CHARACTERIZATION OF INTEGRON-MEDIATED ANTIMICROBIAL RESISTANCE AMONG ESCHERICHIA COLI STRAINS ISOLATED FROM A CAPTIVE POPULATION OF AMUR TIGERS IN CHINA Author(s): Yuan Xue, Ph.D., Jianfei Chen, Ph.D., Yulong Wang, Ph.D., Yanlong Zhang, Ph.D., Dan Liu, Ph.D. and Yuping Hua, Ph.D. Source: Journal of Zoo and Wildlife Medicine, 44(4):951-956. 2013. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0020R2.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0020R2.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 44(4): 951–956, 2013 Copyright 2013 by American Association of Zoo Veterinarians
CHARACTERIZATION OF INTEGRON-MEDIATED ANTIMICROBIAL RESISTANCE AMONG ESCHERICHIA COLI STRAINS ISOLATED FROM A CAPTIVE POPULATION OF AMUR TIGERS IN CHINA Yuan Xue, Ph.D., Jianfei Chen, Ph.D., Yulong Wang, Ph.D., Yanlong Zhang, Ph.D., Dan Liu, Ph.D., and Yuping Hua, Ph.D.
Abstract: The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant Escherichia coli isolates from a captive population of Amur tigers (Panthera tigris altaica) in China. In addition, the prevalence of antimicrobial resistance and class I integrons was assessed in E. coli strains (n ¼ 61) isolated from a captive population of Amur tigers in Heilongjiang Amur Tiger Park, China. Among the isolates, 52.46% (32 of 61) were positive for intI1, but no isolates carried intI2 or intI3. Most isolates were susceptible to amoxicillin/clavulanic acid, aztreonam, and polymyxin B, while they also exhibited high incidence rates of resistance to ampicillin, doxycycline, chloramphenicol, tetracycline, and dihydrofolate reductase. Sequencing analysis revealed three gene cassettes, which encoded resistance to dihydrofolate reductase (dfrA15), dihydrofolate reductase (dfrA12), and adenyltransferase (aadA2). The gene cassette arrays dfrA15 (31%) and dfrA12–aadA2 (19%) were most prevalent among these isolates. Key words: Amur tiger, antimicrobial resistance, Escherichia coli, gene cassette, integron.
INTRODUCTION The Amur tiger (Panthera tigris altaica) is the largest of the five tiger subspecies. They are currently distributed mainly in the eastern mountainous areas of Heilongjiang and Jilin provinces in China, in east Russia, and North Korea.11 Heilongjiang Amur Tiger Park is currently the world’s largest wild natural garden for the Amur tiger, which was established to save and protect some of the world’s endangered species, including the Amur tiger. Although antimicrobial therapy is used to treat clinical infections, antibiotic resistance in pathogenic bacteria from human, animal, and environmental sources is recognized as a global problem in public health. The increasing drug resistance in clinical isolates may be explained by antibiotic selection pressure and the widespread presence of integrons. In particular, over the past decade, the high degree of resistance to common antibiotics and the worldwide emergence of multidrug-resisFrom the College of Wildlife Resources of Northeast Forest University, Harbin 150040, Heilongjiang, People’s Republic of China (Xue, Wang, Zhang, Hua); State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, People’s Republic of China (Chen); and Heilongjiang Amur Tiger Park, Harbin 150400, Heilongjiang, People’s Republic of China (Liu). Correspondence should be directed to Dr. Hua ([email protected]).
tant (MDR) phenotypes are of increasing concern.15 Integrons are genetic structures able to capture, excise, and express genes and are frequently included in mobile elements such as plasmids that allow their dissemination among bacteria.18 They are genetic elements that incorporate an open reading frame or gene cassettes.13 Integrons, which are widely distributed among bacteria, are capable of capturing and, integrating antibiotic-resistance gene cassettes. The class 1 integron is the most numerous, especially in clinical Gram-negative bacterial isolates. Class 1 integrons are assumed to play an important role in the dissemination of antibiotic resistance genes. Class 1 integrons, the major integrons found in clinical isolates, are strongly associated with multiple antibiotics. Those resistances obtained from clinical isolates often have 59- and 39conserved segments flanking the variable region where gene cassettes integrate.10,22 Most genes inserted into integrons lack their own promoters, and expression is dependent on two potential promoters, called P1 and P2, which may differ in sequence and length in different integrons, located in the 59-conserved segment of the integron.9 Currently, there is little information regarding the molecular basis of antibiotic resistance in Escherichia coli strains isolated from captive populations of Amur tigers; therefore, this study was conducted to screen and analyze the antimicrobial resistance in E. coli strains isolated from a
951
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Table 1. Primers
IntI1 IntI2 IntI3 59-CSa 39-CS a
Oligonucleotide primers used for the amplification of integrons. Nucleotide sequences (59 to 39)
Sizes of polymerase chain reaction amplicons (base pair)
IntI1F:CCTCCCGCACGATGATC IntI1R:TCCACGCATCGTCAGGC IntI2F:TTGCGAGTATCCATAACCTG IntI2R:TTACCTGCACTGGATTAAG IntI3F:AGTGGGTGGCGAATGAGTG IntI3R:TGTTCTTGTATCGGCAGGTG 59-CSF:GGCATCCAAGCAGCAAG 39-CSR:AAGCAGACTTGACCTGA
References
280
22
288
8
600
6
variable
10
CS ¼ conserved segment
captive population of Amur tigers in Heilongjiang Amur Tiger Park in China.
