Isolation of Multidrug Resistant and Extended Spectrum β-Lactamase Producing Bacteria from Faecal Samples of Piggery Farms in Anambra State, Nigeria

Kyrian-Ogbonna Evelyn Ada^1, , Ekwealor Chito Clare¹, Okey-Ezeokoli Stella Chidinma¹, Ugwuoke Onyinyechukwuka Goodluck¹ and Ekwealor Ikechukwu A¹

¹Department of Applied Microbiology and Brewing, Faculty of Biosciences, Nnamdi Azikiwe University, Awka, Anambra State

Cite this paper:
Kyrian-Ogbonna Evelyn Ada, Ekwealor Chito Clare, Okey-Ezeokoli Stella Chidinma, Ugwuoke Onyinyechukwuka Goodluck and Ekwealor Ikechukwu A. Isolation of Multidrug Resistant and Extended Spectrum β-Lactamase Producing Bacteria from Faecal Samples of Piggery Farms in Anambra State, Nigeria. American Journal of Infectious Diseases and Microbiology. 2021; 9(4):106-113. doi: 10.12691/ajidm-9-4-1

Abstract

Background: Rising global concern about antimicrobial resistance (AMR) has drawn attention to the use of antibiotics in livestock. This research was aimed at isolation and determination of the total coliform bacteria, characterization of the bacterial isolates, screening for multidrug resistant bacteria and the resistant genes from faecal samples of pig farms in Anambra State. Methods: A total of 400 pig faecal samples collected from 40 farms in three senatorial zones of Anambra State were subjected to microbiological analysis and the total coliform bacteria determined. Enteric bacteria from the faecal samples were isolated and identified based on their morphological and biochemical characteristics. Susceptibility of the isolates to different antibiotics were carried out using the standard Kirby‐Bauer disc diffusion method and isolates resistant to cephalosporins were further subjected to Double Disc Synergy Test (DDST) for the phenotypical detection of extended spectrum beta-lactamase (ESBL) production. The ESBL producers were subjected to molecular studies for the detection of ESBL genes using PCR protocols. Results: Mean Total coliform counts of the bacteria from pig faecal samples varied in the 3 senatorial zones. Gram-negative bacteria from the pig faecal samples include the genera Escherichia, Klebsiella, Citrobacter, Salmonella, Enterobacter and Proteus. There is a significant difference in resistance of the isolates to cefotaxime (p = 0.025) and streptomycin (p = 0.012) but no significant difference (p > 0.05) was observed on the rest of the antibiotics tested. Streptomycin (60%) was the most highly resisted while Imipenem (4%) was the least antibiotics. 54.6% of all the organisms were multidrug resistant while 43.7% were ESBL producers. Bla_CTX was present in ESBL-producing isolates. Conclusion: There is presence of pathogenic enteric organisms in pig faecal samples harbouring antimicrobial resistant genes. Prudent use of antibiotics in pig farms in Anambra State is therefore recommended to reduce the spread of antibiotic resistance.

Keywords:
Antibiotics Antimicrobial resistance coliforms pig farms resistant genes

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References:

