Prevalence and molecular characterization of extended-spectrum β-lactamase (ESBL) producing Escherichia coli isolated from dogs suffering from diarrhea in and around Kolkata

Document Type : Full paper (Original article)


1 Department of Veterinary Biochemistry, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, K. B. Sarani, Belgachia, Kolkata-700 037, West Bengal, India

2 Ph.D. Student in Veterinary Biochemistry, Department of Veterinary Biochemistry, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, K. B. Sarani, Belgachia, Kolkata-700 037, West Bengal, India

3 Eastern Regional Station, Indian Veterinary Research Institute, Kolkata-700 037, West Bengal, India

4 Department of Veterinary Clinical Complex, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, K. B. Sarani, Belgachia, Kolkata-700 037, West Bengal, India


Background: Dogs are the favorite companion animals among humans. The close interaction between dogs and people increases the risk of antibiotic resistance spreading. Surveillance for antimicrobial resistance and the identification of ESBL-producing Escherichia coli as an indicator bacterium is an important tool for managing antimicrobial drug therapy. Aims: The present study targeted to identify and characterize ESBL-producing E. coli among dogs suffering from diarrhea in and around Kolkata. Methods: Isolation and identification of E. coli from dogs suffering from diarrhea (n=70) along with screening for the production of both ESBL and AmpC. The isolates were further characterized through antimicrobial resistance profiling, resistance genes (blaCTX-M, blaTEM, and blaSHV) screening, and phylogenetic group study. Results: Among the 70 isolates, 21 (30%) were confirmed ESBL producers. An antibiogram typing of ESBL-producing E. coli revealed that the majority of them were resistant to norfloxacin (85.7%) followed by tetracycline (61.90%), doxycycline (57.14%), piperacillin/tazobactam (52.38%), cotrimoxazole (47.62%), gentamicin (42.62%), amikacin (23.81%), and chloramphenicol (19.05%). Major resistance genes included blaCTX-M (100%), blaTEM (28.57%), and blaSHV (9.50%). The predominant phylogenetic groups were phylogroup A (76%) followed by phylogroup D (24%). Conclusion: The current investigation reported a high prevalence of both ESBL and AmpC β-lactamase (AmpC) producing E. coli, co-resistance to a distinct group of antibiotics, and co-existence of different ESBL genes in dogs. Our findings highlight the importance of diagnostic antimicrobial susceptibility testing for proper antimicrobial therapy and to prevent antimicrobial resistance from spreading to humans from dogs in Kolkata and the surrounding area.


