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Journal of Clinical Microbiology, January 2007, p. 241-243, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01318-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Prevalence and Characterization of Class I Integrons among Pseudomonas aeruginosa and Acinetobacter baumannii Isolates from Patients in Nanjing, China{triangledown}

Bing Gu,1 Mingqing Tong,1* Wenjun Zhao,1 Genyan Liu,1 Mingzhe Ning,2 Shiyang Pan,1 and Wangsheng Zhao1

Department of Clinical Laboratories, First Affiliated Hospital of Nanjing Medical University, Nanjing, China,1 Department of Clinical Laboratories, Nanjing Drum Tower Hospital, Nanjing, China2

Received 27 June 2006/ Returned for modification 17 August 2006/ Accepted 6 November 2006


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ABSTRACT
 
Class I integrons were detected in 40.8% (40/98) of Pseudomonas aeruginosa strains and 52.8% (56/106) of Acinetobacter baumannii strains in the Nanjing area of China, including several cassette arrays not previously reported.


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TEXT
 
The rapid dissemination of antibiotic resistance genes among bacterial isolates is an increasing problem in infectious disease. Recent studies have shown that a conserved DNA sequence, integron, may be carried on these episomal genetic structures (12, 14). Integrons possess two conserved segments separated by a variable region that includes integrated cassettes, which often include antibiotic resistance genes (11). Many resistance genes are present as gene cassettes within integrons, which may themselves be located on transmissible plasmids and transposons (11). In China, resistance to various antibiotics is common in clinical isolates, often more so than in Western countries, especially among Pseudomonas aeruginosa and Acinetobacter baumannii isolates (7, 8, 16). However, the frequencies, characteristics, and roles of integrons and gene cassettes in the two species have not yet been investigated in a large-scale study.

In order to study the roles of gene cassettes in the two species, we obtained 98 P. aeruginosa isolates and 106 Acinetobacter sp. isolates from four general hospitals in the Nanjing area of China during June 2003 and June 2005. The strains were randomly obtained from a variety of clinical specimens from diverse units of the four hospitals. All bacterial strains were identified by the analytical profile index procedure (API-20NE system; bioMerieux, France). Susceptibility to antimicrobial agents was tested by the disk diffusion method on Mueller-Hinton agar plates, according to Clinical and Laboratory Standards Institute guidelines (2). DNA used for PCR was prepared as described previously (9). All isolates were screened for integrons by PCR using degenerate primers hep35 (5' TGCGGGTYAARGATBTKGATTT 3') and hep36 (5' CARCACATGCGTRTARAT 3') and HinfI restriction analysis of the integrase gene product (18). Class I integron cassette regions were amplified using primers 5'CS and 3'CS as described previously (17). Cassette PCR products were also digested with HinfI. Cassettes with identical profiles were deemed to contain the same gene cassettes. One or two cassette amplicons representative of each restriction profile were selected for DNA sequence characterization. The resulting DNA sequences were analyzed by the BLAST program, available at the NCBI homepage (http://www.ncbi.nlm.nih.gov/BLAST/).

Integrons were widely distributed among clinically isolated P. aeruginosa and A. baumannii strains in Nanjing, China, with integrase gene amplicons obtained from 40.8% (40/98) and 52.8% (56/106), respectively, all of which were class I by restriction fragment length polymorphism analysis. This is comparable with previously reported frequencies of 41.5% in Brazil (P. aeruginosa) (3), 60% in the United Kingdom (A. baumannii) (15), 43% in Europe (gram-negative isolates) (10), >50% in The Netherlands (Enterobacteriaceae) (5), 59% in France (Enterobacteriaceae) (13), and 52% in Taiwan (Escherichia coli) (1). We did not detect class II or class III integrons in our study; nor were they detected in A. baumannii isolates found in the United Kingdom (15) or in P. aeruginosa isolates from clinical settings in Brazil (3).

