doi: 10.14202/vetworld.2018.1082-1088
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Article history: Received: 04-05-2018, Accepted: 29-06-2018, Published online: 09-08-2018
Corresponding author: Mohamed M. Amer
E-mail: profdramer@yahoo.com
Citation: Amer MM, Mekky HM, Amer AM, Fedawy HS (2018) Antimicrobial resistance genes in pathogenic Escherichia coli isolated from diseased broiler chickens in Egypt and their relationship with the phenotypic resistance characteristics, Veterinary World, 11(8): 1082-1088.Aim: The aim of this study was to determine the relationship between phenotypic resistance and genotypic resistance of isolated serotyped pathogenic Escherichia coli isolates from the clinically diseased broiler.
Materials and Methods: A total of 160 samples (heart, liver, kidney, and lung) were collected from 18 to 34 days old clinically diseased broiler from 40 broiler farms (3-5 birds/farm) reared in Giza and Kaluobaia Governorates for the isolation of pathogenic E. coli. Various E. coli isolates were tested for the pathogenicity based on Congo red (CR) dye binding assay. The obtained CR-positive E. coli isolates were subjected to serological identification using slide agglutination test. Disc diffusion test was used to study the sensitivity pattern of E. coli isolates to available 12 antibiotics. Polymerase chain reaction was performed for the detection of antimicrobial resistance genes in the studied pathogenic E. coli isolates.
Results: The results revealed that 56 samples (35 %) were positive for E. coli. The results of the CR assay indicates that 20 isolates of 56 (35.7%) were positive and 36 isolates (64.3%) were negative. Identified E. coli serotypes of CR-positive isolates were 1 (O24), 2 (O44), 2 (O55), 5 (O78), 2 (O86), 1 (124), 3 (O127), 1 (O158), and 3 untyped. Resistance rate in disc diffusion test was 85% to oxytetracycline and kanamycin; 80% to ampicillin (AMP), clindamycin, and streptomycin (S); 75% to enrofloxacin; 65% to chloramphenicol; 55% to cefotaxime and gentamicin (CN); 45% to trimethoprim+sulfamethoxazole; 35% to erythromycin (ERI); and 30% to oxacillin. All strains are multidrug-resistant (MDR). Antibacterial resistance genes CITM, ere, aac (3)-(IV), tet(A), tet(B), dfr(A1), and aad(A1) were detected in 14 (70%), 12 (60%), 12 (60%), 8 (40%), 11 (55%), 8 (40%), and 9 (45%) of tested 20 isolates, respectively. Multidrug resistance was detected in the form of resistance to 42%-83.3% of tested 12 antibiotics. Three isolates (15%) of 20 tested isolates showed a relationship between phenotype and genotype and 17 (85%) showed irregular relation. Strains are sensitive and show resistant gene (P-G+) presented in three isolates for AMP (beta-lactam), one for ERI (Macrolide), as well as five isolates for trimethoprim (pyrimidine inhibitor). E. coli isolates had resistance and lacked gene (P+ G-) reported meanly in one isolate for CN (aminoglycoside), two isolates for tetracycline, four isolates for ERI, seven isolates for trimethoprim, and eight isolates for S (aminoglycoside).
Conclusions: The study demonstrates that E. coli is still a major pathogen responsible for disease conditions in broiler. E. coli isolates are pathogenic and MDR. Responsible gene was detected for six antibiotics in most of the isolates, but some do not show gene expression, this may be due to few numbers of resistance genes tested or other resistance factors not included in this study.
Keywords: antibiotic resistance genes, broiler, Escherichia coli, isolation.
1. Saif, Y.M., Barnes, H.J., Fadly, A.M., Glisson, J.R. and Swayne, D.E. (2003) Colibacillosis. In: Poultry Diseases. 11th ed. Iowa State Press, Iowa.
2. Roy, P., Edwin, P.G. and Purushothaman, V. (2004) Characterization of Escherichia coli isolates from hatchery and breeder hens. Indian Vet. J., 81: 1317-1320.
3. Susantha, M.G., Riddell, C., Andrew, A.P. and Allan, B.J. (2001) Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions. Can. J. Vet. Res., 65: 1-6.
4. CDC. (2009) Antibiotic Resistance Questions and Answers. Get Smart: Know When Antibiotics Work. Centers for Disease Control and Prevention, USA. Available from: https://www.cdc.gov/antibiotic-use/community/about/index.html. Accessed on 30-06-2009.