MATERIALS AND METHODS Bacterial isolates During May and November 2011, 61 E. coli strains were isolated from captive Amur tigers in Heilongjiang Amur Tiger Park. The fecal swabs were inoculated in nutrient broth (OxoidTM, Thermo Fisher Biochemicals Ltd., Beijing 101312, China) and incubated at 378C for 18 hr, purified subsequently on MacConkey agar (Hangzhou Tianhe Microorganism Reagent Co. Ltd., Hangzou 310008, Zhejiang, China) and eosin methylene blue ([EMB]; Hangzhou Tianhe Microorganism Reagent Co. Ltd.) agar plates, and incubated at 378C for 24–48 hr. For selective isolation and identification of E. coli, two blue– black colonies (presumptive E. coli) with a metallic sheen, growing on EMB agar plates, were randomly selected from each plate. Escherichia coli (25922TM, American Type Culture Collection [ATCCt], Manassas, Virgina 20110, USA) was used as the reference strain. The strain was stored at 808C until analysis. Antibiotic resistance test The antimicrobial sensitivity phenotypes of the E. coli strains were determined using a Kirby– Bauer disk diffusion assay, according to the standards and interpretive criteria described by Clinical and Laboratory Standards Institute (CLSI) guidelines.2 Antimicrobial susceptibility testing was performed using the CLSI document on Mueller– Hinton agar (Hangzhou Tianhe Microorganism Reagent Co. Ltd.). The following antimicrobials were tested: ampicillin, amoxicillin/clavulanic acid, cefalotin, aztreonam, gentamicin, kanamycin, apramycin, ofloxacin, ciprofloxacin, norfloxacin, enrofloxacin base, doxycycline, chloramphenicol,
florfenicol, polymyxin B, trimethorpim sulfur, and tetracycline. All antibiotic disks were purchased from Oxoid (Thermo Fisher Biochemicals Ltd.). Quality control for susceptibility testing was performed using E. coli (ATCC 25922), which is a recommended reference strain for antibiotic susceptibility testing. Detection of the integrase genes and gene cassettes The polymerase chain reaction (PCR) amplification of integrase genes was performed using specific primer pairs (Table 1) published in previous reports6,8,10,22 to determine the existence of antimicrobial resistance gene cassettes within the integrons. DNA sequencing and data analysis The PCR products were excised from 1.0% agarose gels and purified using the AxyPrepTM DNA Gel Extraction Kit (Axygen Scientific, Inc., Union City, California 94587, USA), cloned subsequently into a T-tailed vector, pMD18T(TaKaRa Biotechnology Co., Ltd., Dalian, 116600, China), and transformed using JM109 competent cells (TaKaRa Biotechnology Co., Ltd.). Three to five recombinant DNA clones of each E. coli strain were sequenced by Beijing Genomics Institute (Beijing, 101300, China). The nucleotide sequences were edited and analyzed with EditSeq software (version 7.1.0, DNASTAR Inc., Madison, Wisconsin 53705, USA) and the program NCBI-BLAST (www.ncbi.nlm.nih.gov).
RESULTS Antimicrobial resistance rates The antimicrobial susceptibility results of 61 E. coli strains isolated from the captive population of Amur tigers to 17 antimicrobial agents are shown in Figure 1. About 72% isolates were susceptible
XUE ET AL.—INTEGRON-MEDIATED ANTIMICROBIAL RESISTANCE
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Figure 1. In vitro susceptibilities of 61 E. coli strains isolated from captive Amur tiger to 17 antimicrobial agents. R% ¼ resistance rate; I% ¼ intermediate rate; S% ¼ susceptible rate; AMP ¼ ampicillin; AMC ¼ amoxicillin/ clavulanicacid; CEF ¼ cefalotin; ATM ¼ aztreonam; GEN ¼ gentamicin; KAN ¼ kanamycin; APR ¼ apramycin; OF ¼ ofloxacin; NOR ¼ norfloxacin; CIP ¼ ciprofloxacin; EN ¼ enrofloxacin base; DOX ¼ doxycycline; CMP ¼ chloramphenicol; FFC ¼ florfenicol; SXT ¼ trimesulf; PB ¼ polymyxin B; TET ¼ tetracycline.
