain. After overnight incubation, transconjugants were selected by plating the conjugation mixture (donor and recipient strains) onto MacConkey agar supplemented with ceftazidime (10 mg/liter) and rifampin (100 mg/liter) (11). Antimicrobial susceptibility testing results for the two donors and their E. coli J53 transconjugants are shown in Table 1. Both K. pneumoniae isolates demonstrated increased MICs for carbapenems. In contrast, only one strain (VA367) expressed high-level resistance to quinolones, whereas the other (VA375) showed only a small increase in the MICs.


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  		TABLE 1. Susceptibility test results of the parental K. pneumoniae isolates, VA367 and VA375, and the corresponding E. coli J53 transconjugants, VA367-J53 and VA375-J53a

K. pneumoniae VA367 was isolated in November 2007 from a sputum sample from a 75-year-old man admitted to the surgery service of the LSVAMC with a diagnosis of adenocarcinoma of the esophagus. He had not received antibiotics in the preceding 12 months. On the first hospital day, the patient underwent a transhiatal esophagectomy and received piperacillin-tazobactam (3.375 g every 6 h) perioperatively for 36 h. Since fever and leukocytosis developed on the fifth postoperative day, administration of piperacillin-tazobactam was resumed. On the 11th postoperative day, the patient suffered cardiorespiratory arrest. He was intubated and transferred to the intensive care unit. Fever persisted, and on the 16th postoperative day, a sputum culture grew Enterobacter aerogenes, which had intermediate resistance to ceftazidime but was susceptible to piperacillin-tazobactam and carbapenems, and K. pneumoniae VA367 as a coisolate (Table 1). On the 22nd postoperative day, therapy was changed to meropenem (500 mg every 8 h). Fever persisted, and on the 27th postoperative day, tigecycline therapy (100 mg followed by 50 mg every 12 h) was initiated. The patient died on the 28th day after hospital admission.

Molecular analysis of K. pneumoniae VA367 revealed the following resistance determinants: blaKPC-3, qnrB19, blaTEM-1, blaSHV-11, blaSHV-12, and aac(6')-Ib. The E. coli transconjugant of strain VA367 contained the blaKPC-3, qnrB19, blaTEM-1, blaSHV-11, and aac(6')-Ib genes. Analytical isoelectric focusing (aIEF) was performed as previously described (9). aIEF revealed that the donor and transconjugant strains possessed β-lactamases at pIs of 5.4, 6.7, and 7.6; only the donor had an additional β-lactamase at pI 8.2. Strain VA367 and its transconjugant carried one transferable plasmid of approximately 80 kb.

K. pneumoniae VA375 was recovered on August 2007 from a culture of a blood sample obtained from a 58-year-old man admitted to the UHCMC for a kidney transplant. He received intravenous ciprofloxacin (400 mg/day) and amikacin (500 mg after each hemodialysis) treatment for 14 days, with complete resolution of the infection. Molecular analysis demonstrated that the isolate contained blaKPC-3, qnrA1, blaTEM-1, and blaSHV-11. The qnrA1, blaTEM-1, and blaSHV-11 genes were transferred to E. coli J53, whereas blaKPC-3 was not. aIEF showed β-lactamases at pIs of 5.4, 5.8, 6.7, 7.0, and 7.6 in the donor isolate, but the β-lactamases at pIs of 5.4, 5.8, and 7.6 were observed only in the transconjugant strain. VA375 contained at least two plasmids of approximately 80 kb and 130 kb, whereas the transconjugant contained only the larger plasmid.

This is the first molecular epidemiological survey assessing the spread of PMQR genes among blaKPC-containing K. pneumoniae isolates. Our limited survey suggests that the prevalence of these isolates in the United States may be approximately 5%. VA367 represents the first K. pneumoniae isolate reported to carry both the blaKPC-3 and qnrB genes on a single conjugative plasmid. Strain VA375 is the first observed K. pneumoniae clinical isolate possessing both the blaKPC and qnrA genes. In both strains, the qnr genes were cotransferred with other important drug-resistant elements [e.g., β-lactamases and aminoglycoside resistance determinants, such as aac(6')-1b]. These findings warn us that novel combinations of transferable resistance determinants continue to emerge and could seriously undermine therapeutic regimens with β-lactams, fluoroquinolones, and aminoglycosides. The possibility of K. pneumoniae transferring these resistant plasmids to other Enterobacteriaceae and nonfermenting gram-negative bacilli is a serious consideration in the care of hospitalized patients.


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ACKNOWLEDGMENTS
 
This work was supported in part by AstraZeneca, the National Institutes of Health (grant RO1-AI63517), and the Veterans Affairs Merit Review Program.


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FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Section, Department of Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4399. Fax: (216) 231-3482. E-mail for Robert A. Bonomo: robert.bonomo{at}med.va.gov. E-mail for Andrea Endimiani: aendimiani{at}tin.it Back

{triangledown} Published ahead of print on 21 April 2008. Back


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Antimicrobial Agents and Chemotherapy, July 2008, p. 2680-2682, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.00158-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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