Characterization of Class 1 Integrons and Antimicrobial Resistance in CTX-M-3-Producing Serratia marcescens Isolates from Southern Taiwan

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1 Jpn. J. Infect. Dis., 60, , 2007 Original Article Characterization of Class 1 Integrons and Antimicrobial Resistance in CTX-M-3-Producing Serratia marcescens Isolates from Southern Taiwan Chien-Fang Peng 1,2 *, Mei-Feng Lee 3, Hsiao-Ting Fu 4, Yuh-Jyh Chen 4 and Hui-Jine Hsu 4 1 Laboratory of Microbiology and Clinical Microbiology, 4 Faculty of Biomedical Laboratory Science, College of Health Sciences, 3 Department of Laboratory Medicine, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, and 2 Division of Clinical Microbiology, Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan (Received November 27, Accepted May 9, 2007) SUMMARY: The objective of this study was to characterize the gene cassettes of class 1 integrons and antimicrobial resistance among CTX-M-3-producing Serratia marcescens isolates from different specimens in southern Taiwan. One hundred and twenty-two isolates (70.5%) of 173 CTX-M-3-producing S. marcescens isolates were positive for class 1 integrons, including 53.3% of blood isolates, 94.1% of urine isolates, and 87.2% of sputum isolates. No class 2 or class 3 integrons were detected in this study. By PCR with primers 5 -CS and 3 -CS for the amplification of gene cassettes regions, amplicons ranging from 0.7 to 3.0 kb in length were found in 108 (88.5%) of the 122 class 1 integron-containing isolates of CTX-M-3-producing S. marcescens isolates. Ten different types by pattern of amplicons for class 1 integrons were obtained. The Type I amplicon (46.3%) harbors two different class 1 integrons containing the gene cassettes of aada2 and aadb-catb3, respectively, and was most prevalent in the gene cassette region-positive S. marcescens isolates, followed by the Type II amplicon, which harbors one class 1 integron containing the gene cassette dfra12-orff-aada2 (28.7%). Most of the S. marcescens isolates (66.7%, 8/12) harboring three different class 1 integrons (Type IV amplicon) were found in blood isolates. Class 1 integrons were conjugally transferred to recipients in 92.0% of S. marcescens harboring two different class 1 integrons containing the gene cassettes aada2 and aadb-catb3, respectively. The transfer rate of class 1 integron carrying dfra12-orff-aada2 was detected in 77.4% of S. marcescens isolates. The results showed that all those isolates with conjugative transfer of integrons carried their class 1 integrons on the conjugative plasmids. INTRODUCTION Serratia marcescens, which belongs to the family Enterobacteriaceae, is an important nosocomial pathogen and causes many hospital infections, such as septicemia (1-3), respiratory tract infections (4), and urinary tract infections (5). S. marcescens is often resistant to multiple antimicrobial agents, including -lactam antibiotics and aminoglycosides (6). Multiresistance to -lactam antibiotics in S. marcescens is mediated by the expression of acquired enzymes, including high-level production of the chromosomal AmpC-type cephalosporinase (7), plasmid-mediated enzymes including extended-spectrum -lactamases (ESBLs) (8), and metallo- -lactamases (9). Among the groups of ESBLs, CTX-M-type ESBLs emerge rapidly in resistant clinical isolates of Enterobacteriaceae (10). There are now more than 40 CTX-M ESBLs comprising five subgroups. CTX-M ESBL-producing isolates usually exhibit a greater hydrolytic activity against cefotaxime than against ceftazidime. In Taiwan (11) and Korea (12), CTX- M-3 ESBLs were most prevalent in S. marcescens isolates. The molecular mechanisms of the acquisition and dissemination of resistance genes among clinical bacterial isolates have been studied intensively (13,14). The most important *Corresponding author: Mailing address: Laboratory of Microbiology and Clinical Microbiology, Faculty of Biomedical Laboratory Science, College of Health Sciences, Kaohsiung Medical University, No. 