Carbapenem-Resistant Isolates of Klebsiella pneumoniae in China and Detection of a Conjugative Plasmid (bla_KPC-2 plus qnrB4) and a bla_IMP-4 Gene

Wei et al. (13) recently described the emergence of plasmid-mediated KPC-2 carbapenemase in a Klebsiella pneumoniae isolate from China. Interestingly, KPC-producing isolates, which were initially restricted to hospitals located in the New York City area (2), have recently been detected in other areas of the United States (5) and in several nations, including China (13), Colombia (11), and Israel (8). This enzyme, considered a problem for clonal spread, has also been detected among other organisms, such as Pseudomonas aeruginosa (10), Escherichia coli (8), and Serratia marcescens (15), emphasizing the global risk of interspecies dissemination of resistance genes. Furthermore, several IMP-type metallo-β-lactamase (MβL) enzymes have been detected in China but most often among nonfermentative gram-negative bacilli (12).

This study selected Enterobacteriaceae isolated during the 2006 SENTRY Antimicrobial Surveillance Program (Asia-Pacific region) with decreased susceptibility (MIC of ≥2 μg/ml) for imipenem or meropenem. The isolates were tested by reference broth microdilution methods (3, 4), and MIC results were interpreted as specified by the M100-S17 CLSI document (4), apart from tigecycline results, which were interpreted according to the breakpoints approved by the U.S. Federal Drug Administration (≤2 or ≥8 μg/ml for susceptible or resistant, respectively). The isolates were screened for KPC and MβL, as recently described (5, 6), followed by gene sequencing. K. pneumoniae clinical isolate 231-21D was recovered from a 42-year-old male trauma victim with a venous catheter infection on the 77th hospital day in Zhejiang, China. The patient had received multiple antimicrobial treatments, including piperacillin-tazobactam for 9 days, metronidazole for 3 days, vancomycin for 10 days, cefoperazone-sulbactam for 19 days, teicoplanin for 15 days, imipenem for 11 days, and levofloxacin for 6 days, when the isolate 231-21D was recovered. Negative blood cultures were obtained after removing the catheter. K. pneumoniae clinical isolate 234-49C was isolated from the sputum of a 50-day-old female infant with acute bronchopneumonia on the second hospital day in Wuhan, China. This patient received mezlocillin-sulbactam for 3 days prior to the recovery of isolate 234-49C, followed by ceftizoxime for 5 days and imipenem for 3 days. Both cases had favorable clinical outcomes.

Isolate 231-21D showed resistance or decreased susceptibility to several antimicrobials, including carbapenems, but remained susceptible to tigecycline and the polymyxins. Isolate 234-49C also exhibited resistance to several antimicrobials, remaining susceptible to aminoglycosides, fluoroquinolones, tigecycline, and polymyxins (Table 1). The isolates showed positive phenotypic screening results for the presence of MβL or Bush-Jacoby-Medeiros subgroup 2f serine β-lactamase (5). PCR and sequencing analysis identified the presence of bla_KPC-2 and bla_IMP-4 genes in the isolates 231-21D and 234-49C, respectively. No other Enterobacteriaceae harboring such genes were recovered from Zhejiang or Wuhan hospitals during the same period of time. Additionally, carbapenem-resistant Pseudomonas sp. and Acinetobacter sp. isolates recovered from Wuhan were also screened for MβL genes (bla_IMP-like, bla_VIM-like, bla_SPM-1, bla_SIM-1, and bla_GIM-1), but positive results were not obtained, e.g., bla_IMP-4.

TABLE 1.

Antimicrobial susceptibility profiles of K. pneumoniae clinical isolates 231-21D and 234-49C, the E. coli K-12 strain carrying the conjugative plasmid p231-21D, and the recipient E. coli K-12 strain

Antimicrobial agent	MIC (μg/ml)
	K. pneumoniae 234-49C	K. pneumoniae 231-21D	E. coli K-12 (p231-21D)	E. coli K-12
Ampicillin	>16	>16	>16	4
Piperacillin-tazobactam	>64	>64	32	2
Aztreonam	8	>16	>16	0.06
Cefoxitin	>16	>16	>16	4
Cefotaxime	>16	>16	4	≤0.25
Ceftazidime	>32	>32	32	0.25
Ceftriaxone	16	>32	32	0.06
Cefepime	8	8	4	0.06
Imipenem	4	8	4	0.03
Meropenem	8	8	2	0.03
Ertapenem	4	>8	>8	0.06
Amikacin	≤4	>32	>32	≤4
Gentamicin	≤2	>8	>8	≤2
Ciprofloxacin	0.5	>4	0.25	0.06
Levofloxacin	0.5	>4	0.25	0.06
Tigecycline	0.5	0.5	0.12	0.06
Colistin	≤0.5	≤0.5	≤0.5	≤0.5
Polymyxin B	1	≤0.5	≤0.5	≤0.5

The β-lactam resistance determinant from K. pneumoniae clinical isolate 231-21D was transferred by conjugation (7). The E. coli K-12 transconjugant and the E. coli K-12 recipient strains were tested for susceptibility by using E-test strips according to the manufacturer's recommendations (AB Biodisk, Solna, Sweden). The transconjugant strain showed resistance to several β-lactams and aminoglycosides and decreased susceptibility (fourfold) to fluoroquinolones (Table 1), leading to investigations for the presence of qnr-like genes (qnrA, qnrB, and qnrS) and aac(6′)-Ib-cr. PCR and sequencing detected in the index and transconjugant organisms the presence of a qnrB gene, which was identical to the qnrB4 variant found in the K. pneumoniae clinical isolate recovered from China (GenBank accession no. EF683583) (data not shown). The aac(6′)-Ib-cr gene was not detected.

Analysis of plasmid content of the isolate 231-21D revealed the presence of three plasmids (60, 5, and 3.5 kb), while the transconjugant strain showed only the 60-kb plasmid. KPC-2-mediated β-lactam and QnrB-mediated quinolone resistance determinants were selftransferable and located in the 60-kb conjugative plasmid (p231-21D) (Table 1). Conjugation, transformation, and plasmid analysis of the IMP-4-producing K. pneumoniae isolate (234-49C) failed to identify a plasmid-borne location of bla_IMP-4, suggesting a chromosomal location.

Although the previously reported KPC-2-producing K. pneumoniae isolate (ZR01) occurred in the same hospital 20 months earlier (13) and bla_KPC-2 was also located in a 60-kb plasmid, that plasmid was not conjugative. Moreover, it did not appear to harbor the qnrB gene, since the E. coli pYW1 transformant strain did not exhibit decreased susceptibility to fluoroquinolones (13). These findings suggest that recombination events may have mobilized bla_KPC-2 from the ZR01 isolate to p231-21D found in our KPC-2 strain or the gene may have been acquired elsewhere. The detection of the IMP-4-producing K. pneumoniae clinical isolate (234-49C) highlights the continued spread of this gene in this particular geographic region (Asia Pacific) and emphasizes the importance of surveillance programs (1, 9).

Such organisms represent a contemporary diagnostic challenge, because the resistance phenotype may not be obvious and could require further specialized testing. Previous studies have shown the coexistence of MβL-encoding or extended-spectrum β-lactamase-encoding genes with qnr (14). However, this report identifies the first coproduction of KPC-2 and QnrB4 encoded by resistance genes located in the same conjugative plasmid. The plasmid-mediated fluoroquinolone and β-lactam (carbapenems, cephalosporins, and penicillins) resistance determinants may spread further among clinical isolates of enteric bacilli and escalate the resistance rates for these antimicrobial classes.