, Volume 22, Issue 2, pp 402–414 | Cite as

Molecular diversity of class 2 integrons in antibiotic-resistant gram-negative bacteria found in wastewater environments in China

  • Ruirui Xia
  • Ye Ren
  • Xianhu Guo
  • Hai XuEmail author


The molecular architecture of class 2 integrons among gram-negative bacteria from wastewater environments was investigated in Jinan, China. Out of the 391 antibiotic-resistant bacteria found, 38 isolates harboring class 2 integrons encoding potentially transferrable genes that could confer antibiotic resistance were found. These isolates were classified into 19 REP-PCR types. These strains were identified using 16S rRNA gene sequencing and found to be as follows: Proteus mirabilis (16), Escherichia coli (7), Providencia spp. (7), Proteus spp. (2), P. vulgaris (3), Shigella sp. (1), Citrobacter freundii (1), and Acinetobacter sp. (1). Their class 2 integron cassette arrays were amplified and then either analyzed using PCR–RFLP or sequenced. The typical array dfrA1-sat2-aadA1 was detected in 27 isolates. Six atypical arrays were observed, including three kinds of novel arrangements (linF2(∆attC1)-dfrA1(∆attC2)-aadA1-orf441 or linF2(∆attC1)-dfrA1(∆attC2)-aadA1, dfrA1-catB2-sat2-aadA1, and estX Vr -sat2-aadA1) and a hybrid with the 3′CS of class 1 integrons (dfrA1-sat2-aadA1-qacH), and dfrA1-sat1. Twenty-four isolates were also found to carry class 1 integrons with 10 types of gene cassette arrays. Several non-integron-associated antibiotic resistance genes were found, and their transferability was investigated. Results showed that water sources in the Jinan region harbored a diverse community of both typical and atypical class 2 integrons, raising concerns about the overuse of antibiotics and their careless disposal into the environment.


Antibiotic-resistant Hybrid integron Multigene cassette Non-integron associated antibiotic resistance gene Novel gene cassette arrays 



This work was supported by the grants from the National Natural Science Foundation of China (30870084 and 31170112), the Medical and Health Technology Foundation of Shandong (2011HZ050), and the State Key Laboratory of Microbial Technology, Shandong University.We would like to thank Professor Julian Davies and Dr. Vivian Miao from the University of British Columbia for helpful comments on manuscript revision and George A. Jacoby for providing E. coli J53 (Azide R).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10646_2012_1034_MOESM1_ESM.doc (5.1 mb)
Supplementary material 1 (DOC 5237 kb)