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel: ext. 2352, Fax: , chfape@kmu.edu.tw mechanism by which Gram-negative bacteria capture gene cassettes is site-specific recombination catalyzed by specific integrase genes (13), followed by incorporation into resistance integrons. In the structure of class 1 integrons, the 5 conserved segment (5 -CS) includes the integrase gene (inti1) and the recombination site (atti1), and the 3 -CS contains a variable region for gene cassettes, an attc site, a qace 1, an sul gene, and an orf5 gene (13). Among the antibiotic resistance integrons, class 1 integrons are the most important type found in the family Enterobactriaceae, which includes Escherichia coli, Citrobacter spp., Klebsiella spp., Enterobacter spp., Proteus spp., and Salmonella spp. Class 1 integrons are the most studied in the transfer of antibiotic resistance. However, few reports have been performed to elucidate the antibiotic resistance gene cassettes between class 1 integrons and resistance phenotypes in multidrug-resistant S. marcescens isolates (7,15,16). Several different gene cassettes, dhfr1 and ant(3 )-Ia (7), bla VIM-2 (15), and dfra1 and ant(3 )-Ia (16), have been described in class 1 integrons of S. marcescens isolates. In recent years, a high rate of cefotaxime resistance of S. marcescens from blood culture specimens has been reported in southern Taiwan (3). In our university hospital, an increase of multidrug-resistant S. marcescens isolates has been observed in the past few years. In this study, we investigated the prevalence and antimicrobial resistance associated with gene cassettes in class 1 integrons among CTX-M-3-producing S. marcescens isolates in southern Taiwan. 250

2 MATERIALS AND METHODS Isolation of CTX-M-3-producing S. marcescens isolates: A total of 389 isolates of cefotaxime-resistant S. marcescens were isolated at the Kaohsiung Medical University Hospital, Taiwan, during the period from 2003 to These clinical isolates were obtained from patient specimens, including blood (207 strains), urine (75 strains), and sputum (107 strains). In this study, cefotaxime-resistant S. marcescens isolates were screened for the presence of ESBL, and then further investigated for the presence of known CTX-M type ESBLs. ESBL activity was detected by the double-disk diffusion method using cefotaxime or ceftazidime disks with or without clavulanate and interpreted by the Clinical and Laboratory Standards Institute (CLSI) (formerly the National Committee for Clinical Laboratory Standards) criteria (17). For detection of the bla CTX-M gene, polymerase chain reaction (PCR) was carried out using primers MA (5 -CGC TTT GCG ATG TGC AG-3 ) and MB (5 -ACC GCG ATA TCG TTG GT-3 ) (accession no. X92506) (18). The complete coding sequences of the bla CTX-M-3 gene were amplified with the designed primers of CTX-M-3F (5 -GAA CCC ATG GTT AAA AAA TCA CTG C-3 ) and CTX-M-3R (5 -CCG CTA TTA CAA ACC GTC GGT GAC-3 ) (accession no. AF550415). All PCR products were purified and subjected to direct sequencing twice on both strands with an Applied Biosystems sequencer (model ABI 377; Applied Biosystems, Foster City, Calif., USA). The obtained DNA sequences were analyzed using the NCBI (National Center for Biotechnology Information)-BLAST (Basic Local Alignment Search Tool) program with published GenBank DNA Sequences (19). CTX-M-3-producing S. marcescens isolates were selected for the detection of class 1 integrons and antimicrobial resistance genes. Antimicrobial susceptibility testing: In vitro susceptibility testing was carried out by the disk-diffusion method on Mueller-Hinton agar (Difco Laboratories, Detroit, Mich., USA) according to CLSI document M2-A8 (20). Antimicrobial minimum inhibitory concentration (MIC) values were determined with the E-test strip (AB Biodisk, Solna, Sweden) (21). The E-test was performed in duplicate according to the manufacturer s instructions. PCR amplification and DNA sequence analysis of class 1 integrons: Isolates of CTX-M-3-producing S. marcescens were screened