Abram, K; Udaondo, Z; Bleker, C; Wanchai, V; Wassenaar, TM; Robeson, MS and Ussery, DW (2021). Mash-based analyses of Escherichia coli genomes reveal 14 distinct phylogroups. Commun. Biol., 4: 1-2.
Andrews, J (2012). Detection of extended-spectrum beta-lactamases (ESBLs) in E. coli and Klebsiella species. British Society for Antimicrobial Chemotherapy. lla.pdf.
Babic, M; Hujer, AM and Bonomo, RA (2006). What’s new in antibiotic resistance? Focus on beta-lactamases. Drug. Resist. Updat., 9: 142-156.
Bhattacharjee, A; Sen, MR; Anupurba, S; Prakash, P and Nath, G (2007). Detection of OXA-2 group extended-spectrum-β-lactamase-producing clinical isolates of Escherichia coli from India. J. Antimicrob. Chemother., 60: 703-704.
Birgy, A; Mariani-Kurkdjian, P; Bidet, P; Doit, C; Genel, N; Courroux, C; Arlet, G and Bingen, E (2013). Characterization of extended-spectrum-beta-lactamase-producing Escherichia coli strains involved in maternal-fetal colonization: prevalence of E. coli ST131. J. Clin. Microbiol., 51: 1727-1732.
Branger, C; Zamfir, O; Geoffroy, S; Laurans, G; Arlet, G; Thien, HV; Gouriou, S; Picard, B and Denamur, E (2005). Genetic background of Escherichia coli and extended-spectrum β-lactamase type. Emerg. Infect. Dis., 11: 54-61.
Cantón, R and Coque, TM (2006). The CTX-M β-lactamase pandemic. Curr. Opin. Microbiol., 9: 466-475.
Chakraborty, A; Adhikari, P; Shenoy, S and Saralaya, V (2014). Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India. Indian J. Pathol. Microbiol., 57: 255-258.
Chong, Y; Ito, Y and Kamimura, T (2011). Genetic evolution and clinical impact in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Infect. Genet. Evol., 11: 1499-1504.
Ciesielczuk, H; Hornsey, M; Choi, V; Woodford, N and Wareham, DW (2013). Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J. Med. Microbiol., 62: 1823-1827.
Clermont, O; Bonacorsi, S and Bingen, E (2000). Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol., 66: 4555-4558.
Clinical Laboratory Standards Institute (CLSI) (2014). Performance standards for antimicrobial susceptibility testing; Twenty-Fourth Informational Supplement. Clinical and Laboratory Standard Institute, Wayne, PA, CLSI document 2014; M100-S124.
Dahms, C; Hübner, NO; Kossow, A; Mellmann, A; Dittmann, K and Kramer, A (2015). Occurrence of ESBL-producing Escherichia coli in livestock and farm workers in Mecklenburg-Western Pomerania, Germany. PLoS One. 10: e0143326.
Dallenne, C; Da Costa, A; Decre, D; Favier, C and Arlet, G (2010). Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother., 65: 490-495.
Damborg, P; Broens, EM; Chomel, BB; Guenther, S; Pasmans, F; Wagenaar, JA; Weese, JS; Wieler, LH; Windahl, U; Vanrompay, D and Guardabassi, L (2016). Bacterial zoonoses transmitted by household pets: state-of-the-art and future perspectives for targeted research and policy actions. J. Comp. Pathol., 155: S27-S40.
Day, MJ; Hopkins, KL; Wareham, DW; Toleman, MA; Elviss, N; Randall, L; Teale, C; Cleary, P; Wiuff, C; Doumith, M and Ellington, MJ (2019). Extended-spectrum β-lactamase-producing Escherichia coli in human-derived and food chain-derived samples from England, Wales, and Scotland: an epidemiological surveillance and typing study. Lancet Infect. Dis., 19: 1325-1335.
Deepthi, B; Srivani, M; Ramani Pushpa, RN and Chaitanya, Y (2020). Detection of extended spectrum beta-lactamase (ESBL) producing Escherichia coli in companion dogs. J. Pharm. Innov., 9: 189-194.
Dobrindt, U (2005). (Patho-) genomics of Escherichia coli. Int. J. Med. Microbiol. Suppl., 295: 357-371.
Džidić, S; Šušković, J and Kos, B (2008). Antibiotic resistance mechanisms in bacteria: biochemical and genetic aspects. Food Technol. Biotechnol., 46: 11-21.
Ewers, C; Bethe, A; Semmler, T; Guenther, S and Wieler, LH (2012). Extended-spectrum β-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin. Microbiol. Infect., 18: 646-655.
Ewers, C; Grobbel, M; Bethe, A; Wieler, LH and Guenther, S (2011). Extended-spectrum beta-lactamases-producing gram-negative bacteria in companion animals: action is clearly warranted. Berl. Munch. Tierarztl. Wochenschr., 124: 94-101.
Goldstein, RE; Micallef, SA; Gibbs, SG; He, X; George, A; Sapkota, A; Joseph, SW and Sapkota, AR (2012). Methicillin-resistant Staphylococcus aureus (MRSA) detected at four U.S. wastewater treatment plants. Environ. Health Perspect., 120: 1551-1558.
Haenni, M; de Moraes, NA; Châtre, P; Médaille, C; Moodley, A and Madec, JY (2014). Characterisation of clinical canine meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius in France. J. Glob. Antimicrob. Resist., 2: 119-123.