to amoxicillin/clavulanic acid (73.66%), aztreonam (71.46%), and apramycin (71.71%). However, high incidence rates were exhibited for resistance to ampicillin (100.00%), tetracycline (85.12%), and trimesulf (80.24%). In this study, the antimicrobial agents tested were divided into eight classes. Among these, there were four fluoroquinolones, two penicillins, two cephalosporins, one tetracycline, four aminoglycosides, two chloramphenicols, and two sulfonamides. By selecting one representative drug from each class, the most frequent antibiotic resistance patterns of the 61 E. coli strains to the 17 antimicrobial agents are shown in Table 2. Detection of class 1, 2, and 3 integrons by PCR Neither class 2 nor class 3 integrons were detected, while 52.46% (32 of 61) of the isolates were positive for the presence of intI1, which is a marker for class 1 integrons. Gene cassettes detected by PCR The integron-borne gene cassettes detected by PCR were cloned and sequenced. The integrons were found to contain one or two gene cassettes, and the combinations of these gene cassettes are shown in Table 2. Two distinct types of gene cassette array were characterized: dfrA15 and
dfrA12–aadA2, respectively. A search for the published homology sequences in GenBank revealed that 1) the gene cassette array dfrA12– aadA2 presented 99% identity with that found in E. coli isolates from beef cattle in Canada (FJ855131.1), human in Russia (GU001949.1, GQ924767.1, and GQ924766.1), and the Ku¨u¨kcekmece lagoon in Turkey (GQ280258.1); and 2) the gene cassette array dfrA15 presented 100% identity with those found in E. coli isolates from the Ku¨c u¨kcekmece lagoon in Turkey (GQ280260.1 and GQ280262.1). Correlation analysis was performed between the antimicrobial resistance profile and the occurrence of integrons.
DISCUSSION Escherichia coli is commonly found in the gastrointestinal tracts of domestic and wild animals and humans.21 Various selection pressures in these environments favor the development, persistence, and dissemination of robust E. coli strains, some of which may be resistant to antimicrobial agents.4 Antimicrobial susceptibility patterns of E. coli isolates to 17 antimicrobial agents were obtained, and the results are presented in Figure 1 and Table 2. As shown in Figure 1, most of the isolates were susceptible to amoxicillin/clavulanic acid, aztreonam, and apramycin. On the other hand, the
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Table 2. integrons.
JOURNAL OF ZOO AND WILDLIFE MEDICINE
Antibiotic resistance patterns of E. coli isolates and their relationship with occurrence of the
No. of strains
16 07 57 39 37 22 74 55 32 75 29 68 72 23 62 91
Resistance phenotypesa
AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP,
CEF, GEN, DOX, TET, EN, SXT, CMP, PB, FFC AMC, CEF, KAN, DOX, TET, EN, SXT, CMP, NOR CEF, GEN, DOX, TET, EN, SXT, CMP, PB, FFC, CIP CEF, GEN, KAN, TET, EN, SXT, CMP, PB, FFC, CIP, NOR APR, CEF, TET, SXT, CMP AMC, APR, CEF, GEN, KAN, DOX, TET, CIP, EN, OF, SXT, CMP, NOR GEN, DOX, TET, FFC, SXT DOX, TET, SXT CEF, KAN, DOX,TET, SXT, CMP, PB, FFC CEF, KAN, DOX, TET, NOR, SXT, CMP CEF, GEN, KAN, DOX, TET, EN, SXT, CMP, FFC, CIP, NOR CEF, CIP, DOX, TET, KAN, SXT CEF, DOX, TET, EN, SXT,CMP, CIP, KAN CEF, CIP, GEN, KAN, DOX, TET, EN, SXT, CMP, NOR, FFC CEF, DOX, KAN, TET, EN, SXT, CMP, CIP, NOR, FFC ATM, APR, CEF, GEN, KAN, DOX, TET, CIP, EN, OF, SXT, CMP, NOR
Integrons
dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15
a AMP, ampicillin; AMC, amoxicillin/clavulanicacid; CEF, cefalotin; ATM, aztreonam; GEN, gentamicin; KAN, kanamycin; APR, apramycin; OF, ofloxacin; NOR, norfloxacin; CIP, ciprofloxacin; EN, enrofloxacin base; TET, tetracycline; DOX, doxycycline; CMP, chloramphenicol; FFC, florfenicol; SXT, trimesulf; PB, polymyxin B.