  1. Ahmed AM, Nakano H, Shimamoto T (2005) Molecular characterization of integrons in non-typhoid Salmonella serovars isolated in Japan: description of an unusual class 2 integron. J Antimicrob Chemother 55:371–374. doi: 10.1093/jac/dkh534 CrossRefGoogle Scholar
  2. Aminov RI, Mackie RI (2007) Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett 271:147–161. doi: 10.1111/j.1574-6968.2007.00757.x CrossRefGoogle Scholar
  3. Antunes P, Machado J, Peixe L (2007) Dissemination of sul3-containing elements linked to class 1 integrons with an unusual 3′ conserved sequence region among Salmonella isolates. Antimicrob Agents Chemother 51:1545–1548. doi: 10.1128/AAC.01275-06 CrossRefGoogle Scholar
  4. Barlow RS, Gobius KS (2006) Diverse class 2 integrons in bacteria from beef cattle sources. J Antimicrob Chemother 58:1133–1138. doi: 10.1093/jac/dkl423 CrossRefGoogle Scholar
  5. Biskri L, Mazel D (2003) Erythromycin esterase gene ere (A) is located in a functional gene cassette in an unusual class 2 integron. Antimicrob Agents Chemother 47(10):3326–3331. doi: 10.1128/AAC.47.10.3326-3331.2003 CrossRefGoogle Scholar
  6. Biyela PT, Lin Bezuidenhout CC (2004) The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Water Sci Technol 50:45–50Google Scholar
  7. Boonjakuakul JK, Canfield DR, Solnick JV (2005) Comparison of Helicobacter pylori virulence gene expression in vitro and in the Rhesus macaque. Infect Immun 73:4895–4904. doi: 10.1128/IAI.73.8.4895-4904.2005 CrossRefGoogle Scholar
  8. Cao V, Lambert T, Nhu DQ, Loan HK, Hoang NK, Arlet G, Courvalin P (2002) Distribution of extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae in Vietnam. Antimicrob Agents Chemother 46:3739–3743. doi: 10.1128/AAC.46.12.3739-3743.2002 CrossRefGoogle Scholar
  9. Chen B, Zheng W, Yu Y, Huang W, Zheng S, Zhang Y, Guan X, Zhuang Y, Chen N, Topp E (2011) Class 1 integrons, selected virulence genes, and antibiotic resistance in Escherichia coli isolates from the Minjiang River, Fujian Province. China Appl Environ Microbiol 77:148–155. doi: 10.1128/AEM.01676-10 CrossRefGoogle Scholar
  10. Chuanchuen R, Koowatananukul C, Khemtong S (2008) Characterization of class 1 integrons with unusual 3′ conserved region from Salmonella enterica isolates. Southeast Asian J. Trop Med Public Health 39:419–424Google Scholar
  11. Clinical and Laboratory Standards Institute (2007) Performance standards for antimicrobial susceptibility testing: 17th informational supplement. 27: M100-S17. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
  12. Collis CM, Hall RM (1992) Gene cassettes from the insert region of integrons are excised as covalently closed circles. Mol Microbiol 6(19):2875–2885. doi: 10.1111/j.1365-2958.1992.tb01467.x CrossRefGoogle Scholar
  13. Frana TS, Carlson SA, Griffith RW (2001) Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype typhimurium phage type DT104. Appl Environ Microbiol 67:445–448. doi: 10.1128/AEM.67.1.445-448.2001 CrossRefGoogle Scholar
  14. Guerra B, Soto SM, Argüelles JM, Mendoza MC (2001) Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-]. Antimicrob Agents Chemother 45:1305–1308. doi: 10.1128/AAC.45.4.1305-1308.2001 CrossRefGoogle Scholar
  15. Guo X, Xia R, Han N, Xu H (2011) Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China. Lett Appl Microbiol 52:667–675. doi: 10.1111/j.1472-765X.2011.03059.x CrossRefGoogle Scholar
  16. Hall RM, Collis CM (1995) Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol 15:593–600. doi: 10.1111/j.1365-2958.1995.tb02368.x CrossRefGoogle Scholar
  17. Hall RM, Collis CM (1998) Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updates 1:109–119CrossRefGoogle Scholar