Hasman, H; Mevius, D; Veldman, K; Olesen, I and Aarestrup, FM (2005). β-Lactamases among extended-spectrum β-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J. Antimicrob. Chemother., 56: 115-121.
Holten, KB and Onusko, EM (2000). Appropriate prescribing of oral beta-lactam antibiotics. Am. Fam. Physician., 62: 611-620.
Hordijk, J; Schoormans, A; Kwakernaak, M; Duim, B; Broens, E; Dierikx, C; Mevius, D and Wagenaar, JA (2013). High prevalence of fecal carriage of extended spectrum β-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs. Front. Microbiol., 4: 242 (1-5).
Huber, H; Zweifel, C; Wittenbrink, MM and Stephan, R (2013). ESBL-producing uropathogenic Escherichia coli isolated from dogs and cats in Switzerland. Vet. Microbiol., 162: 992-996.
Jacoby, GA (2009). AmpC β-Lactamases. Clin. Microbiol. Rev., 22: 161-182.
Johnson, JR; Delavari, P; Kuskowski, M and Stell, AL (2001). Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli. J. Infect. Dis., 183: 78-88.
Johnson, JR; Kaster, N; Kuskowski, MA and Ling, GV (2003). Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E. coli isolates from dogs with urinary tract infection. J. Clin. Microbiol., 41: 337-345.
Johnson, JR and Stell, AL (2000). Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis., 181: 261-272.
Kar, D; Bandyopadhyay, S; Bhattacharyya, D; Samanta, I; Mahanti, A; Nanda, PK; Mondal, B; Dandapat, P; Das, AK; Dutta, TK and Bandyopadhyay, S (2015). Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect. Genet. Evol., 29: 82-90.
Kuenzli, E (2016). Antibiotic resistance and international travel: Causes and consequences. Travel Med. Infect. Dis., 14: 595-598.
Laxminarayan, R; Duse, A; Wattal, C; Zaidi, AK; Wertheim, HF; Sumpradit, N; Vlieghe, E; Hara, GL; Gould, IM; Goossens, H and Greko, C (2013). Antibiotic resistance—the need for global solutions. Lancet Infect. Dis., 13: 1057-1098.
Ljungquist, O; Ljungquist, D; Myrenås, M; Rydén, C; Finn, M and Bengtsson, B (2016). Evidence of household transfer of ESBL-/pAmpC-producing Enterobacteriaceae between humans and dogs-a pilot study. Infect. Ecol. Epidemiol., 6: 31514 (1-7).
Machado, E; Cantón, R; Baquero, F; Galán, JC; Rollán, A; Peixe, L and Coque, TM (2005). Integron content of extended-spectrum-β-lactamase-producing Escherichia coli strains over 12 years in a single hospital in Madrid, Spain. Antimicrob. Agents Chemother., 49: 1823-1829.
Matloko, K; Fri, J; Ateba, TP; Molale-Tom, LG and Ateba, CN (2021). Evidence of potentially unrelated AmpC beta-lactamase producing Enterobacteriaceae from cattle, cattle products and hospital environments commonly harboring the bla ACC resistance determinant. PLoS One. 16: e0253647.
Mohmid, EA; El-Sayed, ESA and El-Haliem, MFA (2013). Molecular study on extended spectrum-lactamase-producing Gram negative bacteria isolated from Ahmadi hospital in Kuwait. Afr. J. Biotechnol., 12: 5040-5053.
Molina, F; López-Acedo, E; Tabla, R; Roa, I; Gómez, A and Rebollo, JE (2015). Improved detection of Escherichia coli and coliform bacteria by multiplex PCR. BMC Biotechnol., 15: 48 (1-9).
Ng, LK; Martin, I; Alfa, M and Mulvey, M (2001). Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell. Probes. 15: 209-215.
Normand, EH; Gibson, NR; Carmichael, S; Reid, SW and Taylor, DJ (2000). Trends of antimicrobial resistance in bacterial isolates from a small animal referral hospital. Vet. Rec., 146: 151-155.
Nowrouzian, FL; Clermont, O; Edin, M; Östblom, A; Denamur, E; Wold, AE and Adlerberth, I (2019). Escherichia coli B2 phylogenetic subgroups in the infant gut microbiota: predominance of uropathogenic lineages in Swedish infants and enteropathogenic lineages in Pakistani infants. Appl. Environ. Microbiol., 85: e01681-19.
O’Keefe, A; Hutton, TA; Schifferli, DM and Rankin, SC (2010). First detection of CTX-M and SHV extended-spectrum β-lactamases in Escherichia coli urinary tract isolates from dogs and cats in the United States. Antimicrob. Agents Chemother., 54: 3489-3492.
Ovejero, CM; Delgado-Blas, JF; Calero-Caceres, W; Muniesa, M and Gonzalez-Zorn, B (2017). Spread of mcr-1-carrying Enterobacteriaceae in sewage water from Spain. J. Antimicrob. Chemother., 72: 1050-1053.
Park, CH; Robicsek, A; Jacoby, GA; Sahm, D and Hooper, DC (2006). Prevalence of aac (6’) Ib-cr encoding a ciprofloxacin-modifying enzyme in the United States. Antimicrob. Agents Chemother., 50: 3953-3955.
Pérez-Pérez, FJ and Hanson, ND (2002). Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol., 40: 2153-2162.
Picard, B; Garcia, JS; Gouriou, S; Duriez, P; Brahimi, N; Bingen, E; Elion, J and Denamur, E (1999). The link between phylogeny and virulence in Escherichia coli extra intestinal infection. Infect. Immun., 67: 546-553.