[1]	Ezeibe, A.B.C. Profitability analysis of pig production under intensive management systems in Nsukka Local Government Area of Enugu State, Nigeria. International Journal of Economic Development Research and Investment, 1 (2&3): 1- 7. 2010.
[2]	Ironkwe M.O. and Amefule K. U. Appraisal of indigenous pig procution and management practices in Rivers State, Nigeria. Journal of Agriculture and Social Research, 8 (1). Aug. 2008.
[3]	Lekagul, A., Tangcharoensathien, V., Mills, A., Rushton, J. and Yeung, S. How antibiotics are used in pig farming: a mixed-methods study of pig farmers, feed mills and veterinarians in Thailand. BMJ Global Health, 5(2):e001918. Feb. 2020.
[4]	Laxminarayan, R., Duse, A. and Wattal, C. Antibiotic resistance--the need for global solutions. The Lancet InfectiousDiseases, 13 (12): 1057-1098. Dec. 2013.
[5]	Bennani, H., Mateus, A., Mays, N., Eastmure, E., Stärk, K.D.C. and Häsler, B.. Overview of evidence of antimicrobial use and antimicrobial resistance in the food chain. Antibiotics, 9: 49. Jan. 2020.
[6]	Van Boeckel, T.P., Brower, C., Gilbert, M., Grenfell, B.T., Levin, S.A. and Robinson, T.P. Global trends in anti- microbial use in food animals. Proceeding of National Academy of Science USA. 112 (18): 5649-5654. May 2015.
[7]	Filippitzi, M.E., Callens, B. and Pardon, B. Antimicrobial use in pigs, broilers and veal calves in Belgium. Vlaams Diergeneeskundig Tijdschrift, 83: 214-224. Oct. 2014.
[8]	World Health Organization (WHO). Critically important antimicrobials for human medicine [Internet]. Geneva. Available: http://apps.who.int/iris/bitstream/handle/10665/255027/9789241512220eng.pdf; jsessionid=FC65DF119DE54907C6E5D457093EC97E?sequence=1 Accessed 20 Apr 2020.
[9]	Liu, Y.Y, Wang, Y. and Walsh, T.R. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infectious Disease, 16: 161-168. Feb. 2016.
[10]	Looft, T., Johnson, T.A., Allen, H.K., Bayles, D.O., Alt, D.P. and Stedtfeld, R.D. In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences of the United States ofAmerica, 109: 1691-1696. Jan. 2012.
[11]	Johnson, T.A., Stedtfeld, R.D., Wang, Q., Cole, J.R., Hashsham, S.A. and Looft, T. Clusters of antibiotic resistance genes enriched together stay together in swine agriculture. MBio, 7: 2214-2215. April 2016.
[12]	Krehbiel, C. The role of new technologies in global food security: improving animal production efficiency and minimizing impacts. Animal Frontiers. 3(3): 4-7. Oct. 2013.
[13]	Hao, H., Cheng, G., Iqbal, Z., Ai, X., Hussain, H.I. and Huang L. Benefits and risks of antimicrobial use in food-producing animals. Frontiers in Microbiology, 5: 288. June 2014.
[14]	Liu, Z., Klümper, U., Shi, L., Ye, L. and Li, M. From pig breeding environment to subsequently produced pork: Comparative analysis of antibiotic resistance genes and bacterial community composition. Frontiers in Microbiology, 10:43. Jan. 2019.
[15]	Ogunleye, A.O. and Okunlade, O.A. Antibiotic resistance status of Escherichia coli isolated from healthy pigs from some piggery farms in Ibadan, Nigeria. Tropical Veterinarian 33: 3-4. Aug. 2017.
[16]	Zaniani, F. R., Meshkat, Z., Naderi Nasab, M., Khaje-Karamadini, M., Ghazvini, K., Rezaee, A., Esmaily, H., Nabavinia, M. S. and Darban Hoseini, M. The prevalence of TEM and SHV genes among Extended-Spectrum Beta-Lactamases producing Escherichia coli and Klebsiella pneumoniae. Iranian Journal of Basic Medical Sciences, 15(1): 654-660. January 2012
[17]	Dohmen, W., Dorado-García, A., Bonten, M. J., Wagenaar, J. A., Mevius, D. and Heederik, D. J. Risk factors for ESBL-producing Escherichia coli on pig farms: A longitudinal study in the context of reduced use of antimicrobials. PloS one, 12(3), e0174094. March 2017.