5. Bennett, P.M. (2008) Review: Plasmid-encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br. J. Pharmacol., 153: S347-S357. [Crossref] [PubMed] [PMC]
6. Corona, F. and Martinez, L. (2013) Review: Phenotypic resistance to antibiotics. Antibiotics, 2: 237-255. [Crossref] [PubMed] [PMC]
7. Morrison, B.J. and Rubin, J.E. (2015) Carbapenemase producing bacteria in the food supply escaping detection. PLoS One, 10(5): e0126717. [Crossref]
8. WHO. (2014) Antimicrobial Resistance: Global Report on Surveillance. Geneva: World Health Organization.
9. Marshall, B.M. and Levy, S.B. (2011) Food animals and antimicrobials: Impacts on human health. Clin. Microbiol. Rev., 24: 718-733. [Crossref] [PubMed] [PMC]
10. WHO. (2011) Tackling Antibiotic Resistance from a Food Safety Perspective in Europe. Available from: http://www.euro.who.int/__data/assets/pdf_file/0005/136454/e94889.pdf. Last accessed on 08-03-2018.
11. Heuer, H. and Smalla, K. (2007) Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environ. Microbiol., 9: 657-666. [Crossref] [PubMed]
12. Gosh, S. and LaPara, T.M. (2007) The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J., 1: 191-203. [Crossref]
13. Salyers, A.A., Gupta, A. and Wang, Y. (2004) Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol., 12(9): 412-416. [Crossref] [PubMed]
14. Yousef, S.A., Ammar, A.M. and Ahmed, D.A. (2013) Serological and molecular typing of avian pathogenic E. coli originating from outbreaks of colibacillosis in chicken flocks. Internat. J. Sci. Res., 4(2): 2082-2088.
15. Abd El Tawab, A.A., Ammar, M.A., Nasef, A.S. and Reda, M.R. (2015) Prevalence of E. coli in diseased chickens with its antibiogram pattern. Benha Vet. Med. J., 28(2): 224-230.
16. Amer, M.M., Bastamy, M., Elbayoumi, K.M. and Mervat, S. (2015) Isolation and characterization of avian pathogenic Escherichia coli from broiler chickens in some Governorates of Egypt. Vet. Med. J. Giza (VMJG), 61(1): 1-7.
17. Moawad, A.A., Hotzel, H., Awad, O., Tomaso, H., Neubauer, H., Hafez, H.M. and El Adawy, H. (2017) Occurrence of Salmonella enterica and Escherichia coli in raw chicken and beef meat in Northern Egypt and dissemination of their antibiotic resistance markers. Gut Pathog., 9: 57. [Crossref]
18. Konemann, E.W., Allen, S.D., Schrechen, W.M., Berrjer, P.C. and Winn, W.J.P. (1992) Colour Atlas and Textbook of Diagnostic Microbiology. 4th ed. J.B. Lippincott Co., Philadelphia, USA.
19. Quinn, P.J., Carter, M.E., Markey, B.K., Donnoly, W.J. and Leonard, F.C. (2002) Veterinary Microbiology and Microbial Diseases 166-1117 Osney Mead. Oxford First Ltd, Registered in the United Kingdom.
20. Berkhoff, H.A. and Vinal, A.C. (1986) Congo red medium to distinguish between invasive and non-invasive Escherichia coli for poultry. Avian Dis., 30: 117-121. [Crossref] [PubMed]
21. Edward, P.R. and Ewing, W.H. (1972): Identification of Enterobacteriaceae. 3rd ed. Burgess Publishing Co., Minneapolis. p709.
22. CLSI. (2013) Performance Standards for Antimicrobial Susceptibility Testing. CLSI Approved Standard. Clinical and Laboratory Standard Institute, Wayne, PA.
23. Peirano, G., Agerso, Y., Aarestrup, F.M., Falavina dos Reis, F.M. and Rodirgues, D.P. (2006) Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil. J. Antimicrob. Chemother., 58: 305-309. [Crossref] [PubMed]
24. Van, T.T., Chin, J., Chapman, T., Tran, L.T. and Coloe, P.J. (2008) Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes. Internat. J. Food Microbiol., 124: 217-223. [Crossref] [PubMed]
25. Pu-o-Sarmiento, J., Medeiros, L., Chiconi, C., Martins, F., Pelayo, J., Rocha, S., Blanco, J., Blanco, M., Zanutto, M., Kobayashi, R. and Nakazato, G. (2013) Detection of diarrheagenic Escherichia coli strains isolated from dogs and cats in Brazil. Vet. Microbiol., 166: 676-680. [Crossref] [PubMed]
26. Torkan, S., Khamesipour, F. and Anyanwu, M.U. (2015) Detection of virulence and antibacterial resistance genes in Salmonella isolates from diarrhoeic dogs in Iran. Rev. Med. Vet., 166: 221-228.