for the presence of class 1, 2, and 3 resistance integrons (14) using primers specific for the inti1 gene (IntI1A: 5 -AAAACCGCCACTTGCGCCGTTA-3 and IntI1B: 5 - GAAGACGGCTGCACTGAACG-3 ), inti2 gene (IntI2A: 5 -ATGTCTAACAGTCCATTTTTAAATTCT-3 and IntI2B: 5 -AAATCTTTAACCCGCAAACGC-3 ), and inti3 gene (IntI3A: 5 -GTGGCGCAGGGTGTGGAC-3 and IntI3B: 5 - ACAGACCGAGAAGGCTTATG-3 ). PCR amplification was done with commercial kits of PuReTaq Ready-To-Go PCR beads (Amersham Biosciences, Piscataway, N.J., USA). The PCR products were also purified with a QIA quick PCR Purification kit (Qiagen, Valencia, Caif., USA) and subjected to direct sequencing for detection of the integrase genes. The gene cassettes inserted in the variable region of class 1 integrons were amplified using the primer pairs of 5 -CS (5 - GGCATCCAAGCAGCAAG-3 ) and 3 -CS (5 -AAGCAGA CTTGACCTGA-3 ) (14). All PCR amplicons were purified and sequenced. Characterization of the resistance gene cassettes in class 1 integrons was also analyzed using the BLAST suite of programs (19). DNA manipulation, conjugation experiments, and Southern hybridization: Extracts of genomic DNA were performed using a Genomic DNA Purification Kit (Promega Biosciences, Madison, Wis., USA) according to the manufacturer s instructions. DNA concentrations were determined spectrophotometrically with a GeneQuant II (Pharmacia Biotech, Cambridge, UK). Plasmid DNA was prepared by an alkaline lysis method (22). Plasmid DNA sizes were determined in reference to E. coli 39R861 (154, 66, and 38 kb). Conjugation experiments were performed by the filter-mating method (23). The nalidixic acid-resistant strain E. coli-k12 14R525 was used as the recipient strain in mating experiments. Transconjugants were screened on Luria-Bertani (LB) medium (Difco Laboratories) containing sulfamethoxazole (64 mg/l) plus nalidixic acid (30 mg/l). Plasmid DNA obtained from the donor strains and their transconjugant isolates were screened for the presence of class 1 integrons. Southern hybridization was carried out with a digoxigenin (DIG)- labeled probe for integrase gene inti1 using a DIG system (DIG DNA Labeling and Detection Kit; Roche Diagnostics, Mannheim, Germany) according to the manufacturer s instructions (24). The inti1 DNA probe was obtained by PCR amplification with primers INTF (5 -CGCTGAAAGGTCTGGTC ATA-3 ) and INTR (5 -GCCCA GCTTCTGTATGGAAC-3 ) specific for the integrase gene inti1 of class 1 integron (accession no. M95287) (14). Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR): ERIC-PCRs were performed using the primers of ERIC1R (5 -ATGTAAGCTCCTGGGGATTCAC-3 ) and ERIC2 (5 -AAGTAAGTGACTGGGGTG AGCG-3 ) designed by Versalovic et al. (25). Analysis of amplified products was performed in 2% Seakem LE agarose (BMA, Rockland, Maine, USA). A 1-kb DNA ladder (Gibco-BRL, Gaithersburg, Md., USA) was used as a size marker. Electrophoretic DNA patterns that differed in one or more DNA fragments were considered patterns that represented different types. ERIC fingerprints of amplified DNA fragments obtained by agarose gel electrophoresis were analyzed using the GelCompar 4.2 software package (Applied Maths, Kortrijk, Belgium). Nucleotide sequence accession number: The nucleotide sequences of the gene cassettes aadb-catb3, dfra1-un, and oxa31-aada5 in the class 1 integron reported in this study have been deposited in the NCBI-GenBank sequence databases under accession nos. DQ402097, DQ402098, and EF067840, respectively. RESULTS Identification of class 1 integrons in CTX-M-3-producing S. marcescens isolates: Using the PCR assay, 173 (44.5%) of 389 cefotaxime-resistant S. marcescens isolates were found to be positive for the presence of the CTX-M ESBL gene. Based on DNA sequence analysis, all 173 of these isolates (100%) were positive for the bla CTX-M-3 gene, including 92 blood isolates, 34 urine isolates and 47 sputum isolates. One hundred and twenty-two isolates (70.5%) of the 173 CTX- M-3-producing S. marcescens isolates were positive for class 1 integrons, including blood isolates (49/92, 53.3%), urine isolates (32/34, 94.1%), and sputum isolates (41/47, 87.2%) (Table 1). No integrons were found in the remaining 51 isolates of CTX-M-3-producing S. marcescens. No class 2 or class 3 integrons were detected in this study. 251

