1 Department of Clinical Laboratory, Wuxi No.1 Hospital affiliated with Nanjing Medical University, Wuxi; 2 Wuxi Clone Gen-Tech Institute, WuxiChina
Keywords: P. aeruginosa , metallo-ß-lactamases , carbapenems , resistance
Sir,
Carbapenems, mainly imipenem and meropenem, are potent agents for the treatment of infections due to multiresistant pseudomonads because these drugs are stable even with extended-spectrum and AmpC lactamases. High-level resistance to carbapenems (MIC >32 mg/L) is still uncommon in Pseudomonas aeruginosa, but can be conferred by metallo-ß-lactamases (MBLs).1 IMP-1 was the first MBL described in P. aeruginosa.2 To date, 17 IMP enzymes and 10 VIM enzymesa family of MBLs distinct from IMPhave been identified.3 The blaIMP and blaVIM genes are inserted in integrons, and may be horizontally transferable because some of these integrons are located on conjugative plasmids. Because of its ability to spread, carbapenem resistance related to IMP- and VIM-MBL production has become a serious concern.
During 20012002, 24 isolates of P. aeruginosa resistant to imipenem were obtained at Wuxi No.1 Hospital affiliated with Nanjing Medical University in China. Antimicrobial susceptibility tests for MBL-producing Gram-negative bacilli were performed by the microdilution method with cation-adjusted MuellerHinton broth (Oxoid, Basingstoke, UK) according to the recommendations of the NCCLS. A disc-diffusion test, with 2-mercaptopropionic acid as an MBL inhibitor, was used to screen for MBL producers, as described by Arakawa et al.4 Whole cell DNA from P. aeruginosa prepared by a rapid alkaline lysis procedure was used as template in PCR assays. Primers for PCR were designed based on all MBLs published in GenBank and were: blaIMP, 5'-CTG CCK CAG GAG MGK CTT T-3' and 5'-AAC CAG TTT TGC YTT ACY AT-3'; blaVIM, 5'-CTT TAC CAG ATT GCY GAT GG-3' and 5'- CGG YAG RCC GTG CCC SGG AAT-3'. The amplicons were purified with PCR Clean Up Kits (Roche Molecular Biochemicals, Mannheim, Germany) and were sequenced on an ABI PRISM 377 Sequencer Analyzer (Applied Biosystems, Foster City, CA, USA). Genomic DNA for PFGE analysis was digested overnight with 10 U of SpeI (New England Biolabs, Beverly, MA, USA). The samples were electrophoresed with the Pulsaphor Plus System (Amersham Pharmacia Biotech) at 200 V for 30 h, with pulse times in the range 530 s.
Seven of 24 isolates of P. aeruginosa resistant to imipenem showed potentiation of ceftazidime by 2-mercaptopropionic acid, suggesting the production of MBL. Characteristics of these isolates are listed in Table 1. All seven were resistant to imipenem, piperacillin, cefotaxime, cefazolin, cefalothin, cefoxitin and ceftazidime. Their susceptibilities to aztreonam and ciprofloxacin varied.
|
Rasmussen & Bush5 predicted that an increase in MBL-producing organisms was inevitable, given the more frequent use of carbapenems. Our studies indicate the urgent need for action to prevent further spread of MBL-producing organisms. Previous experience indicates that once resistant bacteria become widespread they cannot be controlled.6 Our first task is to detect MBL producers among clinical isolates. Although the NCCLS does not currently recommend procedures for detection, a disc-diffusion test, as used here, is a simple method for screening for MBL producers.4 Laboratories in China and in other countries with carbapenem-resistant organisms should screen for MBL-producing isolates to determine their clinical impact and to prevent further spread.
Acknowledgements
We are grateful to Yu Yun-song (Department of Infectious Diseases, NO.1 Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China) for his generous provision of the blaIMP-positive control strain and technical assistance.
Footnotes
* Corresponding author. Tel: +86-0510-2700778; Fax: +86-0510-2706950; Email: wcx110{at}etang.com
References
1 . Bush, K. (1998). Metallo-ß-lactamases: a class apart. Clinical Infectious Diseases 27, Suppl. 1, S48S53.[ISI][Medline]
2 . Watanabe, M., Iyobe, S., Inoue, M. et al. (1991). Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, 14751.[ISI][Medline]
3 . Jacoby, G. & Bush, K. (2004). Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant ß-lactamases. [Online.] http://www.lahey.org/studies/webt.htm (2 April 2004, date last accessed).
4
.
Arakawa, Y., Shibata, N., Shibayama, K. et al. (2000). Convenient test for screening metallo-ß-lactamase-producing Gram-negative bacteria by using thiol compounds. Journal of Clinical Microbiology 38, 403.
5
.
Rasmussen, B. A. & Bush, K. (1997). Carbapenem-hydrolyzing ß-lactamases. Antimicrobial Agents and Chemotherapy 41, 22332.
6 . Lee, K., Lee, H. S., Jang, S. J. et al. (2001). Antimicrobial resistance surveillance of bacteria in 1999 in Korea with a special reference to resistance of enterococci to vancomycin and gram-negative bacilli to third generation cephalosporin, imipenem, and fluoroquinolone. Journal of Korean Medical Science 16, 26270.[ISI][Medline]