  18. Hansson K, Sundström L, Pelletier A, Roy PH (2002) intI2 integron integrase in Tn7. J Bacteriol 184:1712–1721. doi: 10.1128/JB.184.6.1712-1721.2002 CrossRefGoogle Scholar
  19. Kadlec K, Schwarz S (2008) Analysis and distribution of class 1 and class 2 integrons and associated gene cassettes among Escherichia coli isolates from swine, horses, cats and dogs collected in the BfT-GermVet monitoring study. J Antimicrob Chemother 62:469–473. doi: 10.1093/jac/dkn233 CrossRefGoogle Scholar
  20. Kehrenberg C, Friederichs S, de Jong A, Michael GB, Schwarz S (2006) Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis. J Antimicrob Chemother 58:18–22. doi: 10.1093/jac/dkl213 CrossRefGoogle Scholar
  21. Kojima A, Ishii Y, Ishihara K, Esaki H, Asai T, Oda C, Tamura Y, Takahashi T, Yamaguchi K (2005) Extended-spectrum-β-lactamase-producing Escherichia coli strains isolated from farm animals from 1999 to 2002: report from the Japanese veterinary antimicrobial resistance monitoring program. Antimicrob Agents Chemother 49:3533–3537. doi: 10.1128/AAC.49.8.3533-3537.2005 CrossRefGoogle Scholar
  22. Luo Y, Xu L, Rysz M, Wang Y, Zhang H, Alvarez PJ (2011) Occurrence and transport of tetracycline, sulfonamide, quinolone, and macrolide antibiotics in the Haihe River Basin. China Environ Sci Technol 45:1827–1833CrossRefGoogle Scholar
  23. Márquez C, Labbate M, Ingold AJ, Roy Chowdhury P, Ramírez MS, Centrón D, Borthagaray G, Stokes HW (2008) Recovery of a functional class 2 integron from an Escherichia coli strain mediating a urinary tract infection. Antimicrob Agents Chemother 52(11):4153–4154. doi: 10.1128/AAC.00710-08 CrossRefGoogle Scholar
  24. Marshall B, Tachibana C, Levy SB (1983) Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms. Antimicrob Agents Chemother 24:835–840. doi: 10.1128/AAC.24.6.835 CrossRefGoogle Scholar
  25. Marshall B, Morrissey S, Flynn P, Levy SB (1986) A new tetracycline-resistance determinant, class E, isolated from Enterobacteriaceae. Gene 50:111–117CrossRefGoogle Scholar
  26. Martínez JL (2008) Antibiotics and antibiotic resistance genes in natural environments. Science 321:365–367. doi: 10.1126/science.1159483 CrossRefGoogle Scholar
  27. Mazel D (2006) Integrons: agents of bacterial evolution. Nat Rev Microbiol 4:608–620. doi: 10.1038/nrmicro1462 CrossRefGoogle Scholar
  28. Mendez B, Tachibana C, Levy SB (1980) Heterogeneity of tetracycline resistance determinants. Plasmid 31:99–108CrossRefGoogle Scholar
  29. Nwosu VC (2001) Antibiotic resistance with particular reference to soil microorganisms. Res Microbiol 152:421–430CrossRefGoogle Scholar
  30. Pan JC, Ye R, Meng DM, Zhang W, Wang HQ, Liu KZ (2006) Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri. J Antimicrob Chemother 58:288–296. doi: 10.1093/jac/dkl228 CrossRefGoogle Scholar
  31. Partridge SR, Tsafnat G, Coiera E, Iredell JR (2009) Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev 33(4):757–784. doi: 10.1111/j.1574-6976.2009.00175.x CrossRefGoogle Scholar
  32. Peng X, Zhang K, Tang C, Huang Q, Yu Y, Cui J (2011) Distribution pattern, behavior, and fate of antibacterials in urban aquatic environments in South China. J Environ Monit 13:446–454. doi: 10.1039/C0EM00394H CrossRefGoogle Scholar
  33. Ploy MC, Denis F, Courvalin P, Lambert T (2000) Molecular characterization of integrons in Acinetobacter baumannii: description of a hybrid class 2 integron. Antimicrob Agents Chemother 44:2684–2688. doi: 10.1128/AAC.44.10.2684-2688.2000 CrossRefGoogle Scholar
  34. Ramírez MS, Piñeiro S, Centrón D, Argentinian Integron Study Group (2010) Novel insights about class 2 integrons from experimental and genomic epidemiology. Antimicrob Agents Chemother 54:699–706. doi: 10.1128/AAC.01392-08 CrossRefGoogle Scholar
