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J Med Microbiol 57 (2008), 332-337; DOI: 10.1099/jmm.0.47576-0
© 2008 Society for General Microbiology
ISSN 1473-5644

High-level carbapenem resistance in a Citrobacter freundii clinical isolate is due to a combination of KPC-2 production and decreased porin expression

Rong Zhang1, Lijiang Yang2, Jia Chang Cai1, Hong Wei Zhou1 and Gong-Xiang Chen1

1 Second Affiliated Hospital of Zhejiang University, Zhejiang University, 88 JieFang Road, Hangzhou 310009, PR China

2 Huazhong University of Science & Technology, Wuhan, Hubei 430074, PR China

Correspondence
Gong-Xiang Chen
chengong218{at}163.com

Received 17 August 2007
Accepted 8 November 2007

An imipenem-resistant isolate of Citrobacter freundii ZJ163 (MIC 256 µg ml–1) isolated from a Chinese hospital was investigated. The C. freundii ZJ163 isolate exhibited high-level resistance to carbapenems, penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides. Isoelectric focusing (IEF) demonstrated three β-lactamases with pIs of 5.4 (TEM-1), 6.7 (KPC-2) and 7.9 (CTX-M-14). Two different transconjugants (types A and B) were obtained by conjugation studies. The type A transconjugant exhibited reduced susceptibility or resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of the type B transconjugant were similar to that of type A, except for its significantly reduced carbapenem susceptibility (imipenem MIC 2 µg ml–1). IEF, specific PCRs and DNA sequence analysis indicated that the type A transconjugant produced CTX-M-14 β-lactamase with a pI of 7.9, that the type B transconjugant produced KPC-2 β-lactamase with a pI of 6.7 and that the β-lactamase with a pI of 5.4 was TEM-1. PCR analysis and sequencing confirmed the presence of the ampC gene in the chromosomal DNA from C. freundii ZJ163, although no activity of AmpC β-lactamase was detected by IEF. Urea/SDS-PAGE analysis of outer-membrane proteins revealed that the levels of the 41 and 38 kDa porins were decreased in C. freundii ZJ163. It was concluded that production of KPC-2 combined with decreased expression of porins contributes to high-level resistance to carbapenems in C. freundii ZJ163.

Abbreviations: ESBL, extended-spectrum β-lactamase; pI, isoelectric point; OMP, outer-membrane protein.

The GenBank/EMBL/DDBJ accession numbers for the blaKPC-2 and ampC gene sequences of Citrobacter freundii ZJ163 are EF062508 and EF426097.




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