high incidence rates of resistance to tetracycline, chloramphenicol, and trimethoprim sulfur may be derived from the frequent, long-term, and widespread use of these antimicrobials in the park studied. In the multiresistant strains, the rates of resistance of E. coli to ciprofloxacin, norfloxacin, and ofloxacin were similar. In the past few years, an intensive and largescale pig, chicken, and bovine farming industry in China has developed. Detailed results of antimicrobial resistance testing of E. coli isolates from various food animals, including cattle, swine, camels, sheep, goats, chickens, and foods of animal origin, in China have been reported previously.15,20 Integrons play an important role in the acquisition and dissemination of antimicrobial resistance genes. Integrons that contain gene cassettes provide a powerful vehicle for the rapid horizontal transfer of resistance across bacterial populations and thus may contribute to a sudden increase in the prevalence of MDR infections in a community.12 Class 1 integrons have been detected previously in different E. coli strains. For instance, class 1 integrons were detected in 10 to 65% of the antimicrobial resistant E. coli isolated in Egypt, Spain, Switzerland, China, and the United States.1,3,7,13,18,20 In this study, the prevalence of integron-positive isolates was 52.46%, which is similar to the data obtained from the Beijing area in China, which showed a prevalence of 54.70%.5 It has been indicated that the prevalence of
integrons is related to the antimicrobial pressure in the environment. The investigation of resistance gene cassettes in this study revealed that aminoglycoside resistance determinants (dfrA12–aadA2) and trimethoprim resistance determinants (dfrA15) were prevalent among E. coli strains isolated from a captive population of Amur tigers. To the authors’ knowledge, there are no previous reports in the scientific literature on integrons and associated gene cassettes in E. coli or other bacterial pathogens from the captive population of Amur tigers in Heilongjiang Amur Tiger Park. However, the persistence of these gene cassettes has been reported worldwide in isolates of different origins.7,14,17 Similar to this current study, class 1 integrons, including dfr and aadA gene cassettes, have been found to be the most prevalent type of integron in E. coli isolates from humans, animals, and food in other studies.16,19 In conclusion, this study demonstrated the distribution of the class I integrons among E. coli strains isolated from a captive population of Amur tigers in Heilongjiang Amur Tiger Park. Gene cassettes not previously reported in integrons among E. coli from this captive population of Amur tigers were found. The genetic content, combination, and frequency of emergence of these gene cassettes may reflect the antibiotic selection pressure in Heilongjiang Amur Tiger Park, China, and provide useful surveillance information for the rational use of antimicrobials.
XUE ET AL.—INTEGRON-MEDIATED ANTIMICROBIAL RESISTANCE
The potential role of integrons in the uptake and dissemination of resistance genes may be a continuing threat to the effectiveness of certain antibiotic therapeutic agents in Heilongjiang Amur Tiger Park. Acknowledgments: The authors thank Ming Zhou and Xian-guang Huang for the collection of isolates. This study was supported by the Fundamental Research Funds for the Central Universities (DL12BA04) and the Heilongjiang Postdoctoral Financial Assistance (LBHZ11262).
LITERATURE CITED 1. Ahmed, A. M., E. E. Younis, S. A. Osman, Y. Ishida, S. A. El-Khodery, and T. Shimamoto. 2009. Genetic analysis of antimicrobial resistance in Escherichia coli isolated from diarrheic neonatal calves. Vet. Microbiol. 136: 397–402. 2. Clinical and Laboratory Standards Institute. 2006. Performance Standards for Antimicrobial Susceptibility Testing; 16th Informational Supplement. Document M100-S16. Clinical Laboratory Standards Institute, Wayne, Pennsylvania. p. 32–58. 3. Cocchi, S., E. Grasselli, M. Gutacker, C. Benagli, M. Convert, and J. C. Piffaretti. 2007. Distribution and characterization of integrons in Escherichia coli strains of animal and human origin. FEMS Immunol. Med. Microbiol. 50: 126–132. 4. Davies, J., and S. O’Connor. 1978. Enzymatic modification of aminoglycoside antibiotics: 3-N-acetyltransferase with broad specificity that determines resistance to thenove aminoglycoside apramycin. Antimicrob. Agents Chemother. 14: 69–72. 5. Du, X., Z. Shen, B. Wu, S. Xia, and J. Shen. 2005. Characterization of class integrons-mediated antibiotic resistance among calf pathogenic Escherichia coli. FEMS Microbiol. Lett. 245: 285–289. 6. Goldstein, C., M. D. Lee, S. Sanchez, C. Hudson, B. Phillips, B. Register, M. Grady, C. Liebert, A. O. Summers, D. G. White, and J. J. Maurer. 2001. Incidence of Class 1 and class 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrob. Agents Chemother. 45: 723–726. 7. Hsu, S., T. Chiu, J. Pang, C. Hsuan-Yuan, G. Chang, and H. Tsen. 2006. Characterisation of antimicrobial resistance patterns and class 1 integrons among Escherichia coli and Salmonella enterica serovar Choleraesuis strains isolated from humans and swine in Taiwan. Int. J. Antimicrob. Agents 27: 383–391. 8. Koeleman, J. G., J. Stoof, and M. W. Van. 2001. Identification of epidemic strains of Acine-tobacter baumannii by integrase gene PCR. J. Clin. Microbiol. 39: 8–13. 9. Le´vesque C., S. Brassard, J. Lapointe, and P. H. Roy. 1994. Diversity and relative strength of tandem
