1 Department of Microbiology, 2 Institute of Infectious Diseases and 3 Department of Clinical Pathology, Kyungpook National University School of Medicine, 101 Dongin-dong, Jung-gu, Daegu 700-422, Korea
Received 15 December 2004; returned 18 January 2005; revised 21 January 2005; accepted 26 January 2005
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Abstract |
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Methods: E. coli isolates were examined for susceptibility to antimicrobial agents. Integrase genes were amplified. Gene cassette regions for classes 1 and 2 integrons were amplified and sequenced. Conjugal transfer and Southern hybridization were performed to determine the genetic localization of class 1 integrons. The clonal relationship of E. coli isolates carrying an identical cassette array was analysed by PFGE.
Results: Commensal E. coli isolates from animals were highly resistant to commonly used antimicrobial agents such as tetracycline, sulfamethoxazole, streptomycin, ampicillin and carbenicillin. Integrons were most prevalent in commensal E. coli isolates from poultry (44%), followed by clinical isolates from humans (33%), commensal isolates from swine (23%) and humans (13%). dfrA17-aadA5, dfrA12-orfF-aadA2 and aadA1 were found most frequently in E. coli isolates from humans, poultry and swine, respectively. Class 1 integrons were mostly located in conjugative plasmids. E. coli isolates carrying an identical cassette array were phylogenetically unrelated.
Conclusions: The use of antibiotics is strongly associated with antimicrobial resistance. E. coli isolates from different sources may select a specific gene cassette by antibiotic selective pressure, which results in differences in class 1 integrons. The horizontal transfer of class 1 integrons through conjugative plasmids seems to be responsible for wide dissemination of a particular type of class 1 integron.
Keywords: antibiotics , gene cassettes , plasmids
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Introduction |
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Since many gene cassettes of integrons contain antimicrobial resistance genes in Gram-negative bacteria, the horizontal transfer of integrons through plasmids and transposons has been found to play an important role in the dissemination of antimicrobial resistance genes and the development of multiresistance.6 Three classes of integrons that encode antimicrobial resistance have been described. Class 1 integrons are most commonly found in clinical isolates of Gram-negative bacteria and more than 60 distinct gene cassettes have been identified.7 In our previous studies, class 1 integrons were detected in 54% of urinary E. coli isolates, and the presence of integrons was strongly associated with multiresistance.8
Apart from being carried by clinical pathogens, few studies have reported the prevalence of integrons and gene cassettes in the enteric faecal flora of humans and animals.9,10 The transfer of integrons between human and animal isolates is not fully understood, although it is known that resistance to antimicrobial agents is transferred from animals to humans through transferable plasmids.11 The aim of the current study was to characterize antimicrobial susceptibility and integrons found in E. coli isolates from humans and animals. The study was also to determine if there was any possible transfer of antimicrobial resistance between E. coli isolates from humans and animals.
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Materials and methods |
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A total of 664 E. coli isolates were obtained from four sources: clinical specimens from hospitalized patients at Kyungpook National University Hospital (n=201), the stools of healthy humans (Kyungpook National University students, n=167), large intestine swabs from poultry (n=163) and swine (n=133) grown in Kyungpook province during 20012003.
The isolates from poultry and swine were obtained from their respective slaughterhouses. The animals from which E. coli isolates were obtained consumed feed containing antibiotics. The patients and students from whom isolates were obtained consumed the animal products from these slaughterhouses.
Antimicrobial susceptibility testing
The MICs were determined by agar dilution in a MuellerHinton agar (Difco Laboratories, Detroit, MI, USA) according to the guidelines of the NCCLS.12
The inoculated plates were incubated at 37°C for 20 h, and the MIC defined as the lowest concentration of antimicrobial agent that completely inhibited the growth of the organism. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. Antimicrobial resistance was determined according to their MICs of ampicillin (32 mg/L), carbenicillin (
64 mg/L), piperacillin (
128 mg/L), cefazolin (
32 mg/L), cefoxitin (
32 mg/L), ceftazidime (
32 mg/L), streptomycin (
64 m/L), kanamycin (
64 mg/L), gentamicin (
16 mg/L), tobramycin (
16 mg/L), sulfamethoxazole (
512 mg/L), trimethoprim (
16 mg/L), ciprofloxacin (
4 mg/L), chloramphenicol (
32 mg/L) and tetracycline (
16 mg/L).