  35. Ramírez MS, Quiroga C, Centrón D (2005a) Novel rearrangement of a class 2 integron in two non-epidemiologically related isolates of Acinetobacter baumannii. Antimicrob Agents Chemother 49:5179–5181. doi: 10.1128/AAC.49.12.5179-5181.2005 CrossRefGoogle Scholar
  36. Ramírez MS, Vargas LJ, Cagnoni V, Tokumoto M, Centrón D (2005b) Class 2 integron with a novel cassette array in a Burkholderia cenocepacia isolate. Antimicrob Agents Chemother 49:4418–4420. doi: 10.1128/AAC.49.10.4418-4420.2005 CrossRefGoogle Scholar
  37. Robicsek A, Strahilevitz J, Sahm DF, Jacoby GA, Hooper DC (2006) qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother 50:2872–2874. doi: 10.1128/AAC.01647-05 CrossRefGoogle Scholar
  38. Rodríguez-Martínez JM, Nordmann P, Poirel L (2012) Group IIC intron with an unusual target of integration in Enterobacter cloacae. J Bacteriol 194(1):150–160. doi: 10.1128/JB.05786-11 CrossRefGoogle Scholar
  39. Rowe-Magnus DA, Mazel D (2002) The role of integrons in antibiotic resistance gene capture. Int J Med Microbiol 292:115–125CrossRefGoogle Scholar
  40. Sambrook J, MacCallum P, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  41. Sunde M, Norström M (2006) The prevalence of, associations between and conjugal transfer of antibiotic resistance genes in Escherichia coli isolated from Norwegian meat and meat products. J Antimicrob Chemother 58:741–747. doi: 10.1093/jac/dkl294 CrossRefGoogle Scholar
  42. Van Essen-Zandbergen A, Smith H, Veldman K, Mevius D (2007) Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands. J Antimicrob Chemother 59:746–750. doi: 10.1093/jac/dkl549 CrossRefGoogle Scholar
  43. Waddell CS, Craig NL (1988) Tn7 transposition: two transposition pathways directed by five Tn7-encoded genes. Genes Dev 2:137–149. doi: 10.1101/gad.2.2.137 CrossRefGoogle Scholar
  44. White PA, McIver CJ, Deng Y, Rawlinson WD (2000) Characterization of two new gene cassettes, aadA5 and dfrA17. FEMS Microbiol Lett 182:265–269. doi: 10.1111/j.1574-6968.2000.tb08906.x CrossRefGoogle Scholar
  45. White PA, McIver CJ, Rawlinson WD (2001) Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 45:2658–2661. doi: 10.1128/AAC.45.9.2658-2661.2001 CrossRefGoogle Scholar
  46. Wilson K (2001) Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol 2:2–4. doi: 10.1002/0471142727.mb0204s56 Google Scholar
  47. Wolkow CA, DeBoy RT, Craig NL (1996) Conjugating plasmids are preferred targets for Tn7. Genes Dev 10:2145–2157. doi: 10.1101/gad.10.17.2145 CrossRefGoogle Scholar
  48. Wright GD (2010) Antibiotic resistance in the environment: a link to the clinic? Curr Opin Microbiol 13:589–594CrossRefGoogle Scholar
  49. Xu H, Davies J, Miao V (2007) Molecular characterization of class 3 integrons from Delftia spp. J Bacteriol 189:6276–6283. doi: 10.1128/JB.00348-07 CrossRefGoogle Scholar
  50. Xu H, Broersma K, Miao V, Davies J (2011) Class 1 and class 2 integrons in multidrug-resistant gram-negative bacteria isolated from the Salmon River. British Columbia. Can J Microbiol 57:460–467CrossRefGoogle Scholar
  51. Zhang X, Wu B, Zhang Y, Zhang T, Yang L, Fang HH, Ford T, Cheng S (2009) Class 1 integronase gene and tetracycline resistance genes tetA and tetC in different water environments of Jiangsu Province, China. Ecotoxicology 18:652–660. doi: 10.1007/s10646-009-0332-3 CrossRefGoogle Scholar
  52. Zhao J, Aoki T (1992) Nucleotide sequence analysis of the class G tetracycline resistance determinant from Vibrio anguillarum. Microbiol Immunol 36:1051–1060Google Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial TechnologySchool of Life Science, Shandong UniversityJinanChina
  2. 2.Shandong Longlive Bio-technology Co. Ltd., High-tech ZoneYucheng, DezhouChina

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