Pitout, JD (2012). Extraintestinal pathogenic Escherichia coli: an update on antimicrobial resistance, laboratory diagnosis and treatment. Expert. Rev. Anti. Infect. Ther., 10: 1165-1176.
Polsfuss, S; Bloemberg, GV; Giger, J; Meyer, V; Böttger, EC and Hombach, M (2011). Practical approach for reliable detection of AmpC beta-lactamase-producing Enterobacteriaceae. J. Clin. Microbiol., 49: 2798-2803.
Pupo, GM; Karaolis, DK; Lan, R and Reeves, PR (1997). Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. Infect. Immun., 65: 2685-2692.
Qekwana, DN; Phophi, L; Naidoo, V and Oguttu, JW (2018). Antimicrobial resistance among Escherichia coli isolates from dogs presented with urinary tract infections at a veterinary teaching hospital in South Africa. BMC Vet. Res., 14: 228 (1-6).
Quinn, PJ; Markey, BK; Leonard, FC; Hartigan, P; Fanning, S and Fitzpatrick, E (2011). Veterinary microbiology and microbial disease. 2nd Edn., Wiley-Blackwell, Chichester.
Robicsek, A; Strahilevitz, J; Jacoby, GA; Macielag, M; Abbanat, D; Park, CH; Bush, K and Hooper, DC (2006). Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat. Med., 12: 83-88.
Rossolini, GM; D’andrea, MM and Mugnaioli, C (2008). The spread of CTX-M-type extended-spectrum β-lactamases. Clin. Microbiol. Infect., 14: 33-41.
Saladin, M; Cao, VT; Lambert, T; Donay, JL; Herrmann, JL; Ould-Hocine, Z; Verdet, C; Delisle, F; Philippon, A and Arlet, G (2002). Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol. Lett., 209: 161-168.
Salgado-Caxito, M; Benavides, JA; Adell, AD; Paes, AC and Moreno-Switt, AI (2021). Global prevalence and molecular characterization of extended-spectrum β-lactamase producing-Escherichia coli in dogs and cats-A scoping review and meta-analysis. One Health. 12: 100236 (1-15).
Seguin, MA; Vaden, SL; Altier, C; Stone, E and Levine, JF (2003). Persistent urinary tract infections and reinfections in 100 dogs (1989-1999). J. Vet. Intern. Med., 17: 622-631.
Shaheen, BW; Nayak, R; Foley, SL; Kweon, O; Deck, J; Park, M; Rafii, F and Boothe, DM (2011). Molecular characterization of resistance to extended-spectrum cephalosporins in clinical Escherichia coli isolates from
companion animals in the United States. Antimicrob. Agents Chemother., 55: 5666-5675.
Shahid, M; Sobia, F; Singh, A and Khan, HM (2012). Concurrent occurrence of blaampC families and blaCTX-M genogroups and association with mobile genetic elements ISEcp1, IS26, ISCR1, and sul1-type class 1 integrons in Escherichia coli and Klebsiella pneumoniae isolates originating from India. J. Clin. Microbiol., 50: 1779-1782.
Smet, A; Van Nieuwerburgh, F; Vandekerckhove, TT; Martel, A; Deforce, D; Butaye, P and Haesebrouck, F (2010). Complete nucleotide sequence of CTX-M-15-plasmids from clinical Escherichia coli isolates: insertional events of transposons and insertion sequences. PLoS One. 5: e11202.
Stiffler, KS; Stevenson, MAM; Sanchez, S; Barsanti, JA; Hofmeister, E and Budsberg, SC (2006). Prevalence and characterization of urinary tract infections in dogs with surgically treated type 1 thoracolumbar intervertebral disc extrusion. Vet. Surg., 35: 330-336.
Thakuria, B and Lahon, K (2013). The beta lactam antibiotics as an empirical therapy in a developing country: an update on their current status and recommendations to counter the resistance against them. J. Clin. Diagn. Res., 7: 1207-1214.
Valenza, G; Nickel, S; Pfeifer, Y; Eller, C; Krupa, E; Lehner-Reindl, V and Höller, C (2014). Extended-spectrum-β-lactamase-producing Escherichia coli as intestinal colonizers in the German community. Antimicrob. Agents Chemother., 58: 1228-1230.
Vashist, H; Sharma, D and Gupta, A (2013). A review on commonly used biochemical test for bacteria. Innovare J. Sci., 1: 1-7.
Ventola, CL (2015). The antibiotic resistance crisis: part 1: causes and threats. Pharm. Ther., 40: 277-283.
Walk, ST; Alm, EW; Calhoun, LM; Mladonicky, JM and Whittam, TS (2007). Genetic diversity and population structure of Escherichia coli isolated from freshwater beaches. Environ. Microbiol., 9: 2274-2288.
Zeynudin, A; Pritsch, M; Schubert, S; Messerer, M; Liegl, G; Hoelscher, M; Belachew, T and Wieser, A (2018). Prevalence and antibiotic susceptibility pattern of CTX-M type extended-spectrum β-lactamases among clinical isolates of gram-negative bacilli in Jimma, Ethiopia. BMC Infect. Dis., 18: 524 (1-10).
Zogg, AL; Simmen, S; Zurfluh, K; Stephan, R; Schmitt, SN and Nüesch-Inderbinen, M (2018). High prevalence of extended-spectrum β-lactamase producing entero-bacteriaceae among clinical isolates from cats and dogs admitted to a veterinary hospital in Switzerland. Front. Vet. Sci., 5: 62 (1-8).