Conjugal transfer of plasmids carrying integrons
E. coli RG488 Rifr and RG176 Nalr were used as the recipients for the conjugation experiment.13 Prior to each experiment for conjugation, a single colony from a MacConkey agar plate (Difco Laboratories) was inoculated in trypticase soy broth (TSB, Difco Laboratories) and grown at 37°C for 20 h. The donor and recipient strains in a logarithmic phase were grown in TSB, mixed and incubated at 37°C for 20 h. The transconjugants were selected on MuellerHinton agar supplemented with trimethoprim (50 mg/L) or streptomycin (50 mg/L), and rifampicin (50 mg/L) or nalidixic acid (50 mg/L).
Plasmid DNA isolation and Southern hybridization
The plasmid DNA was isolated using the alkaline extraction method.14 The extracted DNA was separated by electrophoresis in 0.7% agarose gel. After agarose gel electrophoresis of the plasmids, the denatured DNA was transferred onto a positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany) using the capillary method. For the hybridization assays, a DIG DNA labelling and detection kit (Roche, Mannheim, Germany) was used according to the manufacturer's instructions. The purified integrase gene from the PCR products was used as the probe DNA and labelled with digoxigenin-11-dUTP according to the random labelling method.
PCR amplification of integrase genes
The conserved regions of integrase genes were amplified using the degenerate primer hep35 (5'-TGCGGGTYAARGATBTKGATTT-3') and hep36 (5'-CARCACATGCGTRTARAT-3'), and the PCR products were further restricted using either RsaI or HinfI (Boehringer Mannheim) to determine the class of integrons as described previously.15 The template was prepared by suspending a loopful of each isolate, which had been growing on a trypticase soy agar (Difco Laboratories) plate, in 200 µL of sterile water, followed by boiling for 10 min and centrifuging for 5 min.
Amplification and sequencing of gene cassette regions
The gene cassette regions for the class 1 integrons were amplified using hep58 (5'-TCATGGCTTGTTATGACTGT-3') and hep59 (5'-GTAGGGCTTATTATGCACGC-3') as described previously.15,16
The gene cassette regions for the class 2 integrons were amplified using hep74 (5'-CGGGATCCCGGACGGCATGCACGATTTGTA-3') and hep51 (5'-GATGCCATCGCAAGTACGAG-3'). To determine identical arrays of gene cassettes, same-sized amplicons were digested with RsaI and HinfI (Boehringer Mannheim). The PCR products of the gene cassette regions that showed different restriction patterns were ligated with a pGEM T-easy vector (Promega, Madison, WI, USA) and transformed to E. coli DH5 cells. Sequencing reactions were performed with a double-stranded plasmid preparation using dideoxy chain termination with T7 and Sp6 primers.
PFGE
The genomic DNA was digested with XbaI (Boehringer Mannheim) for 18 h and separated on a 1.0% agarose gel using a contour-clamped homogeneous-field apparatus (CHEF DRIII systems; Bio-Rad Laboratories, Hercules, CA, USA) in a 0.5 x TBE buffer.17
The conditions for electrophoresis were 6 V/cm for 20 h with an increasing pulse time of 540 s. A DNA ladder consisting of 48.5 kb concatemers (Bio-Rad Laboratories) was used as the size standard. The PFGE patterns were interpreted using the criteria established by Tenover et al.18
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Results |
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Of the 368 E. coli isolates of human origin, the overall rates of resistance to antimicrobial agents were higher in clinical E. coli isolates than in commensal E. coli isolates (Table 1). Fifty-five (32.9%) commensal E. coli isolates were all susceptible to antimicrobial agents tested, whereas three (1.5%) clinical isolates were all susceptible. The second or third generation of cephalosporins such as cefoxitin, cefuroxime, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone and ceftazidime have been used to treat Gram-negative bacterial infections in humans in Korea. Only one commensal E. coli isolate was resistant to cephalosporins tested, whereas 66 (32.8%) clinical E. coli isolates were resistant to cefazolin.
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Integron carriage and characterization of gene cassettes
Of the 664 E. coli isolates tested, 189 (28.5%) carried an integrase gene (Table 2). Integrase genes were detected in 44.2% (72/163), 32.8% (66/201), 22.6% (30/133) and 12.6% (21/167) of commensal isolates from poultry, clinical isolates from humans, commensal isolates from swine and humans, respectively. Among the 189 integrase-positive isolates, 175 (92.6%) carried intI1, nine (4.8%) carried intI2 and five (2.6%) carried both intI1 and intI2. No class 3 integrons were detected.