3 Table 1. Incidence of inti1-positive and gene cassette region-positive isolates of CTX-M-3-producing S. marcescens from different specimens of blood, urine and sputum No. of CTX-M-3- No. of No. of gene cassette Specimen producing inti1-positive isolate (%) S. marcescens 1) region-positive isolates (%) 2) Blood (53.3) 49 (100.0) Urine (94.1) 27 ( 84.4) Sputum (87.2) 32 ( 78.0) Total (70.5) 108 ( 88.5) 1) indicates the number and percentage of inti1-positive isolate in the total CTX-M-3-producing S. marcescens isolates tested. 2) indicates the number and percentage of gene cassette region-positive isolates in the total inti1- positive isolates. Table 2. Types of gene cassette arrays in class 1 integrons among CTX-M-3-producing S. marcescens isolates from different specimens of blood, urine and sputum No. of strains of Type of gene cassette arrays S. marcescens isolated from Total (%) blood urine sputum I (aada2, aadb-catb3) ( 46.3) II (dfra12-orff-aada2) ( 28.7) III (dfra1-un) ( 0.93) IV (aada2, aadb-catb3, dfra12-orff-aada2) ( 11.1) V (aadb) ( 0.93) VI (oxa31-aada5) ( 0.93) VII (aadb-catb3, dfra12-orff-aada2) ( 0.93) VIII (aadb-catb3) ( 8.3) IX (aada2, dfra12-orff-aada2) ( 0.93) X (aada2) ( 0.93) Total (100) Characterization of gene cassettes in class 1 integrons: By PCR with primers 5 -CS and 3 -CS for the amplification of gene cassette regions, amplicons ranging from 0.7 to 3.0 kb in length were found in 108 (88.5%) of the 122 class 1 integron-containing isolates of CTX-M-3-producing S. marcescens isolates (Fig. 1, Table 1). No gene cassette regions were amplified for the remaining 14 isolates. Ten different types by pattern of amplicons for class 1 integrons were obtained (Fig. 1, Table 2). The Type I amplicon (46.3%) harbors two different class 1 integrons containing the gene cassettes aada2 and aadb-catb3, respectively, and was most prevalent in the gene cassette region-positive S. marcescens isolates, followed by the Type II amplicon, which harbors one type of class 1 integron containing the gene cassette dfra12-orff-aada2 (28.7%) (Table 2). Most of the S. marcescens isolates harboring three different class 1 integrons (Type IV amplicon) were found in blood isolates. The gene cassette dfra12-orff-aada2 was identified in various amplicons of Type II, IV, VII, and IX. All amplicons of Type I, IV, VII, and VIII harbor one class 1 integron containing the gene cassette aadb-catb3. This study is the first to report the presence of the novel gene cassette array of dfra1-un (DQ402098) and oxa31-aada5 (EF067840) in blood isolates of S. marcescens (Table 2). Antimicrobial resistance profiles: The antimicrobial susceptibilities of the 132 isolates of CTX-M-3-producing S. marcescens are compared in Table 3. These strains include 50 isolates harboring two different class 1 integrons containing the gene cassettes aada2 and aadb-catb3, respectively, 31 isolates harboring one class 1 integron containing the gene cassette dfr12-orff-aada2, and 51 isolates that were integronnegative. All strains tested exhibited high levels of resistance Fig. 1. PCR products of 10 different amplicons with gene cassettes array in class 1 integrons among CTX-M-3-producing S. marcescens isolates. Lines 1 to 10 represent amplicons Types I to X, respectively. Type I (aada2, aadb-catb3); Type II (dfra12-orff-aada2); Type III (dfra1-un); Type IV (aada2, aadb-catb3, dfra12-orffaada2); Type V (aadb); 6, Type VI (oxa31-aada5); Type VII (aadbcatb3, dfra12-orff-aada2); Type VIII (aadb-catb3); Type IX (aada2, dfra12-orff-aada2); Type X (aada2). M, molecular weight markers (100 bp-3,000 bp) (Bio-100 TM DNA Ladder; Protech Technology Enterprise Co., Ltd, Taipei, Taiwan). to cefotaxime (MIC > 256 g/ml). All integron-positive isolates showed 100% resistance to streptomycin (MIC > 256 g/ml), spectinomycin (MIC > 512 g/ml), ampicillin (MIC > 256 g/ml), and sulfamethoxazole (MIC > 512 g/ ml). In addition, these isolates carried two different class 1 integrons containing the gene cassettes aada2 and aadbcatb3, respectively, and were associated significantly with 100.0% resistance to chloramphenicol (MIC 64->256 g/ ml). All isolates with integron positive for carrying gene cassettes dfr12-orff-aada2 were also 100% resistant to trimethoprim (MIC16->128 g/ml). In contrast, isolates that were integron-negative generally showed a low rate of resist- 252

