Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli strains in dairy farm wastewater in Chiang Mai

Main Article Content

Prayuth Saekhow
Chayaphon Sriphannam


We investigated the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains in dairy farm wastewater in Chiang Mai, Thailand. We analyzed wastewater samples collected from 150 dairy farms and found that 88.7% of the farms (n = 133) were positive for ESBL-producing E. coli. Multiplex polymerase chain reaction (PCR) amplification was performed to characterize the presence of bla CTX-M, bla TEM, and blaSHV in ESBL-producing isolates. blaCTX-M was found in all isolates (n = 133), followed by blaTEM (80/133, 60.2%), whereas blaSHV was not detected in any isolate. blaCTX-M and blaTEM were present in 60.2% (80/133) of the isolates, and 39.8% (53/133) isolates carried bla CTX-M alone. Subgroup analysis showed that CTX-M-1 was the most prevalent subgroup among the isolates (129/133, 97.0%), followed by CTX-M-8 (2/133, 1.5%) and CTX-M-9 (2/133, 1.5%). The distribution of the phylogenetic groups was as follows: group A (100/133, 75.2%), followed by B1 (14/133, 10.5%), D (6/133, 4.5%), F (6/133, 4.5%), B2 (4/133, 3.0%), and E (3/133, 2.3%). Based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and dendrogram analysis, 24 isolates were classified into clades I (n = 21), II (n =1), and III (n =2). Minor genetic differences were found in all clade I isolates. Our data suggest that the circulating of ESBL-producing E. coli carried at least one bla gene strain distributed in dairy farm wastewater in Chiang Mai.


Download data is not yet available.

Article Details

How to Cite
Saekhow, P., & Sriphannam, C. . (2021). Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli strains in dairy farm wastewater in Chiang Mai: Veterinary Integrative Sciences, 19(3), 349–362. Retrieved from
Research Articles


Anuchatkitcharoen, C., Numee, S., Bender, J., Awaiwanont, N., and Intanon, M. 2020. Prevalence and antimicrobial resistance of Salmonella isolated from backyard pigs in Chiang Mai, Thailand. Vet. Integr. Sci., 18(3), 193-204.

Bar-Yoseph, H., Hussein, K., Braun, E., Paul, M., 2016. Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis. J. Antimicrob. Chemother. 71, 2729–2739.

Bonnedahl, J., Drobni, M., Gauthier-Clerc, M., Hernandez, J., Granholm, S., Kayser, Y., Melhus, Å., Kahlmeter, G., Waldenström, J., Johansson, A., 2009. Dissemination of Escherichia coli with CTX-M type ESBL between humans and yellow-legged gulls in the south of France. PloS One 4, e5958.

Boonyasiri, A., Tangkoskul, T., Seenama, C., Saiyarin, J., Tiengrim, S., Thamlikitkul, V., 2014. Prevalence of antibiotic resistant bacteria in healthy adults, foods, food animals, and the environment in selected areas in Thailand. Pathog. Glob. Health 108, 235–245.

Brisson-Noël, A., Arthur, M., Courvalin, P., 1988. Evidence for natural gene transfer from gram-positive cocci to Escherichia coli. J. Bacteriol. 170, 1739–1745.

Bubpamala, J., Khuntayaporn, P., Thirapanmethee, K., Montakantikul, P., Santanirand, P., Chomnawang, M.T., 2018. Phenotypic and genotypic characterizations of extended-spectrum beta-lactamase-producing Escherichia coli in Thailand. Infect. Drug Resist. 11, 2151.

Cantón, R., González-Alba, J.M., Galán, J.C., 2012. CTX-M enzymes: origin and diffusion. Front. Microbiol. 3, 110.

Casarez, E.A., Pillai, S.D., Di Giovanni, G.D., 2007. Genotype diversity of Escherichia coli isolates in natural waters determined by PFGE and ERIC-PCR. Water Res. 41, 3643–3648.

Chen, J., Griffiths, M., 1998. PCR differentiation of Escherichia coli from other Gram‐ negative bacteria using primers derived from the nucleotide sequences flanking the gene encoding the universal stress protein. Letters in applied microbiology 27, 369-371.

Clermont, O., Christenson, J.K., Denamur, E., Gordon, D.M., 2013. The Clermont Escherichia coli phylo‐typing method revisited: improvement of specificity and detection of new phylo‐groups. Env. Microbiol. Rep. 5, 58–65.

Clinical and Laboratory Standards Institute, 2013. Performance standards for antimicrobial susceptibility testing; Twenty-third informational supplement M100-S23. USA: CLSI.