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promoters for the antibiotic-resistance genes of several integrons. Gene 142: 49–54. 10. Le´vesque, C., L. Piche, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39: 185–195. 11. Luan, X., Y. Qu, D. Li, S. Liu, X. Wang, B. Wu, and C. Zhu. 2011. Habitat evaluation of wild Amur tiger (Panthera tigris altaica) and conservation priority setting in north-eastern China. J. Environ. Manag. 92: 31–42. 12. Ruiz-Martı´nez, L., L. Lo´pez-Jime´nez, E. Fuste´, T. Vinuesa, J. P. Martı´nez, and M. Vinas. 2011. Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa. Int. J. Antimicrob. Agents 38: 398–402. 13. Shaheen, B. W., O. A. Oyarzabal, and D. M. Boothe. 2010. The role of class 1 and 2 integrons in mediating antimicrobial resistance among canine and feline clinical E. coli isolates from the US. Vet. Microbiol. 144: 363–370. 14. Shriparna, M., and C. Ranadhir. 2006. Incidence of class 1 integrons in multiple resistant Gram-negative copiotrophic bacteria from the River Torsa in India. Res. Microbiol. 157: 220–226. 15. Tang, X., C. Tan, X. Zhang, Z. Zhao, X. Xia, B. Wu, A. Guo, R. Zhou, and H. Chen. 2011. Antimicrobial resistances of extraintestinal pathogenic Escherichia coli isolates from swine in China. Microb. Pathog. 50: 207–212. 16. Trobos, M., L. Jakobsen, K. E. Olsen, N. Frimodt-Møller, M. A. Hammerum, K. Pedersen, Y. Agersø, L. J. Porsbo, and J. E. Olsen. 2008. Prevalence of sulphonamide resistance and class 1 genes in Escherichia coli isolates obtained from broilers, broilermeat, healthy humans and urinary infections in Denmark. Int. J. Antimicrob. Agents 32: 367–369. 17. Van Essen-Zandbergen, A., H. Smith, K. Veldman, and D. Mevius. 2009. In vivo transfer of an incFIB plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1. Vet. Microbiol. 137: 402–407. 18. Vinue´, L., Y. Sa´enz, B. Rojo-Bezares, I. Olarte, E. Undabeitia, S. Somalo, M. Zarazaga, and C. Torres. 2010. Genetic environment of sul genes and characterisation of integrons in Escherichia coli isolates of blood origin in a Spanish hospital. Int. J. Antimicrob. Agents 35: 492–496. 19. Vinue´, L., Y. Sa´enz, S. Somalo, E. Escudero, M. A. Moreno, F. Ruiz-Larrea, and C. Torres. 2008. Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain. J. Antimicrob. Chemother. 62: 934–937. 20. Wang, G., C. Wu, X. Du, Z. Shen, L. Song, X. Chen, and J. Shen. 2008. Characterization of integronsmediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis. Vet. Microbiol. 127: 73–78. 21. Zhang, X., L. Ding, and M. Fan. 2009. Resistance patterns and detection of aac (3)-IV gene in
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apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China. Res. Vet. Sci. 87: 449–454. 22. Zhao, S., D. G. White, B. Ge, S. Ayers, S. Friedman, L. English, D. Wagner, S. Gaines, and J. Meng. 2001. Identification of integron-mediated anti-
biotic -resistance among Shigatoxin-Producing Escherichia coli isolates. Appl. Environ. Microbiol. 67: 1558– 1564.
Received for publication 18 March 2013
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 44(4): 951–956, 2013 Copyright 2013 by American Association of Zoo Veterinarians
CHARACTERIZATION OF INTEGRON-MEDIATED ANTIMICROBIAL RESISTANCE AMONG ESCHERICHIA COLI STRAINS ISOLATED FROM A CAPTIVE POPULATION OF AMUR TIGERS IN CHINA Yuan Xue, Ph.D., Jianfei Chen, Ph.D., Yulong Wang, Ph.D., Yanlong Zhang, Ph.D., Dan Liu, Ph.D., and Yuping Hua, Ph.D.
Abstract: The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant Escherichia coli isolates from a captive population of Amur tigers (Panthera tigris altaica) in China. In addition, the prevalence of antimicrobial resistance and class I integrons was assessed in E. coli strains (n ¼ 61) isolated from a captive population of Amur tigers in Heilongjiang Amur Tiger Park, China. Among the isolates, 52.46% (32 of 61) were positive for intI1, but no isolates carried intI2 or intI3. Most isolates were susceptible to amoxicillin/clavulanic acid, aztreonam, and polymyxin B, while they also exhibited high incidence rates of resistance to ampicillin, doxycycline, chloramphenicol, tetracycline, and dihydrofolate reductase. Sequencing analysis revealed three gene cassettes, which encoded resistance to dihydrofolate reductase (dfrA15), dihydrofolate reductase (dfrA12), and adenyltransferase (aadA2). The gene cassette arrays dfrA15 (31%) and dfrA12–aadA2 (19%) were most prevalent among these isolates. Key words: Amur tiger, antimicrobial resistance, Escherichia coli, gene cassette, integron.
