Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 2012 Aug;78(15):5444–5447. doi: 10.1128/AEM.00660-12

Identification and Molecular Characterization of Class 1 Integrons in Multiresistant Escherichia coli Isolates from Poultry Litter

Elizabeth Ponce-Rivas a,, María-Enriqueta Muñoz-Márquez b, Ashraf A Khan c
PMCID: PMC3416406  PMID: 22635994

Abstract

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant Escherichia coli isolates from chicken litter. qnrS and qnrA were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of aacA4-catB3-dfrA1 gene cassettes from a class1 integron was found. Additionally, aadA1 and dfrA1 gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry E. coli isolates.

TEXT

Despite having been known of for over a century, avian colibacillosis caused by avian-pathogenic Escherichia coli (APEC) remains one of the major endemic diseases afflicting the poultry industry worldwide (2). During the last 3 decades, resistance to quinolones (QR) and fluoroquinolones (FQR) has been increasingly reported among human and veterinary isolates, related to their wide clinical use. It has been suggested that the emergence and dissemination of FQR E. coli strains that infect humans are a consequence of the intensive use of these drugs in poultry farming (10). Apparently, FQR strains can contaminate poultry by-products and be acquired by human beings via the food supply. The mechanisms attributed to resistance to quinolones in Enterobacteriaceae, related to modifications of the molecular targets (DNA gyrase and topoisomerase IV), decreased outer membrane permeability (porin defect), and overexpression of naturally occurring efflux, were believed to be only chromosomally encoded. However, to date, several plasmid-mediated quinolone resistance (PMQR) mechanisms have been identified, and they currently comprise different types of genes, including the quinolone resistance genes qnrA, qnrB, qnrC, qnrD, and qnrS; the aac(6′)-Ib-cr gene, which encodes a variant of the widespread aminoglycoside acetyltransferase ACC(6′)-Ib; and the efflux pump genes qepA (for quinolone efflux pump) and oqxAB (for olaquindox resistance) (5, 8, 9, 15).

A substantial proportion of resistance determinants in Gram-negative bacteria reside on class 1 integrons that are capable of capturing and expressing genes contained in cassette-like structures (10). To date, there are few reports related to the molecular basis of resistance in avian E. coli isolates in United States. Therefore, the aim of this study was to evaluate the prevalence of class 1 integron cassettes and Qnr determinants in high-level-fluoroquinolone-resistant E. coli isolates from broiler chicken litter. The genetic structures of the class 1 integron cassettes were also investigated.

We screened 19 fluoroquinolone-resistant E. coli strains isolated during 2002 and 2003 from 200 poultry house litter samples (University of Arkansas, Fayetteville, AR) (12). The isolates were assayed for susceptibility to a panel of 14 antibiotics used by NARMS (National Antibiotic Resistance Monitoring System) on Mueller-Hinton agar (Difco Laboratories, Detroit, MI) by a disk agar diffusion method (4). The sensitivity and resistance were determined by the criteria of the Clinical and Laboratory Standards Institute (6).

The presence of qnr genes (qnrA, qnrB, and qnrS) and class 1 integron resistance gene (intI) cassettes was determined by PCR amplification from all 19 isolates using the primers shown in Table 1. Plasmids pMG252 (qnrA1), pMG298 (qnrB1), and pMG306 (qnrS1) were used as positive controls for the PCRs. intI genes were PCR amplified using the method described previously (13). For sequencing, amplified integrons and qnr genes were purified and cloned in vector pCR 2.1 (Invitrogen Corp., Carlsbad, CA). DNA sequences were analyzed with Lasergene DNASTAR (Madison, WI).

Table 1.