Coque, T., Baquero, F., Canton, R., 2008. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Eurosurveillance 13, 19044.

Cormier, A.C., Chalmers, G., McAllister, T.A., Cook, S., Zaheer, R., Scott, H.M., Booker, C., Read, R., Boerlin, P., 2016. Extended-spectrum-cephalosporin resistance genes in Escherichia coli from beef cattle. Antimicrob. Agents Chemother. 60, 1162–1163.

Coura, F.M., de Araújo Diniz, S., Mussi, J.M.S., Silva, M.X., Lage, A.P., Heinemann, M.B., 2017. Characterization of virulence factors and phylogenetic group determination of Escherichia coli isolated from diarrheic and non-diarrheic calves from Brazil. Folia Microbiol. 62,139-144.

Coura, F.M., de Araújo Diniz, S., Silva, M.X., Mussi, J.M.S., Barbosa, S.M., Lage, A.P., Heinemann, M.B., 2015. Phylogenetic group determination of Escherichia coli isolated from animals samples. Sci. World J. 2015

Courvalin, P., 1994. Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Antimicrob. Agents Chemother. 38, 1447.

da Silveira, W.D., Ferreira, A., Lancellotti, M., Barbosa, I.A., Leite, D.S., de Castro, A.F., Brocchi, M., 2002. Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR. Vet. Microbiol. 89, 323–328.

Dhillon, R.H.-P., Clark, J., 2012. ESBLs: a clear and present danger? Crit. Care Res. Pract. 2012.

Dogan, B., Rishniw, M., Bruant, G., Harel, J., Schukken, Y.H., Simpson, K.W., 2012. Phylogroup and lpfA influence epithelial invasion by mastitis associated Escherichia coli. Vet. Microbiol. 159, 163–170.

Duriez, P., Clermont, O., Bonacorsi, S., Bingen, E., Chaventre, A., Elion, J., Picard, B., Denamur, E., 2001. Commensal Escherichia coli isolates are phylogenetically distributed among geographically distinct human populations. Microbiology 147, 1671–1676.

Escobar‐Páramo, P., Le Menac’h, A., Le Gall, T., Amorin, C., Gouriou, S., Picard, B., Skurnik, D., Denamur, E., 2006. Identification of forces shaping the commensal Escherichia coli genetic structure by comparing animal and human isolates. Env. Microbiol. 8, 1975–1984.

Feizabadi, M.M., Delfani, S., Raji, N., Majnooni, A., Aligholi, M., Shahcheraghi, F., Parvin, M., Yadegarinia, D., 2010. Distribution of blaTEM, blaSHV, blaCTX-M genes among clinical isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran. Microb. Drug Resist. 16, 49–53.

Figueira, V., Serra, E., Manaia, C.M., 2011. Differential patterns of antimicrobial resistance in population subsets of Escherichia coli isolated from waste-and surface waters. Sci. Total Env. 409, 1017–1023.

Goldstone, R.J., Harris, S., Smith, D.G., 2016. Genomic content typifying a prevalent clade of bovine mastitis-associated Escherichia coli. Sci. Rep. 6, 1–15.

Gordon, D.M., Cowling, A., 2003. The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects. Microbiology 149, 3575–3586.

Hardy, B., 2002. The issue of antibiotic use in the livestock industry: what have we learned? Anim. Biotechnol. 13, 129–147.

Horcajada, J.P., Soto, S., Gajewski, A., Smithson, A., De Anta, M.T.J., Mensa, J., Vila, J., Johnson, J.R., 2005. Quinolone-resistant uropathogenic Escherichia coli strains from phylogenetic group B2 have fewer virulence factors than their susceptible counterparts. J. Clin. Microbiol. 43, 2962–2964.

Landers, T.F., Cohen, B., Wittum, T.E., Larson, E.L., 2012. A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep. 127, 4–22.

Le Gall, T., Clermont, O., Gouriou, S., Picard, B., Nassif, X., Denamur, E., Tenaillon, O., 2007. Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains. Mol. Biol. Evol. 24, 2373–2384.

Leung, K.T., Mackereth, R., Tien, Y.-C., Topp, E., 2004. A comparison of AFLP and ERIC-PCR analyses for discriminating Escherichia coli from cattle, pig and human sources. FEMS Microbiol. Ecol. 47, 111–119.

Lewis, J.S., Herrera, M., Wickes, B., Patterson, J.E., Jorgensen, J.H., 2007. First report of the emergence of CTX-M-type extended-spectrum β-lactamases (ESBLs) as the predominant ESBL isolated in a US health care system. Antimicrob. Agents Chemother. 51, 4015–4021.