4 Table 3. Comparison of antimicrobial resistance of integron-positive S. marcescens and integron-negative isolates % of resistance (MIC, g/ml) for S. marcescens with: Antimicrobial agents integron-positive integron-positive negative for integron harboring Type I harboring Type II (51 stains) amplicon 1) (50 strains) amplicon 2) (31 strains) amikacin 18.0 (8-64) 54.8 (8-64) 3.1 (0.5-32) gentamicin 54.0 (16-32) 51.6 (4-64) 3.1 (0.5-8) kanamycin 94.0 (32-128) 90.3 (16-128) 3.1 (1-32) streptomycin (>256) (>256) 27.7 (32-128) spectinomycin (>512) (>512) 9.2 (64-128) aztreonam 54.0 (8-32) 77.4 (8-64) 0 (1-8) ceftriaxone 70.6 (8-128) 90.3 (8-256) 0 (0.5-8) ceftazidime 6.0 (1.5-64) 6.5 (1.5-64) 7.7 (1.5-32) cefotaxime (>256) (>256) (>256) ampicillin (>256) (>256) 90.7(16-256) piperacillin 98.0 (64->256) 93.6 (64->256) 9.2 (25-128) ciprofloxacin 90.0 ( ) 51.6 ( ) 4.6 (0.01-4) sulfamethoxazole (>512) (>512) 92.3 (128->512) trimethoprim 94.0 ( ) (16->128) 4.6 ( ) chloramphenicol (64->256) 54.8 (8->256) 4.6 (8-32) 1) :Type I amplicon harbors two different class 1 integrons containing gene cassette of aada2 and aadb-catb3, respectively. 2) : Type II amplicon harbors one class 1 integron containing gene cassette of dfr12-orff-aada2. Table 4. Antimicrobial susceptibility profiles of S. marcescens 540, S. marcescens 468, E. coli Type I, E. coli Type II and recipient strain E. coli 14R525 used in conjugation experiments Antimicrobial drugs MIC ( g/ml) for: S. marcescens S. marcescens E. coli E. coli E. coli 540 1) 468 2) 14R525 Type I 3) Type II 4) amikacin gentamicin kanamycin streptomycin >512 >512 2 >512 >512 spectinomycin >512 >512 4 >512 >512 aztreonam ceftriaxone ceftazidime cefotaxime >256 > ampicillin >256 > piperacillin >256 > ciprofloxacin sulfamethoxazole >512 > >512 >512 trimethoprim chloramphenicol ) : S. marcescens 540 harboring two different class 1 integrons with gene cassette of aada2 and aadb-catb3, respectively. 2) : S. marcescens 468 harboring one class 1 integron with gene cassette of dfr12-orff-aada2. 3) : E. coli Type I is a transconjugant containing two different class 1 integrons with gene cassette of aada2 and aadb-catb3, respectively. 4) : E. coli Type II is a transconjugant containing one class 1 integron with gene cassette of dfr12- orff-aada2. ance (0-27.7%) to aminoglycosides, aztreonam, ceftriaxone, ceftazidime, piperacillin, ciprofloxacin, trimethoprim, and chloramphenicol. There was a good correlation between the presence of a resistance gene cassette and the corresponding antimicrobial resistance phenotype, suggesting that the resistance genes in class 1 integrons were expressed. Transfer of resistance: Eighty-one S. marcescens isolates, including 50 isolates carrying two different class 1 integrons haboring aada2 and aadb-catb3, and 31 isolates carrying one class 1 integron with the gene cassette dfra12-orff-aada2, were analyzed to determine the transfer of resistance of class 1 integrons. Conjugation experiments, plasmid DNA analysis and Southern hybridization with an inti1 probe were performed. Class 1 integrons were conjugally transferred to recipients in 46 (92.0%) of the 50 isolates of S. marcescens carrying two different class 1 integrons with the gene cassettes aada2 and aadb-catb3, respectively. Meanwhile, the transfer rates of class 1 integrons with the gene cassette dfra12-orffaada2 were detected in 77.4% (24/31) of S. marcescens isolates. These results demonstrated that all isolates showing conjugative transfer of integrons successfully transferred their class 1 integrons through the conjugative plasmids. The 253