Oligonucleotide primers for qnr and int1 genes

Primer Locus Sequence (5′ to 3′) Product size (bp) Reference
QnrA1F 5′ region of E. coli qnrA CAG CAA GAG GAT TTC TCA CG This work
QnrA1R 3′ region of E. coli qnrA CCG GCA GCA CTA TTA CTC C 627 This work
QnrBF Nucleotide 158 of E. coli qnrB TCG GCTGTCAGTTCTATGATC This work
QnrBR 3′ region of E. coli qnrB TCC ATG AGC AAC GAT GCC T 500 11
QnrSF Nucleotide 58 of Salmonella qnrS TGA TCT CAC CTT CAC CGC TTG 11
QnrSR 3′ region of Salmonella qnrS GAA TCA GTT CTT GCT GCC AAG 563 11
CSL1 5′ region of class 1 integron GGC ATC CAA GCA GCA AGC 13
CSR1 3′ region of class 1 integron AAG CAG ACT TGA CCT GAT 1,000–2,655 13

The genetic characterization of the isolates was performed by pulsed-field gel electrophoresis (PFGE) and plasmid analysis of the 19 isolates using methods and software described previously (16) (Fig. 1). Southern blot hybridization was carried out by the method described previously (14), using the 1.5-kb PCR int1 fragment.

Fig 1.

Fig 1

XbaI PFGE dendrogram generated by BioNumerics software showing relationships of 19 representative fingerprints of fluoroquinolone-resistant E. coli isolates.

Multidrug resistance was observed in 58% of the E. coli isolates, which showed resistance to 9 to 11 different antibiotics (Table 2). Only half (52.3%) of the nalidixic acid-resistant E. coli strains were resistant to ciprofloxacin and norfloxacin. Additionally, 73.68% of isolates had one of the Qnr determinants. qnrA and qnrS PCR products were observed in 52.63 and 68% of the isolates, respectively. However, qnrB was not detected in any of the isolates. The prevalence of qnrS in quinolone-resistant poultry isolates has also been detected in other poultry and clinical E. coli isolates (7, 19). Qnr determinants were present in isolates with both low and high antibiotic resistance frequencies and were not dependent on the presence of megaplasmids. In some of the isolates, the qnr genes were detected in association with class 1 integrons (Table 2).

Table 2.

Correlation among quinolone genes, the int1 integron, plasmids, PFGE results, and antibiotic resistance of E. coli isolates

E. coli strainc Presence of:
Plasmid groupa PFGE group Antibiotic resistanceb
qnrA qnrB qnrS int1 (1 kb) int1 (1.5 kb) int1 (>2 kb)
329 + 2B A Nar Kmr Amcr Norr Cipr Ter Gmr Amr Atmr Sxtr
330 + NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr
331 NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
334 NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
335 + + + + NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
339 + + + + NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
346 NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
347 + + + + NP D Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
383 + + 1A E Nar Kmr Amcr Norr Cipr Ter Gr Amr Cr Sxtr Sr
386 + + + 6B H Nar Amcr Ter Gr Cazr Gmr Amr Atmr Sr
393 + + 5A H Nar Amcr Ter Gr Cazr Gmr Amr Atmr Sr
33 + + 4A C Nar Amcr Ter Gr Amr Atmr Sr
384 + + + + 2A I Nar Norr Cipr Ter Gr Sr
55 + + + + 5B B Nar Gr Cfr Sxtr Sr
357 + + + 6A F Nar Ter Sr
361 + + + + 6A F Nar Ter Sr
362 + 6A F Nar Ter Sr
368 7A F Nar Ter Sr
62 7B G Nar Ter
a

NP, no plasmid.

b

Nalidixic acid (Na, 30 μg), kanamycin (Km 30 μg), amoxicillin (Amc, 30 μg), norfloxacin (Nor, 10 μg), ciprofloxacin (Cip, 5 μg), tetracycline (Te, 30 μg), sulfisoxazole (G, 0.25 μg), ceftazidime (Caz, 30 μg), gentamicin (Gm, 10 μg), ampicillin (Am, 10 μg), chloramphenicol (C, 30 μg), aztreonam (Atm, 30 μg), trimethoprim-sulfamethoxole (Sxt, 1.25/23.75 μg), streptomycin (S, 5 μg).

c

Bold type indicates the Int1 PCR products that were sequenced.