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To determine the genetic localization of class 1 integrons, 42 E. coli isolates, comprising 16 isolates carrying dfrA1-aadA2, 14 isolates carrying dfrA12-orfF-aadA2 and 12 isolates carrying dfrA17-aadA5, were selected on the basis of isolation date and origin. Conjugation experiment, plasmid analysis and Southern hybridization with intI1 probe were performed. Class 1 integrons were conjugally transferred to recipients in 32 (76.2%) of the 42 isolates. The transfer rates of class 1 integrons carrying dfrA1-aadA2, dfrA12-orfF-aadA2 and dfrA17-aadA5 were 68.8% (11/16), 85.7% (12/14) and 75.0% (9/12), respectively. To determine the genetic localization of non-transferable class 1 integrons, Southern hybridization with an intI1 probe was performed, which revealed that all class 1 integrons were located in plasmids, except for two class 1 integrons carrying dfrA1-aadA2 and dfrA12-orfF-aadA2 that were located in a chromosome. The plasmid profiles of original isolates carrying identical gene cassettes were very different from each other, but intI was hybridized at plasmids of similar sizes (7385 kb) (data not shown).
Epidemiological typing of E. coli isolates carrying identical cassette arrays
To determine whether the high prevalence of a particular type of class 1 integron in E. coli isolates from different sources was caused by the spread of a specific clone, 42 E. coli isolates, which had been selected previously, were analysed by PFGE profiles. E. coli isolates carrying the identical type of class 1 integrons showed distinct patterns (Figure 1). This finding suggested that the spread of a specific clone did not contribute to a wide dissemination of a particular type of class 1 integron.
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Discussion |
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Class 1 integrons are widely distributed in E. coli from humans and animals and directly contribute to the resistance to trimethoprim, aminoglycosides and sulphonamides. Differences in gene cassette arrays of class 1 integrons were found in E. coli isolates from different sources. dfrA17-aadA5, dfrA12-orfF-aadA2 and aadA1 were the most prevalent in E. coli isolates from humans, poultry and swine, respectively. In our previous study, dfrA17-aadA5 has been the most prevalent in urinary E. coli isolates from humans since 1994.8,20 dfrA1-aadA2 first appeared in clinical E. coli isolates from the mid-1990s and the incidence of this type of class 1 integron was maintained at < 10% after 1996.8 This type of class 1 integron was prevalent among commensal E. coli isolates from humans in the current study. Class 1 integrons carrying a single gene cassette such as aadA1 were the most prevalent in commensal E. coli isolates from swine, whereas most of the isolates carried multigene cassettes in clinical isolates from humans. Class 1 integrons carrying single gene cassettes were prevalent in clinical E. coli isolates from the 1980s, whereas class 1 integrons carrying multigene cassettes were prevalent in clinical isolates from the 1990s and 2000s.8 Moreover, the incidence of each class 1 integron in commensal E. coli isolates from swine and poultry was similar to that of clinical E. coli isolates from humans in the 1980s and 1990s, respectively.8 Accordingly, it would seem that E. coli isolates from different sources selected a specific gene cassette by antibiotic selective pressure. The prevalence of class 1 integrons carrying multigene cassettes was explained by the introduction of a new gene cassette encoding resistance to antimicrobial agents commonly used.
Wide dissemination of class 1 integrons carrying identical gene cassettes was observed in E. coli isolates from human and animal origins. We assessed whether wide dissemination of class 1 integrons carrying identical gene cassettes was due to clonal spread or horizontal transfer of plasmids carrying class 1 integrons. E. coli isolates carrying identical cassette arrays showed distinct PFGE patterns, which indicated little clonal relatedness between the E. coli isolates. Leverstein-van Hall et al.21,22 proposed that the worldwide distribution of identical gene cassettes in multiresistant Enterobacteriaceae from clinical specimens and food-producing animals was due to the transfer of class 1 integrons between isolates from animals and humans via the food chain. Three prevalent class 1 integrons were mostly located in plasmids and conjugally transferable, which suggests the horizontal transfer of class 1 integrons through conjugative plasmids. However, the plasmids of the transconjugants carrying identical cassette arrays showed different restriction fragment length polymorphism patterns with HindIII, and the intI probe was hybridized with different-sized fragments of plasmids (data not shown). Accordingly, we could not directly demonstrate evidence for horizontal transfer of class 1 integrons between human and animal E. coli isolates.
In conclusion, the use of antibiotics is strongly associated with the prevalence of antimicrobial resistance in commensal E. coli isolates in food-producing animals. Class 1 integrons were found to be widely disseminated among E. coli isolates from humans and animals. E. coli isolates from different sources selected a specific gene cassette by antibiotic selective pressure. The prevalence of a particular type of class 1 integron in different sources may not be directly associated with clonal spread, but may be associated with horizontal transfer of conjugative plasmids. Therefore, the sequential analysis of class 1 integrons in Enterobacteriaceae from clinical specimens, faecal flora and environmental samples can provide information regarding the evolutionary changes of gene cassettes in class 1 integrons.
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Acknowledgements |
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