5 Fig. 2. Hybridization analysis of class 1 integrons found on plasmids carried by S. marcescens isolates. (A) Plasmid profiles. Lane 1, S. marcescens isolate contains 150, 70 and 50 kb plasmids. Lane 2, reference strain E. coli 39R861 (containing 154-, 66-, and 38-kb plasmids) was used as negative control. Lane 3, Plasmid pub2401:: Tn21 carrying the Tn21 integron was used as positive control for the presence of the inti1 gene. Lane 4, S. marcescens 468 harboring gene cassette of dfr12-orff-aada2 (plasmid profile containing 150-, 90- and 50- kb plasmid) used as a donor strain for mating experiments. Lane 5, transconjugant E. coli Type II containing gene cassette of dfr12-orffaada2 in conjugative 90-kb plasmid transferred from isolate of S. marcescens 468. Chr, chromosomal DNA. (B) Hybridization of (A) with specific inti1 gene probe (lanes 1 to 5). Class 1 integrons presenting on 70-kb plasmid 50-kb plasmid (lane 1) and 90-kb plasmid (lanes 4 and 5). plasmid DNA profiles of the transconjugants were similar to the original isolates carrying identical gene cassettes, and an inti1 probe was hybridized at plasmids of similar sizes (Figure 2). Table 4 shows that the transfer of resistance from S. marcescens 540 (harboring two different class 1 integrons with the gene cassettes aada2 and aadb-catb3, respectively) and S. marcesencs 468 (harboring one class 1 integron with the gene cassette dfr12-orff-aada2) to recipient strain E. coli 14R525. The results indicated that resistance to streptomycin, trimethoprim, spectinomycin, sulfamethoxazole, and chloramphenicol was co-transferred to the recipient strain via the resistance gene cassettes in class 1 integrons. ERIC-PCR patterns of CTX-M-3-producing S. marcescens isolates: ERIC-PCR analysis was used to analyze the molecular epidemiology of the 50 isolates of CTX- M-3-producing S. marcescens isolates harboring two different class 1 integrons with the gene cassette arrays aada2 and aadb-catb3, respectively (Type I amplicon) (Figure 3) and 31 isolates of CTX-M-3-producing S. marcescens isolates harboring one class 1 integron with the gene cassette array dfra12-orff-aada2 (Type II amplicon) (Figure 4). Distinct ERIC-PCR patterns, ranging in size from kb and varying from 2 to 11 bands, were obtained. These S. marcescens isolates were assigned to three major groups that showed 20-30% similarity. Based on the results of the cluster analysis, these isolates demonstrated no related clonal outbreaks in this experiment. These strains were isolated from different patients, with no apparent relationships in source. DISCUSSION This study demonstrates that class 1 integrons have a high rate of occurrence in resistant isolates of S. marcescens. Although class 2 and class 3 integrons were not detected in this study, the class 3 integron was found; this integron was previously detected in -lactam-resistant isolates of S. marcescens (26). A comparison of integron-positive and integron-negative Fig. 3. ERIC fingerprinting analysis of CTX-M-3-producing S. marcescens harboring Type I amplicon. Key indicates the codeword for 50 clinical strains of S. marcescens isolated from various different clinical specimens. blood isolates of S. marcescens revealed that the former showed a higher incidence than the latter in the multidrugresistance phenotypes, particularly in aminoglycoside and trimethoprim resistance. The results suggest that the multidrug resistance in S. marcescens isolates is associated with the presence of antibiotic resistance gene cassettes in class 1 integrons. The genetic diversity of the gene cassette array in class 1 integrons found in S. marcescens isolates has also been reported in other microorganisms (27,28). Interestingly, the frequency of multidrug-resistant S. marcescens isolates harboring two or more integrons was much higher in the present study than in other studies (29,30). In addition, a higher prevalence of multidrug-resistant S. marcescens found in blood isolates carrying more than one integron was noted. The prevalence and diversity of the genetic structure of class 1 integrons were higher in blood S. marcescens isolates than those of isolates from other specimens. This result suggests that septicemia in the antibiotic selective pressure may enhance the capture of new gene cassettes in class 1 integrons (31). Thus, the genetic diversity of integron-borne gene cassettes has a high association with the presence of antimicrobial 254