Liu, Y., Liu, G., Liu, W., Liu, Y., Ali, T., Chen, W., Yin, J., Han, B., 2014. Phylogenetic group, virulence factors and antimicrobial resistance of Escherichia coli associated with bovine mastitis. Res. Microbiol. 165, 273–277.

Livermore, D., Hawkey, P., 2005. CTX-M: changing the face of ESBLs in the UK. J. Antimicrob. Chemother. 56, 451–454.

M’Zali, F.H., Chanawong, A., Kerr, K.G., Birkenhead, D., Hawkey, P.M., 2000. Detection of extended-spectrum betalactamases in members of the family enterobacteriaceae: comparison of the MAST DD test, the double disc and the Etest ESBL. J. Antimicrob. Chemother. 45, 881–885.

Madec, J.Y., Haenni, M., Nordmann, P., Poirel, L., 2017. Extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans? Clin. Microbiol. Infect. 23, 826–833.

Madico, G., Akopyants, N.S., Berg, D.E., 1995. Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157: H7 strains by using templates from boiled cultures. J. Clin. Microbiol. 33, 1534-1536.

Marshall, B.M., Levy, S.B., 2011. Food animals and antimicrobials: impacts on human health. Clin. Microbiol. Rev. 24, 718–733.

McDanel, J., Schweizer, M., Crabb, V., Nelson, R., Samore, M., Khader, K., Blevins, A.E., Diekema, D., Chiang, H.-Y., Nair, R., 2017. Incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella infections in the United States: a systematic literature review. infect. Control Hosp. Epidemiol. 38, 1209–1215.

Meacham, K.J., Zhang, L., Foxman, B., Bauer, R.J., Marrs, C.F., 2003. Evaluation of genotyping large numbers of Escherichia coli isolates by enterobacterial repetitive intergenic consensus-PCR. J. Clin. Microbiol. 41, 5224–5226.

Médigue, C., Rouxel, T., Vigier, P., Hénaut, A., Danchin, A., 1991. Evidence for horizontal gene transfer in Escherichia coli speciation. J. Mol. Biol. 222, 851–856.

Mellata, M., 2013. Human and avian extraintestinal pathogenic Escherichia coli: infections, zoonotic risks, and antibiotic resistance trends. Foodborne Pathog. Dis. 10, 916–932.

Michael, G.B., Kaspar, H., Siqueira, A.K., de Freitas Costa, E., Corbellini, L.G., Kadlec, K., Schwarz, S., 2017. Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates collected from diseased food-producing animals in the GERM-Vet monitoring program 2008–2014. Vet. Microbiol. 200, 142–150.

Monstein, H.J., Östholm‐Balkhed, Å., Nilsson, M., Nilsson, M., Dornbusch, K., Nilsson, L., 2007. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX‐M genes in Enterobacteriaceae. Apmis 115, 1400-1408.

Ngammuangtueng, P., Jakrawatana, N., Nilsalab, P., Gheewala, S.H., 2019. Water, energy and food nexus in rice production in Thailand. Sustainability 11, 5852.

Niumsup, P., Tansawai, U., Na-Udom, A., Jantapalaboon, D., Assawatheptawee, K., Kiddee, A., Romgaew, T., Lamlertthon, S., Walsh, T., 2018. Prevalence and risk factors for intestinal carriage of CTX-M-type ESBLs in Enterobacteriaceae from a Thai community. Eur. J. Clin. Microbiol. Infect. Dis. 37, 69–75.

Nuangmek, A., Rojanasthien, S., Chotinun, S., Yamsakul, P., Tadee, P., Thamlikitkul, V., Tansakul, N., Patchanee, P., 2018. Antimicrobial resistance in ESBL-producing Escherichia coli isolated from layer and pig farms in Thailand. Acta Sci. Vet. 46, 8.

Orsi, R.H., Stoppe, N.C., Sato, M.I.Z., Gomes, T.A., Prado, P.I., Manfio, G.P., Ottoboni, L.M., 2007. Genetic variability and pathogenicity potential of Escherichia coli isolated from recreational water reservoirs. Res. Microbiol. 158, 420–427.

Pavel, A.B., Vasile, C.I., 2012. PyElph-a software tool for gel images analysis and phylogenetics. BMC Bioinformatics 13, 1–6.

Pitout, J.D., Laupland, K.B., 2008. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8, 159–166.

Price, M.N., Dehal, P.S., Arkin, A.P., 2008. Horizontal gene transfer and the evolution of transcriptional regulation in Escherichia coli. Genome Biol. 9, 1–20.

