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Jpn. J. Infect. Dis., 62, 155-157, 2009 Short Communication Colonization of a Medical Center in Southern Taiwan by Epidemic Strains of Carbapenem- and Multidrug-Resistant Acinetobacter baumannii and the Genetic Organization of Their Integrons Yao-Shen Chen1,2, Hsi-Hsun Lin3, Chia-Hsuang Wu4, Yu-Shan Hsiao4, Ning-Shan Hsu4, and Ya-Lei Chen4* 1 Section of Infectious Disease, Kaoshiung Veterans General Hospital, Kaoshiung; 2National Yang-Ming Medical University, Taipei; 3Department of Infectious Disease, E-DA Hospital/I-Shou University, Kaoshiung; and 4Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung, Taiwan (Received August 1, 2008. Accepted January 19, 2009) SUMMARY: A total of 46 carbapenem- and multidrug-resistant (CR- and MDR-)Acinetobacter baumannii bacteremic isolates from a Taiwanese medical center were investigated over the period 2000 to 2006 using randomly amplified polymorphic DNA (RAPD) profiling and by analysing the genetic organization of their integrons. The results of RAPD patterns revealed that before 2003 each CR- and MDR-A. baumannii bacteremic isolate was independent, but after 2003 the isolates appeared to belong in four epidemic strains and persisted in the hospital. All the CR- and MDR-A. baumannii strains harbored class I integron (intI1) genes. PCR amplification and nucleotide sequencing showed that the cassette genes of intI1 were found to form four different antibioticresistant gene alignments in those strains. The blaIMP-1 gene in the cassette genes of intI1 was identified in a clone, which raised great concern that clonal spread of this strain or of an integron-mediated horizontal gene may have occurred. The medical records of 40 of these patients were available for review, and 87.5% (35/40) of cases were mechanical ventilation users. All the CR-A. baumannii bacteremic isolates were also MDR strains as defined. Those strains were confirmed by an automatic system (BD Phoenix 100 Automated Microbiology System; Becton, Dickinson and Company, Franklin Lakes, N.J., USA) as well as by the nucleotide sequences of their 16S-23S rRNA gene spacer region (7). An antimicrobial susceptibility test was performed against a range of commercially available antibiotics, and the identification of this resistance profile followed the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (8). MDR strains were defined as resistant to at least four of the five following groups: (i) gentamicin or amikacin; (ii) cefepime or ceftazidime; (iii) piperacillin, piperacillin--tazobactam or ticarcillin--clavulanic acid; (iv) ciprofloxacin or levofloxacin; (v) meropenem or imipenem. Each strain was grown in 3 ml of Luria-Bertani (LB) broth medium at 37°C. After 16 h, the genomic DNA of the bacteria was isolated using a genomic DNA extraction kit (Promega, Madison, Wis., USA). The primer designs for randomly amplified polymorphic DNA (RAPD) (5´-CCTCATGACC3´) and differential integron (intIf: 5´-CAGTGGACATAAGC CTGTTC-3´ and intIr: CCCGAGGCATAGACTGTA; intIIf: 5´-TTGCGAGTATCCATAACCTG and intIIr: TTACCTGCA CTGGATTAAGC; 5´-CS: 5´-GGCATCCAAGCAGCAAG3´ and 3´-CS 5´-AAGCAGACTTGACCTGA-3´) and PCR conditions were followed as standard protocols (9). The reproducible PCR-RAPD amplicon profiles were visualized by 1.5% agarose electrophoresis by UV illumination. The RAPD profiles were captured and analyzed using the Quantity One software, version 4.4.0 (BioRad, Hercules, Calif., USA). If the isolates had the same RAPD profiles, the second random primer (5´-TGCGCGCGGG-3´) was used to confirm their identical genetic backgrounds. Carbapenem-resistant (CR-) or multidrug-resistant (MDR-) Acinetobacter baumannii has emerged as a major nosocomial pathogen worldwide. The prevalence of nosocomial infection in hospital respiratory care wards due to CR- or MDRA. baumannii was 32.1 or 8.9%, respectively, of all Gramnegative bacterial infection in Taiwan (1). Such infections are beset by the problem that these bacteria offer very few therapeutic options and sometimes can develop into an epidemic strain, which produces repeated outbreaks over a long time period in a hospital (2). Developing MDR strains have been suggested to acquire their antibiotic-resistant genes via class I integrons (IntI1) that carry within them a single or multiple gene cassette(s); most often among MDR-A. baumannii isolates, these are found in the internal variable regions between the integron’s two conserved regions (the 5´-CS and the 3´-CS) (3). Aminoglycoside resistance genes, such as aac (acetyltransferase), aph (phosphotransferase), and aad (adenylyltransferase), which contribute to the enzymatic inactivation of the aminoglycoside antibiotics, are the genes usually found among the cassette genes (4). In special instances, A. baumannii can give rise to the spread of β-lactam antibiotic drug resistance in hospital if the bacteria harbor a bla gene variant among the cassette genes (5,6). In this report, we identified colonization by epidemic strains of CRand MDR-A. baumannii in a 1,400-bed teaching hospital, Kaoshiung Veterans General Hospital (KVGH), and investigated the prevalence of antibiotic-resistant genes within the IntI1 cassettes from those isolates. A total of 46 CR-A. baumannii strains were isolated from individual bacteremic patients over the period 2000 to 2006. *Corresponding author: Mailing address: Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung, Taiwan. Tel: +886-7-6051366, Fax: +886-7-6051353, E-mail: [email protected] 155 The CR- and MDR-A. baumannii bacteremic strains isolated at KVGH included as few as four isolates per year before 2003, but this dramatically increased to over 10 after 2003. A total of 46 individual isolates of CR- and MDR-A. baumannii were obtained over the period from 2000 to 2006 and were analyzed for genetic differentiability (Fig. 1). The results indicated that the RAPD patterns of each CR- and MDR-A. baumannii isolate before 2003 were independent, but after 2003, the isolates appeared to be grouped into four identical patterns (A-D). An epidemic strain (pattern A) initially appeared in 2003, reached a peak in 2004, but then decreased in 2005. The epidemic strain (pattern B) was found in 2004 and continuously appeared in the hospital. Two emerging epidemic strains (patterns C and D) only occurred in 2006 (Table 1). These results indicate that the origin of the CRand MDR-A. baumannii strains in KVGH changed over time. Integron-specific PCR amplification revealed that all the CR- and MDR-A. baumannii strains harbored class 1 integrons and that no class 2 integrons were present in the chromosomes of these strains. The cassette genes found within those strains were shown to form four types by PCR amplification and nucleotide sequencing (Table 1). All types harbored aadA1a, and Types I and IV harbored aacA4. This result was associated with the known resistance of these A. baumannii isolates to aminoglycoside antibiotics. The open reading frames coding for as-yet-undetermined products were presented in all types. An open reading frame orfC in Type III was identical to the sequence of an unknown functional protein which was found in environmental isolates of Acinetobacter spp. (3). It is noted that one strain harbored an intI1 encoding the blaIMP-1 gene. Our group and others (10) have reported that MDR strains harboring blaIMP-1 existed where this gene forms part of the intI1 cassette genes found in A. baumannii. This finding raised great concern because integron-associated imipenem-resistant A. baumannii has been repeatedly found in Taiwanese hospitals, and it suggested that antibiotic resistance might be disseminated throughout populations by the epidemic spread of this clone or through the exchange of genetic material. However, due to natural transformation, loss or altered outer-mem- Fig. 1. Dendrogram and RAPD patterns of A. baumannii isolates. The PCR-RAPD amplicon profiles were amplified with random primer (5´-CCTCATGACC-3´). The RAPD profiles were captured and analyzed using the software of Quantity One, version 4.4.0 (BioRad). The typical RAPD patterns (A-D) of related strains and the profiles of independent strains (strain number) were presented in this figure. Table 1. Summary for RAPD typing and integron organization analysis of