6 results suggest the wide distribution of the gene cassette dfra12-orff-aada2 located on class 1 integrons among members of the family Enterobacteriaceae under the selective pressure for intensive use of aminoglycosides, trimethoprim and sulphonamide were reported in Korea (29) and Taiwan (34,35). In this study, we were unable to determine the precise location of the bla CTX-M-3 gene on the variable region of class 1 integrons among CTX-M-3-producing S. marcescens isolates. Further research will be needed to clarify this location. Finally, while it is generally considered that S. marcescens isolates are of nosocomial origin, the multiresistance encoded by these genes in their integrons is still an important factor for the spread of infection among hospitalized patients. ACKNOWLEDGMENTS We thank our colleagues of the Division of Clinical Microbiology at the Department of Laboratory Medicine in Kaohsiung Medical University Hospital for their help in collecting isolates of S. marcescens. This work was supported partly by grant 93-ND-018 from the Medical Research Council of Kaohsiung Medical University Hospital, No. 100 Tzyou 1st Road, Kaohsiung 807, Taiwan. Fig. 4. ERIC fingerprinting analysis of CTX-M-3-producing S. marcescens harboring Type II amplicon. Key indicates the codeword for 31 clinical strains of S. marcescens isolated from various different clinical specimens. resistance genes in bacterial pathogens obtained from hospital patients in which the particular antibiotics are commonly used (3,5,8). Furthermore, bacterial portals of entry in septicemia cases (32) and prophylactic use of antibiotics (33) may contribute to a high degree of diversity in bacterial resistance. This study demonstrated that the aad gene (aada2, aada5, aadb) and dfr gene (dfra1, dfra12) were the most prevalent gene cassettes in class 1 integrons from CTX-M-3- producing S. marcescens. The same trend has also been reported in class 1 integrons from E. coli (28,29). Although a high variability of gene cassettes was observed within the variable region of class 1 integrons in CTX-M-3-producing S. marcescens isolates, all the gene cassettes found in this study mainly contribute to the resistance to trimethoprim, aminoglycosides, chloramphenicol, and oxacillin. To our knowledge, the three class 1 integrons containing the gene cassette arrays of dfra1-un, aadb-catb3, and oxa31-aada5, respectively, have not previously been described in S. marcescens isolates. In this study, the genetic structure of two different class 1 integrons containing the gene cassettes aada2 and aadb-catb3, respectively, and one class 1 integron containing the gene cassette dfra12-orff-aada2 were found most frequently in S. marcescens isolates. However, the gene cassette dfra12-orff-aada2 was commonly found not only in S. marcescens isolates but also in Salmonella isolates and E. coli isolates described by other investigators (29,34,35). These REFERENCES 1. Hejazi, A. and Falkiner, F.R. (1997): Serratia marcescens. J. Med. Microbiol., 46, Yu, W.L., Lin, C.W. and Wang, D.Y. (1998): Serratia marcescens bacteremia: clinical features and antimicrobial susceptibilities of the isolates. J. Microbiol. Immunol. Infect., 31, Shih, H.I., Lee, H.C., Lee, N.Y., et al. (2005): Serratia marcescens bacteremia at a medical center in southern Taiwan: high prevalence of cefotaxime resistance. J. Microbiol. Immunol. Infect., 38, Byrne, A.H., Boyle, B., Herra, C.M., et al. 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