In P. aeruginosa strains, clinically significant resistance to antibiotics including cefotaxime, trimethoprim-sulfamethoxazole, tetracycline, and chloramphenicol was found (Table 1). In A. baumannii strains, clinically significant resistance to antibiotics including piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, norfloxacin, trimethoprim-sulfamethoxazole, tetracycline, and chloramphenicol was found (Table 2). Most of these strains were resistant to more than one antibiotic. In China, carbapenems (e.g., imipenem), beta-lactam antibiotics plus beta-lactamase inhibitors (e.g., piperacillin-tazobactam), aminoglycosides (e.g., amikacin), and quinolines (e.g., levofloxacin) have often been used to cure P. aeruginosa and A. baumannii infections (7, 8). We compared susceptibility data from integron-positive and -negative P. aeruginosa and A. baumannii isolates, respectively. The chi-square test was used to calculate the P value in terms of resistant, intermediate, and susceptible numbers of integron-positive and -negative isolates (2 degrees of freedom). Integrons were significantly associated with resistance to certain antibiotics, including aminoglycosides, quinolones, and beta-lactam agents, in both types of isolates. This is not surprising, since many antibiotic resistance gene cassettes encoding resistance to a wide range of antibiotics have been reported previously (12, 13). In this study, we detected many aminoglycoside resistance genes, including aadA1, aadA2, aadA5, aadA6, aadB, accA4, and aac(6')-IIa, within the integron structures. The most frequently detected resistance genes (aadA family) were aminoglycoside adenylyltransferase genes that confer resistance to streptomycin and spectinomycin. In addition, we identified three beta-lactamase genes within integrons: blaoxa-10, blaP1, and blaCARB-8.


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  		TABLE 1. Association between antibiotic profile and integrons in 98 P. aeruginosa strainsa


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  		TABLE 2. Association between antibiotic profile and integrons in 106 A. baumannii strainsa

Multiple antibiotic resistance, defined as resistance to six or more antibiotics, correlated strongly with the presence of integrons. The multidrug resistance rates of integron-positive and -negative isolates, respectively, were 77.5% and 8.6% for P. aeruginosa isolates and 98.2% and 12.0% for A. baumannii isolates. The presence of integrons was more pronounced in our study than in previous studies (6, 10).

Most of the integron arrays contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. Thirteen different gene cassettes were identified by DNA sequencing (Table 3). To our knowledge, three cassette arrays, including aadA6-orfD, aadB-blaP1, and aadB-aac(6')-IIa-blaCARB-8 in P. aeruginosa and orfI-aadA1 and aadB-catB-like-blaoxa-10/aadA1 in A. baumannii, have not been reported previously, although all of these genes have been seen in other contexts. Interestingly, a similar array, aadB-blaoxa-10/aadA (GenBank accession no. AY536742), was reported in P. aeruginosa from Shanghai, China (4). The cassette array aadB-catB-like-blaoxa-10/aadA1 identified in our study has an additional catB-like gene; therefore, we infer that our newly discovered array may be formed from the capture of a catB-like gene on the basis of the aadB-blaoxa-10/aadA array. The catB-like gene in this study has 97.2% identity to catB8 (GenBank accession no. AY033653), which was not previously detected within integrons. Some cassette array combinations were particularly common in P. aeruginosa and A. baumannii isolates, such as aadA6-orfD and aacA4-catB8-aadA1, respectively (Table 3).


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  		TABLE 3. Sizes, numbers, gene cassette(s), and new GenBank accession numbers of amplicons amplified from P. aeruginosa and A. baumannii isolates with primers 5'CS and 3'CS

This study demonstrates the wide distribution of integron-like structures in P. aeruginosa and A. baumannii isolates within the hospital environment throughout Nanjing, China.

Nucleotide sequence accession numbers. GenBank accession numbers are as follows: for P. aeruginosa aadA6-orfD, DQ091179; for P. aeruginosa aadB-blaP1, DQ141316; for P. aeruginosa aadB-aac(6')-IIa-blaCARB-8, DQ288251; for A. baumannii orfI-aadA1, DQ092497; and for A. baumannii aadB-catB-like-blaoxa-10/aadA1, DQ288250.


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ACKNOWLEDGMENTS
 
We are grateful to Chun Lu (Nanjing Medical University) for helpful suggestions on this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Laboratories, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. Phone: 86-025-83705234. Fax: 86-025-83705264. E-mail: mqtong{at}163.com. Back

{triangledown} Published ahead of print on 22 November 2006. Back


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Journal of Clinical Microbiology, January 2007, p. 241-243, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01318-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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