Class 1 integron cassettes were found in 52.63% of the isolates. Eight of the 19 strains amplified a 1.0-kb PCR product, strain 55 amplified a 1.5-kb PCR product, and five isolates amplified a 2.6-kb PCR product. Based on the size of the PCR product and the difference in the number of antibiotic resistances, four intI PCR products were selected for further cloning and sequencing analysis: the 1.5-kb product of isolate 55, the 2.6-kb PCR product of isolate 347, and the 1.0-kb PCR product of isolates 361 and 386. As shown in Fig. 2, the 2,655-bp int1 sequence contained an uncommon array that showed 100% identity with the class I integron aminoglycoside 6′-N-acetyltransferase (aacA4), chloramphenicol acetyltransferase (catB3), and dihydrofolate reductase (dfrA1) genes, encoding tobramycin, chloramphenicol, and trimethoprim-sulfamethoxazole resistance, respectively. Additionally, a putative cassette between two 59-base elements that had a unique coding sequence was observed in the array (GenBank accession number EF516991). This uncommon array has also been found in E. coli strains isolated from bovine mastitis in Inner Mongolia (18) and showed 99% identity with sequences from clinical and veterinary E. coli and Pseudomonas aeruginosa isolates from China (GenBank accession numbers DQ836057, HQ880283, and AB195796). These results indicate that int1 genes play an important role in the horizontal dissemination of antibiotic resistance in the same or different bacterial species or host or in different geographic areas.

Fig 2.

Fig 2

Map of variant class 1 integrons of multiresistant E. coli strains 347 (A), 55 (B), and 386 and 361 (C). Empty box, conserved region (CR) containing elements for insertion; filled box, 59-base element (integration site).

The 1,586-bp sequence of isolate 55 demonstrated that it contained the dfrA1 gene cassette and an aadA1 gene cassette encoding resistance to streptomycin and spectinomycin (GenBank accession number EF527229). The aadA1 gene cassette was also found in the 1,009-bp sequences (Fig. 2). Class 1 integrons have been found in resistant E. coli isolates from chickens (retail or farms) in different countries (1, 3, 20), and the resistance genes aadA1 and dfrA1 have frequently been found in class 1 integrons in poultry E. coli isolates (17). Southern blot hybridization showed that aadA1 and dfrA1 gene cassettes were located on mega plasmids as well on chromosome DNA of fluoroquinolone-resistant E. coli isolates in poultry.

Fingerprinting of the 19 E. coli isolates allowed the definition of nine XbaI macrorestriction profiles containing 16 to 21 restriction fragments of total DNA (A to I) (Fig. 1); meanwhile, isolates were differentiated into 10 different plasmid groups with one or two megaplasmids and zero to five different small plasmids (Table 2). The PFGE analysis of these isolates provided much better discrimination than the ribotype pattern obtained (12) with 17 of the 19 fluoroquinolone-resistant isolates used in this study. Interestingly, PFGE group D (seven isolates) presented 100% similarity, showed resistance to 10 or 11 different antibiotics, and did not show any plasmid, suggesting that the antibiotic resistance cassettes of these isolates are part of the chromosomal DNA. The 14 qnr-positive isolates gave eight different PFGE and plasmid groups, while the 10 int1-positive isolates gave six PFGE groups and seven plasmid groups, indicating that resistance has emerged among multiple avian pathogenic E. coli chromosomal backgrounds. These results are in agreement with other studies with E. coli of poultry and human origin in which PMQR or int1-positive isolates were genetically diverse (7, 17). However, some of the qnr- and int1-positive isolates (335, 339, and 347) could be clonally related (Table 2).

These results shows that Qnr determinants and int1 genes are present in both low- and high-frequency antibiotic-resistant poultry E. coli isolates and that the positive isolates are genetically diverse. The evidence of class 1 integron cassettes associated with megaplasmids as well as with chromosomal DNA and the presence of an uncommon int1 sequence highlight the importance of surveillance programs that focus on detection of genetic elements related to horizontal transfer of genes and contribute to the understanding of the resistance mechanisms and the prudent use of antimicrobials.

ACKNOWLEDGMENTS

Positive controls were donated by George Jacoby.

This work was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.

