ISOLATION METHODS FOR MOLECULAR DETECTION AND ANTIBIOTIC RESISTANCE PATTERN OF CAMPYLOBACTER SPP IN LAYER CHICKENS

Authors

  • O.O. KEHINDE Department of Veterinary Public Health & Preventive Medicine, Federal University of Agriculture, Abeokuta
  • M.A. DIPEOLU Department of Veterinary Public Health & Preventive Medicine, Federal University of Agriculture, Abeokuta
  • O.J., AWOYOMI Department of Veterinary Public Health & Preventive Medicine, Federal University of Agriculture, Abeokuta
  • M. AGBAJE Department of Veterinary Microbiology & Parasitology, Federal University of Agriculture, Abeokuta
  • O.G. FASANMI Department of Production Animal Studies, University of Pretoria, South Africa & Federal College of Animal health & Production Tech., Ibadan
  • ISSMAT I. KASSEM Food Animal Health Research Program, Ohio Agricultural Research Development Center, Department of Veterinary Preventive Medicine. The Ohio State University, Wooster, OH 44691, USA
  • O.E., OJO Department of Veterinary Microbiology & Parasitology, Federal University of Agriculture, Abeokuta
  • O.O., ADEBOWALE Department of Veterinary Public Health & Preventive Medicine, Federal University of Agriculture, Abeokuta
  • E.O. OMOSHABA Department of Veterinary Microbiology & Parasitology, Federal University of Agriculture, Abeokuta
  • J. NWANTA Department of Veterinary Public Health & Preventive Medicine, Federal University of Agriculture, Abeokuta

DOI:

https://doi.org/10.51406/jnset.v19i1.2111

Keywords:

antibiotic resistance, campylobacter, layer chickens, polymerase chain reaction, prevalence

Abstract

This study was conducted to compare two culture methods for the isolation of Campylobacter spp from commercial layer chickens and subsequently confirmed by Polymerase Chain Reaction assays (PCR). Furthermore, the antimicrobial resistance profiles of PCR positive Campylobacter isolates were determined.Cloacal swab samples (550) from chickens randomly selected from five poultry farms in the four geographical zones in Ogun State were cultured for Campylobacter using modified charcoal Cefoperazone deoxycholate agar (MCCDA) and an improved culture method involving Preston broth pre-enrichment and subsequent subculture on Mueller Hinton agar with Campylobacter growth supplements. Putative isolates were later confirmed by PCR assay and sequencing analysis.Other isolates that grew on MCCDA and confirmed by sequencing analysis are Enterococcus faecalis, Escherichis coli, Comamonas kerstli and Pseudomonas aeroginusa . The antibiotic resistant profile of all the isolates were evaluated genotypically for resistance genes to tetracyclines (tetO), multiclasses (cmeB), aminoglycosides (aphA-3-1) and β-lactams (Blaoxa-61) using multiplex PCR (mPCR), and phenotypically for chlortetracycline, tylosin, streptomycin, ciprofloxacin and erythromycin resistance by microbroth dilution method which correspond to the antibiotic resistance genes. The apparent prevalence of Campylobacter was 16.8% by MCCDA while none of the isolates was positive to PCR. Meanwhile, prevalence rate of 26% was obtained using Preston broth pre-enrichment and Mueller Hinton agar with Campylobacter growth supplements, of which 11/50 (22%) of the isolates was confirmed positive by PCR. Genotypic characterization of PCR positive isolates showed 10/11(90%) were C. coli, 1/11(10%) other Campylobacter species and 0% C. jejuni. All the isolates carried both tetO and cmeB resistant genes. The results of minimum inhibitory concentration presented all PCR positive isolates had resistance of 10/10(100%), 9/10(90%), 6/10(60%), 9/10(90%), and 8/10(80%) to tetracycline, ciprofloxacin, erythromycin, spectinomycin and tylosin respectively. In addition, all isolates carried multiple resistance to most antibiotics tested which are commonly used in poultry practice in Nigeria. Campylobacter spp in the study areas showed diverse genotypic characteristics, and gene mediated multidrug resistance.

 

 

References

Adegbola, R, A., Akinkuade, F. O., Alabi, S. A., Coker, A. O., Odugbemi, T., 1991: Prevalence, biotype and serogroup of Campylobacter jejuni and campylobacter coli in domesticated animals in Nigeria. Trop. Vet., 9, 43-45.

Adekeye, J. O., Abdu, P. A., Bawa, E. K., 1989: Campylobacter fetus subsp. jejuni in poultry reared under different management systems in Nigeria. Avian Dis., 33, 801–803.

Atabay, H. I., Corry, J. E. L., 1998: The isolation and prevalence of Campylobacters from dairy cattle using variety methods. J. Appl. Microbiol., 84,:733 - 740.

Barrow, G. I., Feltham, R. K. A., 2003: Cowan and Steel’s Manual for the identification of medical bacteria. 3rd edn., Cambridge University Press, Cambridge, 352 pp.

Butzler, J. P., 2004: Campylobacter, from obscurity to celebrity. Clin. Microbiol. Infect., 10, 868–876.

Corry, J. E., Atabay, H. I., 2001: Poultry as a source of Campylobacter and related organisms. J. Appl. Microbiol., 90, 96–114.