27. Sharada, R., Wilfred, R.S. and Thiyageeswaran, M. (2010) Isolation, characterization and antibiotic resistance pattern of Escherichia coli isolated from poultry. Am. Eur. J. Sci. Res., 5: 18-22.
28. Hussein, A.H., Ghanem, I.A., Eid, A.A., Ali, M.A., Sherwood, J.S., Li, G., Nolan, L.K. and Logue, C.M. (2013) Molecular and phenotypic characterization of Escherichia coli isolated from broiler chicken flocks in Egypt. Avian Dis., 57(3): 602-611. [Crossref] [PubMed]
29. Abd El Tawab, A.A., Maarouf, A.A.A., Abd El Al, S.A., El Hofy, F.I. and El Mougy, E.E.A. (2014) Detection of some virulence genes of avian pathogenic E. coli by polymerase chain reaction. Benha Vet. Med. J., 26(2): 159-176.
30. El- sayed, M.E., Shabana, I.I., Esawy, A.M. and Rashed, A.M. (2015) Detection of virulence-associated genes of avian pathogenic Escherichia coli (APEC) isolated from broilers. J. Gen., 1(1): 4.
31. El-Gaber, G.A. and El-Gohary, A.A. (1995) Studies on pyocin typing, dermonecrosis and pathogenicity of Escherichia coli recovered from septicaemic broiler chickens. Vet. Med. J., 43(2): 213-218.
32. Singh, M.P. and Gupta, R.S. (1996) Congo red binding test - A marker for avian pathogenic E. coli strains. Indian J. Comp. Microbiol. Immunol. Infect. Dis., 17: 83-84.
33. Abhilasha, S.S.P. and Gupta, R.S. (2001) Pathogenicity and in vitro drug resistance of Escherichia coli isolated from colibacillosis cases in chicks of Tarai Region. Indian J. Comp. Microbiol., Immunol. Inf. Dis., 22(2): 166-167.
34. Yoder, H.W. (1989) Congo red binding by Escherichia coli isolates from chickens. Avian Dis., 33: 502-505. [Crossref] [PubMed]
35. Taha, M., Ibrahim, R.S. and Hussien, A.A.A. (2002) Studying the pathogenicity and RAPD PCR analysis of different Escherichia coli serotypes isolated from broilers and layer chickens. Assiut Vet. Med. J., 46(92): 224-236.
36. Salama, S.S., Afaf, A.K., Elham, A.E. and Taha, M.M. (2007) Molecular strategies for the differentiation and identification of local Escherichia coli isolated from chicken: I. characterization of protein profile. B. S. Vet. Med. J., 17(1): 25-28.
37. Guerra, B., Junker, E., Schroeter, A., Malorny, B., Lehmann, S. and Helmuth, R. (2003) Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J. Antimicrob. Chemother., 52(3): 489-492. [Crossref] [PubMed]
38. Zhao, S., Maurerb, J.J., Hubert, S., Villena, J.F.D., Dermott, P.F.M., Meng, J., Ayersa, S., English, L. and White, D.G. (2005) Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates. Vet. Microbiol., 107: 215-224. [Crossref] [PubMed]
39. Gyles, L.C. (2008) Antimicrobial resistance in selected bacteria from poultry. Anim. Health Res. Rev., 9(2): 149-158. [Crossref] [PubMed]
40. Xin-sheng, L., Gui-Qin, W., Xiang-Dang, D., Bao-An, C., Su-Mei, Z. and Jian-Zhong, S. (2007) Antimicrobial susceptibility and molecular detection of chloramphenicol and florfenicol resistance among Escherichia coli isolates from diseased chickens. J. Vet. Sci., 8(3): 243-247. [Crossref] [PMC]
41. Dai, L., Lu, L.M., Wu, C.M., Li, B.B., Huang, S.Y., Wang, S.C., Qi, Y.H. and Shen, J.Z. (2008) Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006. FEMS Microbiol. Lett., 286: 178-183. [Crossref] [PubMed]
42. Ozawa, M., Harada, K., Kojima, A., Asi, T. and Sameshima, T. (2008) Antimicrobial susceptibilities, serogroups, and molecular characterization of avian pathogenic Escherichia coli isolates in Japan. Avian Dis., 52: 392-397. [Crossref] [PubMed]
43. Zanatta, G.F., Kanashiro, A.M.I., Castro, A.G.M., Cardoso, A.L.S.P., Tessari, E.N.C. and Pulic, S.C.P. (2004) Susceptibility of Escherichia coli strains of avian origin to antimicrobials. Arquivo Inst. Biol., 71(3): 283-286.
