JAC Advance Access originally published online on June 8, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):454-455; doi:10.1093/jac/dkm208
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Correspondence |
Widespread occurrence of aminoglycoside resistance due to ArmA methylase in imipenem-resistant Acinetobacter baumannii isolates in China
The Key Laboratory of Infectious Disease of Public Health Ministry, First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79, Qing Chun Road, Hangzhou, Zhejiang 310003, China
* Corresponding author. Tel: +86-571-8723-6423; Fax: +86-571-8723-6421; E-mail: zycyg1959{at}sina.com
Keywords: pulsed-field gel electrophoresis , PFGE , homology , minimal inhibitory concentrations , MICs
Imipenem-resistant Acinetobacter baumannii (IRAB) infections and nosocomial outbreaks have been reported in many countries. Epidemic strains of IRAB are often resistant to many other antimicrobial agents, including aminoglycosides. One of the most important mechanisms of aminoglycoside resistance is post-transcriptional rRNA methylation by 16S rRNA methylases. The first 16S rRNA methylase gene, rmtA, was reported in a strain of Pseudomonas aeruginosa in Japan in 2002.1 Other methylase genes (rmtB, rmtC, rmtD and armA) were discovered sequentially, and emerged in isolates from both patients and animals.1–6
Three hundred and forty-two non-duplicate IRABs, screened by agar dilution method according to the CLSI 2006 guidelines,7 were recovered from 19 hospitals of six provinces in China from December 2004 to December 2005. Among these IRABs, 271 isolates were collected from 14 hospitals in 11 cities representative of the 11 districts of Zhejiang province and 71 isolates from five hospitals of five other provinces (Table 1). All isolates were assigned to the Acinetobacter calcoaceticus–A. baumannii complex by Vitek GNI+ card (bioMérieux, France). Species were identified by sequence analysis of the 16S–23S rRNA gene spacer region.8
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The MICs of five aminoglycoside agents against 342 IRABs were determined by agar dilution method as defined by the CLSI.7 Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as reference strains. The aminoglycoside agents tested in this study were amikacin (Sigma, Germany), gentamicin (Sigma, Germany), tobramycin (Sigma, Germany), isepamicin (Hisun Pharmaceutical Co. Ltd, China) and netilmicin (Sigma, Germany). Results were analysed according to the CLSI 2006 guidelines.7 The breakpoint of netilmicin was used for isepamicin. Resistance rates to amikacin, gentamicin, tobramycin, isepamicin and netilmicin were 92.6%, 98.6%, 87.4%, 90.9% and 92.4%, respectively. Two hundred and eighty-seven out of the 342 IRABs were found to be resistant to all of the five tested aminoglycoside agents.
The armA, rmtA and rmtB genes were detected by PCR using the primers as previously described.1,2,4 The rmtC and rmtD genes were screened using the following primers: rmtC, P1: 5'-CAGCCTCCGTAAAGAATG-3' and P2: 5'-CGAAGAAGTAACAGCCAAAG-3' rmtD, P1: 5'-ATGAGCGAACTGAAGGAAAAACTGC-3' and P2: 5'-GCTCCAAAAGCGGCAGCACCTTA-3'. PCR products were sequenced on an ABI 3730 automatic sequencer. Among 342 IRABs, 221 isolates (64.6%) were positive for the armA gene. All armA-positive isolates were distributed among the 287 isolates that exhibited resistance to the five aminoglycosides. Sequencing analyses confirmed that the PCR products were 100% identical to the armA gene (GenBank accession no. AY220558 [GenBank] ). All isolates were negative for rmtA, rmtB, rmtC and rmtD genes.
PFGE of ApaI-digested DNA was performed. Isolates showing an identical banding pattern were considered indistinguishable, while those showing differences of no more than three bands were considered closely related.
PFGE patterns showed that 207 of 221 armA-positive IRABs could be classified into three major pulsotypes, which were designated as pulsotypes A, B and C. The other 14 strains had different ApaI-digested DNA patterns from each other. Pulsotype A was distributed in six hospitals of three different provinces. Pulsotype B was distributed in three hospitals of three provinces. Pulsotype C was distributed in a hospital in Shanghai and 10 hospitals of eight cities in Zhejiang (Table 1).
In conjugation experiments, rifampicin-resistant E. coli 600 strain was used as a recipient. MH agar plates containing 512 mg/L rifampicin and 128 mg/L amikacin were used to select transconjugants. QIAGEN Plasmid Kits (QIAGEN, Germany) were used to isolate plasmids from selected ArmA-producing IRABs. Plasmids and ApaI-digested PFGE DNA were transferred to nylon membranes and were hybridized with a v-dCTP (DuPont Corporation, USA)-labelled armA gene probe. Repeated attempts to transfer the armA gene by conjugation failed. Repeated attempts failed to detect the presence of a plasmid capable of hybridizing with the armA probe, while the fragments of the ApaI-digested PFGE DNA of pulsotypes A, B and C showed hybridization bands at 
220, 300 and 220 kb, respectively (data not shown).
Our study suggested that IRABs were highly resistant to different aminoglycoside agents. A methylase gene (armA), which confers high levels of resistance to aminoglycosides, was detected in the majority of our isolates. This suggests that aminoglycosides may no longer be recommended as a first-line treatment for IRAB infections in China.
ArmA methylase was first characterized in Klebsiella pneumoniae isolates in France5 and thereafter ArmA-producing IRABs were detected in Korea.3 The gene was distributed widely in IRABs in China. armA-positive IRABs were detected in 17 of the 19 hospitals. PFGE analysis suggested that 93.7% of armA-positive A. baumannii belonged to three pulsotypes that were spread widely in China. The armA-coding gene may be located on the chromosomes of the isolates. Pulsotype dissemination occurred not only within or among hospitals but also among cities and possibly played the most important role in the prevalence of the armA gene.
None to declare.
Acknowledgements
We are grateful to Wang Hui, PhD, the Peking Union Medical College Hospital, for kindly providing the rifampicin-resistant E. coli 600 strain. This work was supported by a research grant from the National Basic Research Program 973 of China (no. 2005CB523101), a grant from the High Technology Research and Development Program of China (863 program, no. 2006AA02Z413) and by a grant from the Program for New Century Excellent Talents in University (no. NCET-04-0552).
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Chen L, Chen ZL, Liu JH, et al. Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. J Antimicrob Chemother (2007) 59:880–5.
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Chang HC, Wei YF, Dijkshoorn L, et al. Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J Clin Microbiol (2005) 43:1632–9.
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