[18]	Liebana, E., Carattoli, A., Coque, T. M., Hasman, H., Magiorakos, A. P., Mevius, D., Peixe, L., Poirel, L., Schuepbach-Regula, G., Torneke, K., Torren-Edo, J., Torres, C. and Threlfall, J. Public health risks of enterobacterial isolates producing extended-spectrum β-lactamases or AmpC β-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 56(7), 1030-1037. April 2013.
[19]	Mesa, R. J., Blanc, V., Blanch, A. R., Cortés, P., González, J. J., Lavilla, S., Miró, E., Muniesa, M., Saco, M., Tórtola, M. T., Mirelis, B., Coll, P., Llagostera, M., Prats, G. and Navarro, F. Extended-spectrum beta-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). The Journal of antimicrobial chemotherapy, 58(1), 211-215. July 2006.
[20]	Hammerum AM, Larsen J, Andersen VD, Lester CH, Skytte TS, Hansen F, Olsen , S.S, Mordhorst , H., Skov , R.L., Aarestrup, F.M., Agers, Y. Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins. Journal of Antimicrobial Chemotherapy, 69: 2650-2657. Oct. 2014.
[21]	Oloso, N. O., Fagbo, S., Garbati, M., Olonitola, S. O., Awosanya, E. J., Aworh, M. K., Adamu, H., Odetokun, I. A. and Fasina, F. O. Antimicrobial resistance in food animals and the environment in Nigeria: A Review. International Journal of Environmental Research and Public Health, 15(6): 1284. June 2018
[22]	Ifeka, A.C. and Akinbobola, A. Land use/land cover change detection in some selected stations in Anambra State. Journal of Geography and Regional Planning, 18 (1): 1-11. Jan. 2015.
[23]	Nijsten, R., London, N., van den Bogaard, A. and Stobberingh, E. Resistance in faecal Escherichia coli isolated from pig farmers and abattoir workers. Epidemiology and Infection, 113: 45-52. June 1994.
[24]	Noel, K.R. and John, H.G. Bergey’s Manual of Systematic Bacteriology, Barbara T. edition. William and Wilkins publishers, Baltimore, USA. pp 85-115. 1984.
[25]	Oyeleke, S.B. and Manga, S.B. Essentials of laboratory practicals in microbiology. Tobest Publishers Minna, Nigeria, pp.36-75. Dec. 2008.
[26]	Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk susceptibility test; approved standard. M02-A11: 35 (3): 25-35. 2015.
[27]	Ejikeugwu, C., Iroha, I., Adikwu, M. and Esimone, C. Susceptibility and detection of extended spectrum β-Lactamase enzymes from Otitis Media pathogens. American Journal of Infectious Diseases, 9(1): 24-29. Jan. 2013.
[28]	Kumar, S., Stecher, G. and Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7):1870-1887. July, 2016.
[29]	Wang, L., Mankin, K. R. and Marchin, G. L. Survival of faecal bacteria in Dairy cow manure American Society of Agricultural and Biological Engineers. 47(4): 1239-1246. 2004.
[30]	Chinivasagam, H. N., Thomas, R.J., Casey, K., McGahan, E., Gardner, E.A., Rafiee., M. and Blackall, P.J. Microbiological status of piggery effluent from 13 piggeries in the south east Queensland region of Australia. Journal of Applied Microbiology 97: 883-891. 2004.
[31]	Binh, C.T., Heuer, H., Kaupenjohann, M. and Smalla, K. Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. Microbiology and Ecology, 66: 25-37. 2008.
[32]	Amador, P., Duarte, I.M., Roberto da Costa, R.P., Fernandes, R. and Prudêncio, C. Characterization of antibiotic resistance in Enterobacteriaceae from agricultural manure and soil in Portugal. Soil Science, 182: 292-301. Oct. 2018.
[33]	Martin, B.S., Campos, L., Bravo, V., Adasne, M., and Borie, C. (2005). Evaluation of antimicrobial resistance using indicator bacteria isolated from pigs and poultry in Chile. International Journal of Applied Research in Veterinary Medicine, 3 (2). 2005.