INTRODUCTION The Amur tiger (Panthera tigris altaica) is the largest of the five tiger subspecies. They are currently distributed mainly in the eastern mountainous areas of Heilongjiang and Jilin provinces in China, in east Russia, and North Korea.11 Heilongjiang Amur Tiger Park is currently the world’s largest wild natural garden for the Amur tiger, which was established to save and protect some of the world’s endangered species, including the Amur tiger. Although antimicrobial therapy is used to treat clinical infections, antibiotic resistance in pathogenic bacteria from human, animal, and environmental sources is recognized as a global problem in public health. The increasing drug resistance in clinical isolates may be explained by antibiotic selection pressure and the widespread presence of integrons. In particular, over the past decade, the high degree of resistance to common antibiotics and the worldwide emergence of multidrug-resisFrom the College of Wildlife Resources of Northeast Forest University, Harbin 150040, Heilongjiang, People’s Republic of China (Xue, Wang, Zhang, Hua); State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, People’s Republic of China (Chen); and Heilongjiang Amur Tiger Park, Harbin 150400, Heilongjiang, People’s Republic of China (Liu). Correspondence should be directed to Dr. Hua ([email protected]).
tant (MDR) phenotypes are of increasing concern.15 Integrons are genetic structures able to capture, excise, and express genes and are frequently included in mobile elements such as plasmids that allow their dissemination among bacteria.18 They are genetic elements that incorporate an open reading frame or gene cassettes.13 Integrons, which are widely distributed among bacteria, are capable of capturing and, integrating antibiotic-resistance gene cassettes. The class 1 integron is the most numerous, especially in clinical Gram-negative bacterial isolates. Class 1 integrons are assumed to play an important role in the dissemination of antibiotic resistance genes. Class 1 integrons, the major integrons found in clinical isolates, are strongly associated with multiple antibiotics. Those resistances obtained from clinical isolates often have 59- and 39conserved segments flanking the variable region where gene cassettes integrate.10,22 Most genes inserted into integrons lack their own promoters, and expression is dependent on two potential promoters, called P1 and P2, which may differ in sequence and length in different integrons, located in the 59-conserved segment of the integron.9 Currently, there is little information regarding the molecular basis of antibiotic resistance in Escherichia coli strains isolated from captive populations of Amur tigers; therefore, this study was conducted to screen and analyze the antimicrobial resistance in E. coli strains isolated from a
951
952
JOURNAL OF ZOO AND WILDLIFE MEDICINE
Table 1. Primers
IntI1 IntI2 IntI3 59-CSa 39-CS a
Oligonucleotide primers used for the amplification of integrons. Nucleotide sequences (59 to 39)
Sizes of polymerase chain reaction amplicons (base pair)
IntI1F:CCTCCCGCACGATGATC IntI1R:TCCACGCATCGTCAGGC IntI2F:TTGCGAGTATCCATAACCTG IntI2R:TTACCTGCACTGGATTAAG IntI3F:AGTGGGTGGCGAATGAGTG IntI3R:TGTTCTTGTATCGGCAGGTG 59-CSF:GGCATCCAAGCAGCAAG 39-CSR:AAGCAGACTTGACCTGA
References
280
22
288
8
600
6
variable
10
CS ¼ conserved segment
captive population of Amur tigers in Heilongjiang Amur Tiger Park in China.
MATERIALS AND METHODS Bacterial isolates During May and November 2011, 61 E. coli strains were isolated from captive Amur tigers in Heilongjiang Amur Tiger Park. The fecal swabs were inoculated in nutrient broth (OxoidTM, Thermo Fisher Biochemicals Ltd., Beijing 101312, China) and incubated at 378C for 18 hr, purified subsequently on MacConkey agar (Hangzhou Tianhe Microorganism Reagent Co. Ltd., Hangzou 310008, Zhejiang, China) and eosin methylene blue ([EMB]; Hangzhou Tianhe Microorganism Reagent Co. Ltd.) agar plates, and incubated at 378C for 24–48 hr. For selective isolation and identification of E. coli, two blue– black colonies (presumptive E. coli) with a metallic sheen, growing on EMB agar plates, were randomly selected from each plate. Escherichia coli (25922TM, American Type Culture Collection [ATCCt], Manassas, Virgina 20110, USA) was used as the reference strain. The strain was stored at 808C until analysis. Antibiotic resistance test The antimicrobial sensitivity phenotypes of the E. coli strains were determined using a Kirby– Bauer disk diffusion assay, according to the standards and interpretive criteria described by Clinical and Laboratory Standards Institute (CLSI) guidelines.2 Antimicrobial susceptibility testing was performed using the CLSI document on Mueller– Hinton agar (Hangzhou Tianhe Microorganism Reagent Co. Ltd.). The following antimicrobials were tested: ampicillin, amoxicillin/clavulanic acid, cefalotin, aztreonam, gentamicin, kanamycin, apramycin, ofloxacin, ciprofloxacin, norfloxacin, enrofloxacin base, doxycycline, chloramphenicol,
florfenicol, polymyxin B, trimethorpim sulfur, and tetracycline. All antibiotic disks were purchased from Oxoid (Thermo Fisher Biochemicals Ltd.). Quality control for susceptibility testing was performed using E. coli (ATCC 25922), which is a recommended reference strain for antibiotic susceptibility testing. Detection of the integrase genes and gene cassettes The polymerase chain reaction (PCR) amplification of integrase genes was performed using specific primer pairs (Table 1) published in previous reports6,8,10,22 to determine the existence of antimicrobial resistance gene cassettes within the integrons. DNA sequencing and data analysis The PCR products were excised from 1.0% agarose gels and purified using the AxyPrepTM DNA Gel Extraction Kit (Axygen Scientific, Inc., Union City, California 94587, USA), cloned subsequently into a T-tailed vector, pMD18T(TaKaRa Biotechnology Co., Ltd., Dalian, 116600, China), and transformed using JM109 competent cells (TaKaRa Biotechnology Co., Ltd.). Three to five recombinant DNA clones of each E. coli strain were sequenced by Beijing Genomics Institute (Beijing, 101300, China). The nucleotide sequences were edited and analyzed with EditSeq software (version 7.1.0, DNASTAR Inc., Madison, Wisconsin 53705, USA) and the program NCBI-BLAST (www.ncbi.nlm.nih.gov).