44. Ying, H.J., Chen, S.J., Chang, H., Dong, L., Yang, M. and Kamagata, Y. (2008) Phenotyping and genotyping of antibiotic-resistant Escherichia coli isolated from a natural river basin. Environ. Sci. Technol., 42(9): 3415-3420. [Crossref]
45. Xiu, M.W., Xiao, P.L., Wan, J.Z., Hong, X.J., Jian, S., Mei, J.Z., Xue, F.H., Dong, X.L. and YaHong, L. (2010) Prevalence of serogroups, virulence genotypes, antimicrobial resistance, and phylogenetic background of avian pathogenic Escherichia coli in south of China. Foodborne Pathog. Dis., 7(9): 1099-1106. [Crossref] [PubMed]
46. Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson-Liljequist, B., Paterson, D.L., Rice, L.B., Stelling, J., Struelens, M.J., Vatopoulos, A., Weber, J.T. and Monnet, D.L. (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect., 18: 268-281. [Crossref]
47. Szmolka, A., Anjum, M.F., Ragione, R.M., Kaszanyitzky, E.J. and Nagy, B. (2012) Microarray-based comparative genotyping of gentamicin-resistant Escherichia coli strains from food animals and humans. Vet. Microbiol., 156(1/2): 110-118. [Crossref] [PubMed]
48. Zhao, J.Y. and Dang, H. (2012) Coastal seawater bacteria harbor a large reservoir of plasmid-mediated quinolone resistance determinants in Jiaozhou Bay, China. Microb. Ecol., 64, 187-199. [Crossref] [PubMed]
49. Alm, E.W., Zimbler, D., Callahan, E. and Plomaritis, E. (2014). Patterns and persistence of antibiotic resistance in faecal indicator bacteria from freshwater recreational beaches. J. Appl. Microbiol., 117: 273-285. [Crossref] [PubMed]
50. Henriques, I.S.1., Fonseca, F., Alves, A., Saavedra, M.J. and Correia, A. (2008) Tetracycline-resistance genes in gram-negative isolates from estuarine waters. Lett. Appl. Microbiol., 47(6): 526-533. [Crossref] [PubMed]
51. Scaria, J., Warnick, D.L., Kaneene, B.J., May, K., Teng, C.H. and Chang, Y.F. (2010) Comparison of phenotypic and genotypic antimicrobial profiles in Escherichia coli and Salmonella enterica from the same dairy cattle farms. Mol. Cell Probes, 24(6): 325-345. [Crossref] [PubMed] [PMC]
52. Suzuki, S., Horinouchi, T. and Furusawaa, C. (2014) Prediction of antibiotic resistance by gene expression profiles. Nat. Commun., 5: 5792. [Crossref] [PubMed] [PMC]
53. Lewin, B. (1997) Genes. Oxford University Press, New York.
54. Petrova, M., Gorlenko, Z. and Mindlin, S. (2009) Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain. FEMS Microbiol. Lett., 296: 190-197. [Crossref] [PubMed]
55. Sundin, G.W. (2000) Examination of base pair variants of the strA-strB streptomycin resistance genes from bacterial pathogens of humans, animals and plants. J. Antimicrob. Chemother., 46: 848-849. [Crossref]
56. Daniels, J.B., Call, D.R. and Besser, T.E. (2007) Molecular epidemiology of blaCMY-2 plasmids carried by Salmonella enterica and Escherichia coli isolates from cattle in the Paci?c Northwest. Appl. Environ. Microbiol., 73: 8005-8011. [Crossref]
57. Mulvey, M.R., Susky, E., McCracken, M., Morck, D.W. and Read, R.R. (2009) Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources. Vet. Microbiol., 134: 279-287. [Crossref] [PubMed]
58. Escudero, E., Vinue, L., Teshager, T., Torres, C. and Moreno, M.A. (2010) Resistance mechanisms and farm-level distribution of fecal Escherichia coli isolates resistant to extended-spectrum cephalosporins in pigs in Spain. Res. Vet. Sci., 88: 83-87. [Crossref]
59. Zdolec, N., Racic, I., Vujnovic, A., Zdelar-Tuk, M., Matanovic, K., Filipovic, I. and Spicic, S. (2013) Antimicrobial resistance of coagulase-negative staphylococci isolated from spontaneously fermented sausages. Food Tech. Biotech., 51(2): 240.?
60. Osman, K., Badr, J., Al-Maary, K.S., Moussa, I.M., Hessain, A.M., Girah, Z.M.A., and Saad, A. (2016) Prevalence of the antibiotic resistance genes in coagulase-positive-and negative-Staphylococcus in chicken meat retailed to consumers. Front. Microbial., 7: 1846. [Crossref] [PubMed] [PMC]