[34]	Valiakos, G., Vontas, A., Constantina, N. T., Alexios, G., Dimitrios, C. and Charalambos, B. Resistance in Escherichia coli strains isolated from pig faecal samples and pig farm workers in Greece. American Journal of Animal and Veterinary Sciences, 11 (4):142-144. Nov. 2016.
[35]	Chah, K. F., Ugwu, I. C., Okpala, A., Adamu, K. Y., Alonso, C. A., Ceballos, S., Nwanta, J. N. and Torres, C. Detection and molecular characterisation of extended-spectrum β-lactamase-producing enteric bacteria from pigs and chickens in Nsukka, Nigeria. Journal of Global Antimicrobial Resistance, 15: 36-40. Dec. 2018.
[36]	Paterson, D. L. Resistance in gram-negative bacteria: Enterobacteriaceae. American Journal of Infection Control, 34(5 Suppl 1): 20-28; discussion S64-73. June 2006.
[37]	Vico, J.P., Lorenzutti, A.M., Zogbi, A.P., Aleu, G., Sánchez, I.C., Caffer, M.I., Rosmini, M.R. and Mainar-Jaime, R.C. Prevalence, associated risk factors, and antimicrobial resistance profiles of non-typhoidal Salmonella in large scale swine production in Córdoba Argentina. Research in Veterinary Science, 130: 161-169. June 2020.
[38]	Davies, R. and Wales, A. Antimicrobial resistance on farms: a review including biosecurity and the potential role of disinfectants in resistance selection. Comprehensive Reviews in Food Science and Food Safety, 18: 753-774. April 2019.
[39]	Pires, D., de Kraker, M.E.A., Tartari, E., Abbas, M. and Pittet, D. Fight antibiotic resistance—It’s in your hands: Call from the World Health Organization for 5th May 2017. Clinical Infectious Disease, 64: 1780-1783. May 2017
[40]	Shrivastava, S., Shrivastava, P. and Ramasamy, J. World health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. Journal of Medical and Society, 32: 67-76. June 2018.
[41]	Page, S. W. and Gautier, P. Use of antimicrobial agents in livestock,” Revue Scientifique et Technique, 31 (1): 145-188. April 2012.
[42]	Abdalla, S.E., Abia, A.L.K., Amoako, D.G., Perrett, K., Bester, L.A. and Essack, S.Y. From farm-to-fork: E. coli from an intensive pig production system in South Africa shows high resistance to critically important antibiotics for human and animal use. Antibiotics, 10: 178. Feb. 2021.
[43]	Osterberg, J., Wingstrand, A., Nygaard-Jensen, A., Kerouanton, A., Cibin, V., Barco, L., Denis, M., Aabo, S. and Bengtsson, B. Antibiotic resistance in Escherichia coli from pigs in organic and conventional farming in four European countries. PLoS ONE, 11: e0157049. June 2016.
[44]	Chantziaras, I., Boyen, F., Callens, B. and Dewulf, J. Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. Journal of Antimicrobial Chemotherapy, 69: 827-834. March, 2014.
[45]	Adenipekun, E.O., Jackson, C.R., Oluwadun, A., Iwalokun, B.A., Frye, J.G. and Barrett J.B. Prevalence and antimicrobial resistance in Escherichia coli from food animals in Lagos, Nigeria. Microbiology and Drug Resistance, 21: 358-365. June, 2015.
[46]	Pitout, J.D. and Laupland, K.B. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public health concern. Lancet Infectious Disease, 8: 159-66. March, 2008.
[47]	Ejikeugwu, C., Iroha, I., Orji, J., Ugwu, M., Okonkwo, E., Ikegbunam, M., Ugbo, E., Moses, I., and Nwakaeze, E. Antibiogram of ESBL-producing Pseudomonas aeruginosa isolates of nosocomial origin. European Journal of Pharmaceutical and Medical Research, 2(4): 92-99. June, 2015.
[48]	Samanta, A., Mahanti, A., Chatterjee, S., Joardar, S. N., Bandyopadhyay, S., Sar, T. K., Mandal, G. P., Dutta, T. K. and Samanta, I. Pig farm environment as a source of beta-lactamase or AmpC-producing Klebsiella pneumoniae and Escherichia coli. Annals of Microbiology, 68 (11), 781-791. Oct. 2020.