Rawat, D., Nair, D., 2010. Extended-spectrum β-lactamases in gram negative bacteria. J. Global Infect. Dis. 2, 263.

Rehman, M.A., Yin, X., Lepp, D., Laing, C., Ziebell, K., Talbot, G., Topp, E., Diarra, MS, 2017. Genomic analysis of third generation cephalosporin resistant Escherichia coli from dairy cow manure. Vet. Sci. 4, 57.

Rodroo, J., Intanon, M., Kreausukon, K., Kongkaew, A., Bender, J., Awaiwanont, N., 2021. Occurrence of extended-spectrum beta-lactamase producing E. coli in broiler farm workers and the farm environment in Chiang Mai-Lamphun, Thailand. Veter. Integrat. Sci. 19.

Runcharoen, C., Raven, K.E., Reuter, S., Kallonen, T., Paksanont, S., Thammachote, J., Anun, S., Blane, B., Parkhill, J., Peacock, S.J., 2017. Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand. Genome Med. 9, 1–11.

Sasaki, T., Hirai, I., Niki, M., Nakamura, T., Komalamisra, C., Maipanich, W., Kusolsuk, T., Sa-Nguankiat, S., Pubampen, S., Yamamoto, Y., 2010. High prevalence of CTX-M β-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand. J. Antimicrob. Chemother. 65, 666–668.

Schauss, T., Glaeser, S.P., Gütschow, A., Dott, W., Kämpfer, P., 2015. Improved detection of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli in input and output samples of German biogas plants by a selective pre-enrichment procedure. PloS One 10, e0119791.

Seenama, C., Thamlikitkul, V., Ratthawongjirakul, P., 2019a. Multilocus sequence typing and bla (ESBL) characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolated from healthy humans and swine in Northern Thailand. Infect. Drug. Resist. 12, 2201–2214.

Sooksai, N., Ratbamroong, N., Suwannaprom, P., Chowwanapoonpohn., H., 2019. Antibiotic use in livestock farming: a case study in Chiang Mai. Thai J. Pharm. Pract. 8, 283–294.

Suriyasathaporn, W., Chupia, V., Sing-Lah, T., Wongsawan, K., Mektrirat, R., Chaisri, W., 2012. Increases of antibiotic resistance in excessive use of antibiotics in smallholder dairy farms in Northern Thailand. Asian-Australas. J. Anim. Sci. 25, 1322–1328.

Tamang, M.D., Nam, H.-M., Gurung, M., Jang, G.-C., Kim, S.-R., Jung, S.-C., Park, Y.H., Lim, S.-K., 2013. Molecular characterization of CTX-M β-lactamase and associated addiction systems in Escherichia coli circulating among cattle, farm workers, and the farm environment. Appl. Env. Microbiol. 79, 3898–3905.

Tomazi, T., Coura, F., Gonçalves, J., Heinemann, M., Santos, M., 2018. Antimicrobial susceptibility patterns of Escherichia coli phylogenetic groups isolated from bovine clinical mastitis. J. Dairy Sci. 101, 9406–9418.

Umpiérrez, A., Bado, I., Oliver, M., Acquistapace, S., Etcheverría, A., Padola, N.L., Vignoli, R., Zunino, P., 2017. Zoonotic potential and antibiotic resistance of Escherichia coli in neonatal calves in Uruguay. Microbes Environ. 32, 275–282.

Watson, E., Jeckel, S., Snow, L., Stubbs, R., Teale, C., Wearing, H., Horton, R., Toszeghy, M., Tearne, O., Ellis-Iversen, J., 2012. Epidemiology of extended spectrum beta-lactamase E. coli (CTX-M-15) on a commercial dairy farm. Vet. Microbiol. 154, 339–346.

West, B.M., Liggit, P., Clemans, D.L., Francoeur, S.N., 2011. Antibiotic resistance, gene transfer, and water quality patterns observed in waterways near CAFO farms and wastewater treatment facilities. Water Air Soil Pollut. 217, 473–489.

Wilson, L.A., Sharp, P.M., 2006. Enterobacterial repetitive intergenic consensus (ERIC) sequences in Escherichia coli: Evolution and implications for ERIC-PCR. Mol. Biol. Evol. 23, 1156-1168.

Woerther, P.-L., Burdet, C., Chachaty, E., Andremont, A., 2013. Trends in human fecal carriage of extended-spectrum β-lactamases in the community: toward the globalization of CTX-M. Clin. Microbiol. Rev. 26, 744–758.

Woodford, N., Fagan, E.J., Ellington, M.J., 2005. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J. Antimicrob.Chemother. 57, 154–155.