RESULTS Antimicrobial resistance rates The antimicrobial susceptibility results of 61 E. coli strains isolated from the captive population of Amur tigers to 17 antimicrobial agents are shown in Figure 1. About 72% isolates were susceptible
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Figure 1. In vitro susceptibilities of 61 E. coli strains isolated from captive Amur tiger to 17 antimicrobial agents. R% ¼ resistance rate; I% ¼ intermediate rate; S% ¼ susceptible rate; AMP ¼ ampicillin; AMC ¼ amoxicillin/ clavulanicacid; CEF ¼ cefalotin; ATM ¼ aztreonam; GEN ¼ gentamicin; KAN ¼ kanamycin; APR ¼ apramycin; OF ¼ ofloxacin; NOR ¼ norfloxacin; CIP ¼ ciprofloxacin; EN ¼ enrofloxacin base; DOX ¼ doxycycline; CMP ¼ chloramphenicol; FFC ¼ florfenicol; SXT ¼ trimesulf; PB ¼ polymyxin B; TET ¼ tetracycline.
to amoxicillin/clavulanic acid (73.66%), aztreonam (71.46%), and apramycin (71.71%). However, high incidence rates were exhibited for resistance to ampicillin (100.00%), tetracycline (85.12%), and trimesulf (80.24%). In this study, the antimicrobial agents tested were divided into eight classes. Among these, there were four fluoroquinolones, two penicillins, two cephalosporins, one tetracycline, four aminoglycosides, two chloramphenicols, and two sulfonamides. By selecting one representative drug from each class, the most frequent antibiotic resistance patterns of the 61 E. coli strains to the 17 antimicrobial agents are shown in Table 2. Detection of class 1, 2, and 3 integrons by PCR Neither class 2 nor class 3 integrons were detected, while 52.46% (32 of 61) of the isolates were positive for the presence of intI1, which is a marker for class 1 integrons. Gene cassettes detected by PCR The integron-borne gene cassettes detected by PCR were cloned and sequenced. The integrons were found to contain one or two gene cassettes, and the combinations of these gene cassettes are shown in Table 2. Two distinct types of gene cassette array were characterized: dfrA15 and
dfrA12–aadA2, respectively. A search for the published homology sequences in GenBank revealed that 1) the gene cassette array dfrA12– aadA2 presented 99% identity with that found in E. coli isolates from beef cattle in Canada (FJ855131.1), human in Russia (GU001949.1, GQ924767.1, and GQ924766.1), and the Ku¨u¨kcekmece lagoon in Turkey (GQ280258.1); and 2) the gene cassette array dfrA15 presented 100% identity with those found in E. coli isolates from the Ku¨c u¨kcekmece lagoon in Turkey (GQ280260.1 and GQ280262.1). Correlation analysis was performed between the antimicrobial resistance profile and the occurrence of integrons.
DISCUSSION Escherichia coli is commonly found in the gastrointestinal tracts of domestic and wild animals and humans.21 Various selection pressures in these environments favor the development, persistence, and dissemination of robust E. coli strains, some of which may be resistant to antimicrobial agents.4 Antimicrobial susceptibility patterns of E. coli isolates to 17 antimicrobial agents were obtained, and the results are presented in Figure 1 and Table 2. As shown in Figure 1, most of the isolates were susceptible to amoxicillin/clavulanic acid, aztreonam, and apramycin. On the other hand, the
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Table 2. integrons.