Acinetobacter baumannii isolates Isolation Year No. 2000 2001 2002 2003 2004 3 3 0 2 13 2005 2006 10 15 pattern A Epidemic strain (Strain no.integron type) pattern B pattern C pattern D Independent strain (Strain no.integron type) 1II 2I 3IV 4III5III7III 9III*10III** 11 13III14III15III 16III17III19III20III22III 27III29III 30III 31III III 23IV 12III18III21IV 25IV26IV32IV 24IV28IV33IV III 46IV III III III 34 35 36 39 43III44III45III47III 48III49III 37III41III 40III42III *Roman letters indicated the genetic organization of integron type following as Type I IntI1 arrA3 accA4 ORF aadAla qacEΔ1 Type II IntI1 blaIMP-1 ORF aadAla qacEΔ1 dfrA1 ORFC ORF aadAla qacEΔ1 Type IV IntI1 accA4 catB8 ORF aadAla qacEΔ1 **The underline indicated that the clinical isolates were obtained from mechanical ventilator users. Type III IntI1 156 5. Zarrilli, R., Casillo, R., Di Popolo, A., et al. (2007): Molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a university hospital in Italy. Clin. Microbiol. Infect., 13, 481-489. 6. Canduela, M.J., Gallego, L., Sevillano, E., et al. (2006): Evolution of multidrug-resistant Acinetobacter baumannii isolates obtained from elderly patients with respiratory tract infections. J. Antimicrob. Chemother., 57, 1220-1222. 7. Chang, H.C., Wei, Y.F., Dijkshoorn, L., et al. (2005): 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., 43, 1632-1639. 8. Clinical and Laboratory Standards Institute (2006): Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. CLSI document M100-S16. Clinical and Laboratory Standards Institute, Wayne, Pa. 9. Koeleman, J.G., Stoof, J., Van Der Bijl, M.W., et al. (2001): Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J. Clin. Microbiol., 39, 8-13. 10. Liu, S.Y., Lin, J.Y., Chu, C., et al. (2006): Integron-associated imipenem resistance in Acinetobacter baumannii isolated from a regional hospital in Taiwan. Int. J. Antimicrob. Agents, 27, 81-84. 11. Bergogne-Bérézin, E. and Towner, K.J. (1996): Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev., 9, 148-165. 12. Ruzin, A., Keeney, D. and Bradford, P.A. (2007): AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Antimicrob. Chemother., 59, 1001-1004. 13. Afzal-Shah, M., Woodford, N. and Livermore, D.M. (2001): Characterization of OXA-25, OXA-26, and OXA-27, molecular class D betalactamases associated with carbapenem resistance in clinical isolates of Acinetobacter baumannii. Antimicrob. Agents Chemother., 45, 583-588. 14. Corbella, X., Montero, A., Pujol, M., et al. (2000): Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J. Clin. Microbiol., 38, 4086-4095. brane protein and endogenous metallo-β-lactamases variants, CR strains were probably generated (11-13). Cross-transmission of colonized MDR-A. baumannii between patients who are hospitalized in intensive-care units has been well documented (14). In this study, we reported an epidemic strain that caused a hospital-wide outbreak over the period 2003 to 2005. The results of molecular typing revealed that the origin of epidemic strains after 2005 may have changed in this hospital. The restriction of antibiotics use, the intensification and modification of the cleaning procedures for contaminated equipment and the cohorting of patients infected or colonized with MDR-A. baumannii need to be seriously considered as control measures to confine the spread of these bacteria. ACKNOWLEDGMENTS This project was supported by the grants (DOH95-DC-1023; DOH96DC-1203). REFERENCES 1. Kuo, L.C., Yu, C.J., Kuo, M.L., et al. (2008): Antimicrobial resistance of bacterial isolates from respiratory care wards in Taiwan: a horizontal surveillance study. Int. J. Antimicrob. Agents, 31, 420-426. 2. Hsueh, P.R., Teng, L.J., Chen, C.Y., et al. (2002): Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg. Infect. Dis., 8, 827-832. 3. Petersen, A., Guardabassi, L., Dalsgaard, A., et al. (2000): Class I integrons containing a dhfrI trimethoprim resistance gene cassette in aquatic Acinetobacter spp. FEMS Microbiol. Lett., 182, 73-76. 4. Nemec, A., Dolzani, L., Brisse, S., et al. (2004): Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones. J. Med. Microbiol., 53, 1233-1240. 157