Denis, M., Soumet, C., Rivoal, K., Erml, G., Blivet, D., Salvat, G., 1999: Development of an m-PCR assay for simultaneous identification of Campylobacter jejuni and Campylobacter coli. Letter Appl. Microbiol., 29, 406-410.

Diergaardt, S. M. S., Venter, N., Spreeth, A., Theron, J., Brozel, V. S., 2001: The occurrence of Campylobacters in water sources in South Africa. Water Res., 38, 2589-2595.

Engberg, J., Aarestrup, F. M., Taylor, D. E., Gerner-Smidt, P., Nachamkin, I., 2001: Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg. Infect. Dis., 7, 24–341.

Friedman, C. R., Neimann, J., Wegener, H. C., Tauxe, R. V., 2000: Epidemiology of Campylobacter jejuni infection in the United States and other industrialized nations. In: Nachamkin, I., Blaser, M. J.: Campylobacter. 2nd edn. ASM Press. Washington DC., 121-138.

Ge, B., White, D. G., McDermott, P. F., Girard, W., Zhao, S., Hubert, S., 2003: Antimicrobial –resistant Campylobacter species from retail raw meats. Appl. Environ. Microbiol., 69, 3005-3007.

Gonzalez, I., Grant, K. A., Richardson, P. T., Park, S. F., Collins, M. D., 1997: Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clinic. Microbiol., 35, 759–763.

Humphrey, T., O’Brien, S., Madsen, M., 2007: “Campylobacter as zoonotic pathogens: A food production perspective”. Int J. food microbiol., 117, 237-245.

Jackson, P. D., Hayden, J. D., Quirke, P., 1993: Extraction of nucleic acid from fresh and archival material. In: Mcpherson, M. J., Quirke, P., Taylor, G. R.: PCR, A Practical Approach. Oxford University Press, USA. Pp 29-50.

Janshidi, A., Bassami, M. R., Farkhondeh, T., 2008: Isolation and identification of Campylobacter spp. and Campylobacter coli from poultry carcasses by conventional and multiplex PCR method in Mashhad, Iran. Iranian J. Vet. Res. Shiraz. Uni., 92, 23-37.

Linton, D., Lawson, A. J., Owen, R. J., Stanley, J., 1997: PCR detection, identification to species level and finger printing of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. J. Clinic. Microbiol., 2568 – 2572. 0095.0095-1137/97

Magisrado, P. A., Garcia, M. M., Raymond, A. K., 2001: Isolation and polymerase chain reaction based detection of Campylobacter jejuni and Campylobacter coli from poultry in the Philipines. Int. J. Food Microbiol., 70, 197-206.

Moore, J. E., Corcoran, D., Doole, J. S., Fanning, S., Lucey, B., Matsuda, M. 2005: “Campylobacter”. Vet. Res., 36, 351-382.

Morris, G. K., El Sherbeeny, M. R., Patton, C. M., Kodaka, H., Lombard, G. L., Edmond, P. 1985: Comparison of four hippurate hydrolysis methods for identification of thermophilic Campylobacter spp. J. Clinic. Microbiol., 22, 714-718.

Nayak, R., Stewart, T. M., Nawas, M. S., 2005: PCR identification of Campylobacter coli and Campylobacter jejuni by partial sequencing of virulence genes. Mol. Cell. Probes., 19, 187-193.

Newell, D.G., Frost, J. A., Duim, B., Wagenaar, J. A., Madden, R. H., Van der Plas, J., 2000: New developments in the subtyping of Campylobacter strains. In: Nachamkin, I., Blaser, M. J.: Campylobacter. 2nd edn. ASM Press. Washington DC.,

Newell, D. G., Fearnley, C., 2003: Sources of Campylobacter colonization in broiler chicks. Appl. Environ. Microbiol., 69, 4343-4351.

Obeng, A. S., Rickard, H., Sexton, M., Pang, Y., Peng, H., M. Barton, M., 2012: Antimicrobial susceptibilities and resistance genes in Campylobacter strains isolated from poultry and pigs in Australia. J. Appl. Microbiol., 113, 294-307.

Olubunmi, P. A., Adeniran, M. O. A., 1986: Isolation of Campylobacter species from man and domestic animals in western Nigeria. Bull. Anim. Hlth. Prod. Afr., 32, 224-248.

Silva, J., Leite, D., Fernandes, M., Mena, C., Gibbs, P. A., Teixeira, P., 2011: Campylobacter spp. as a Foodborne Pathogen: A Review. Front. Microbiol., 2, 200. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180643/

Thrusfield, M. 2005. Survey. in Veterinary Epidemiology. 3rd Ed. Blackwell Publishing, 232pp.

Yamazaki-Matsune, W., Taguchi, M., Seto, K., Kawahara, R., Kawatsu, K., Kumeda, Y, 2007: Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyoointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J. Med. Microbiol., 56, 1467-1473.

Zhu, J., Zhang, Y., Hua, X., Hou, J., Jiang, Y., 2006: Antibiotic resistance in Campylobacter. J. Med. Microbiol., 17, 107-112.

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Published

2021-12-02

Issue

Vol. 19 No. 1 (2020)

Section

Articles