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Antibiotic resistance patterns of E. coli isolates and their relationship with occurrence of the
No. of strains
16 07 57 39 37 22 74 55 32 75 29 68 72 23 62 91
Resistance phenotypesa
AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP, AMP,
CEF, GEN, DOX, TET, EN, SXT, CMP, PB, FFC AMC, CEF, KAN, DOX, TET, EN, SXT, CMP, NOR CEF, GEN, DOX, TET, EN, SXT, CMP, PB, FFC, CIP CEF, GEN, KAN, TET, EN, SXT, CMP, PB, FFC, CIP, NOR APR, CEF, TET, SXT, CMP AMC, APR, CEF, GEN, KAN, DOX, TET, CIP, EN, OF, SXT, CMP, NOR GEN, DOX, TET, FFC, SXT DOX, TET, SXT CEF, KAN, DOX,TET, SXT, CMP, PB, FFC CEF, KAN, DOX, TET, NOR, SXT, CMP CEF, GEN, KAN, DOX, TET, EN, SXT, CMP, FFC, CIP, NOR CEF, CIP, DOX, TET, KAN, SXT CEF, DOX, TET, EN, SXT,CMP, CIP, KAN CEF, CIP, GEN, KAN, DOX, TET, EN, SXT, CMP, NOR, FFC CEF, DOX, KAN, TET, EN, SXT, CMP, CIP, NOR, FFC ATM, APR, CEF, GEN, KAN, DOX, TET, CIP, EN, OF, SXT, CMP, NOR
Integrons
dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA12–aadA2 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15 dfrA15
a AMP, ampicillin; AMC, amoxicillin/clavulanicacid; CEF, cefalotin; ATM, aztreonam; GEN, gentamicin; KAN, kanamycin; APR, apramycin; OF, ofloxacin; NOR, norfloxacin; CIP, ciprofloxacin; EN, enrofloxacin base; TET, tetracycline; DOX, doxycycline; CMP, chloramphenicol; FFC, florfenicol; SXT, trimesulf; PB, polymyxin B.
high incidence rates of resistance to tetracycline, chloramphenicol, and trimethoprim sulfur may be derived from the frequent, long-term, and widespread use of these antimicrobials in the park studied. In the multiresistant strains, the rates of resistance of E. coli to ciprofloxacin, norfloxacin, and ofloxacin were similar. In the past few years, an intensive and largescale pig, chicken, and bovine farming industry in China has developed. Detailed results of antimicrobial resistance testing of E. coli isolates from various food animals, including cattle, swine, camels, sheep, goats, chickens, and foods of animal origin, in China have been reported previously.15,20 Integrons play an important role in the acquisition and dissemination of antimicrobial resistance genes. Integrons that contain gene cassettes provide a powerful vehicle for the rapid horizontal transfer of resistance across bacterial populations and thus may contribute to a sudden increase in the prevalence of MDR infections in a community.12 Class 1 integrons have been detected previously in different E. coli strains. For instance, class 1 integrons were detected in 10 to 65% of the antimicrobial resistant E. coli isolated in Egypt, Spain, Switzerland, China, and the United States.1,3,7,13,18,20 In this study, the prevalence of integron-positive isolates was 52.46%, which is similar to the data obtained from the Beijing area in China, which showed a prevalence of 54.70%.5 It has been indicated that the prevalence of
integrons is related to the antimicrobial pressure in the environment. The investigation of resistance gene cassettes in this study revealed that aminoglycoside resistance determinants (dfrA12–aadA2) and trimethoprim resistance determinants (dfrA15) were prevalent among E. coli strains isolated from a captive population of Amur tigers. To the authors’ knowledge, there are no previous reports in the scientific literature on integrons and associated gene cassettes in E. coli or other bacterial pathogens from the captive population of Amur tigers in Heilongjiang Amur Tiger Park. However, the persistence of these gene cassettes has been reported worldwide in isolates of different origins.7,14,17 Similar to this current study, class 1 integrons, including dfr and aadA gene cassettes, have been found to be the most prevalent type of integron in E. coli isolates from humans, animals, and food in other studies.16,19 In conclusion, this study demonstrated the distribution of the class I integrons among E. coli strains isolated from a captive population of Amur tigers in Heilongjiang Amur Tiger Park. Gene cassettes not previously reported in integrons among E. coli from this captive population of Amur tigers were found. The genetic content, combination, and frequency of emergence of these gene cassettes may reflect the antibiotic selection pressure in Heilongjiang Amur Tiger Park, China, and provide useful surveillance information for the rational use of antimicrobials.
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The potential role of integrons in the uptake and dissemination of resistance genes may be a continuing threat to the effectiveness of certain antibiotic therapeutic agents in Heilongjiang Amur Tiger Park. Acknowledgments: The authors thank Ming Zhou and Xian-guang Huang for the collection of isolates. This study was supported by the Fundamental Research Funds for the Central Universities (DL12BA04) and the Heilongjiang Postdoctoral Financial Assistance (LBHZ11262).
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Received for publication 18 March 2013
