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

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 bla KPC-2 and ampC gene sequences of Citrobacter freundii ZJ163 are EF062508 and EF426097.