Footnotes

Published ahead of print 25 May 2012

REFERENCES

  • 1. Altalhi AD, Gherbawy YA, Hassan SA. 2010. Antibiotic resistance in Escherichia coli isolated from retail raw chicken meat in Taif, Saudi Arabia. Foodborne Pathog. Dis. 7:281–285 [DOI] [PubMed] [Google Scholar]
  • 2. Barnes HJ, Nolan LK, Vaillancourt JF. 2008. Colibacillosis, p 691–732 In Saif YM, et al. (ed), Diseases of poultry, 12th ed. Blackwell Publishing, Ames, IA [Google Scholar]
  • 3. Bass L, et al. 1999. Incidence and characterization of integrons, genetic elements mediating multiple-drug resistance, in avian Escherichia coli. Antimicrob. Agents Chemother. 43:2925–2929 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Bauer A, Kirby WW, Sherris J, Turek M. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493–496 [PubMed] [Google Scholar]
  • 5. Cattoir V, Nordmann P. 2009. Plasmid-mediated quinolone resistance in gram-negative bacterial species: an update. Curr. Med. Chem. 16:1028–1046 [DOI] [PubMed] [Google Scholar]
  • 6. Clinical and Laboratory Standards Institute 2006. Performance standards for antimicrobial susceptibility testing: sixteenth informational supplement. M100–S16. CLSI, Wayne, PA [Google Scholar]
  • 7. Huang SY, et al. 2009. Increased prevalence of plasmid-mediated quinolone resistance determinants in chicken Escherichia coli isolates from 2001 to 2007. Foodborne Pathog. Dis. 6:1203–1209 [DOI] [PubMed] [Google Scholar]
  • 8. Jacoby GA. 2008. qnr gene nomenclature. Antimicrob. Agents Chemother. 52:2297–2299 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Jacoby GA, Gacharna N, Black TA, Miller GH, Hooper DC. 2009. Temporal appearance of plasmid-mediated quinolone resistance genes. Antimicrob. Agents Chemother. 53:1665–1666 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Johnson JR, et al. 2006. Similarity between human and chicken Escherichia coli isolates in relation to ciprofloxacin resistance status. J. Infect. Dis. 194:71–78 [DOI] [PubMed] [Google Scholar]
  • 11. Kehrenberg C, Friederichs S, de Jong A, Brenner MG, Schwarz S. 2006. Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis. J. Antimicrob. Chemother. 58:18–22 [DOI] [PubMed] [Google Scholar]
  • 12. Khan AA, Nawaz MS, Summage West C, Khan SA, Lin J. 2005. Isolation and molecular characterization of fluoroquinolone-resistant Escherichia coli from poultry litter. Poultry Sci. 84:61–66 [DOI] [PubMed] [Google Scholar]
  • 13. Khan AA, Cheng CM, Khanh TV, Summage-West C, Nawaz MS, Khan SA. 2006. Characterization of class 1 integron resistance gene cassettes in Salmonella enterica serovars Oslo and Bareily from imported seafood. J. Antimicrob. Chemother. 58:1308–1310 [DOI] [PubMed] [Google Scholar]
  • 14. Khan AA, et al. 2009. Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from imported seafood. Appl. Environ. Microbiol. 75:1192–1196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Poirel L, Cattoir V, Nordmann P. 2012. Plasmid-mediated quinolone resistance; interactions between human, animal, and environmental ecologies. Front. Microbiol. 3:24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Ponce E, Khan AA, Cheng C, Summage-West C, Cerniglia CE. 2008. Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood. Food Microbiol. 25:29–35 [DOI] [PubMed] [Google Scholar]
  • 17. Vasilakopoulous A, et al. 2009. Prevalence and characterization of class 1 integrons in Escherichia coli of poultry and human origin. Foodborne Pathog. Dis. 6:1211–1218 [DOI] [PubMed] [Google Scholar]
  • 18. Wang GQ, et al. 2008. Characterization of integrons-mediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis. Vet. Microbiol. 127:73–78 [DOI] [PubMed] [Google Scholar]
  • 19. Yue L, et al. 2008. Prevalence of plasmid-mediated quinolone resistance qnr genes in poultry and swine clinical isolates of Escherichia coli. Vet. Microbiol. 132:414–420 [DOI] [PubMed] [Google Scholar]
  • 20. Zhang XY, Ding LJ, Yue J. 2009. Occurrence and characterization of class 1 and class 2 integrons in resistant Escherichia coli isolates from animals and farm workers in Northeastern China. Microb. Drug Resist. 15:323–328 [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES