Nidhi RAWAT, Fateh SINGH, Sadanand Devidas HIRPURKAR, Chandrahas SANNAT, Nitin Eknath GADE

Detection and characterization of extended-spectrum beta-lactamase genes ( bla TEM and bla SHV ) among beta-lactam-resistant fecal coliforms of dairy cattle from Chhattisgarh, India

The aim of this study was to isolate the antimicrobial-resistant enterobacteria from cattle feces and to identify the extended-spectrum beta-lactamase (ESBL) and carbapenemase gene among them. A total of 70 cattle fecal swab samples were collected from Durg, Chhattisgarh, India, and cultured in nutrient broth. Bacterial isolation recovered 120 gram-negative bacteria. Antimicrobial susceptibility testing showed 90 (75%) coliform isolates (66 Escherichia coli, 20 Klebsiella sp., and two each of Enterobacter sp. and Citrobacter sp.) resistant to amoxicillin, amoxicillin-clavulanic acid, cefixime, ceftazidime, imipenem, or meropenem. Bacterial isolates showed the highest resistance rate for ceftazidime (61.6%), followed by cefixime and amoxicillin-clavulanic acid (60%). The ESBL phenotype, assessed by combination disk method, was shown by 36 (40%) of the resistant isolates. PCR amplification revealed 20% and 31.6% positivity of blaTEMTEM_{TEM} and blaSHVSHV_{SHV} genes, respectively. blaTEMTEM_{TEM} and blaSHVSHV_{SHV} coexpression was recorded among eight (12.1%) E. coli and two (10%) Klebsiella sp. isolates. Restriction endonuclease analysis indicated clonal blaTEM and clonal and nonclonal blaSHV genes. Nucleotide sequencing confirmed the blaTEMTEM_{TEM} and blaSHVSHV_{SHV} genotypes. None of the isolates showed amplification of the carbapenemase gene. Phenotypic and genetic detection and characterization of ESBLs among Enterobacteriaceae bacteria illustrate the importance of planning and execution of effective control programs for emerging antimicrobial-resistant pathogens.

Keywords:

Beta-lactam resistance, cattle, coliform, Enterobacteriaceae, extended-spectrum beta-lactamase,

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Livermore D. β-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995; 8: 557-584.
Schwarz S, Kehrenberg C, Walsh TR. Use of antimicrobial agents in veterinary medicine and food animal production. Int J Antimicrob Agents 2001; 17: 431-437.
Kang CI, Kim SH, Park WB, Lee KD, Kim HB, Kim EC, Oh MD, Choe KW. Bloodstream infections due to extended- spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae : risk factors for mortality and treatment outcome, with special emphasis on antimicrobial therapy. Antimicrob Agents Chemother 2004; 48: 4574-4581.
Talbot GH, Bradley J, Edwards JE, Gilbert D, Scheld M, Bartlett JG. Bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the Infectious Diseases Society of America. Clin Infect Dis 2006; 42: 657-668.
Kwak YK, Colque P, Byfors S, Giske CG, Mollby R, Kuhn I. Surveillance of antimicrobial resistance among Escherichia coli in wastewater in Stockholm during 1 year: does it reflect the resistance trends in the society? Int J Antimicrob Agents 2015; 45: 25-32.
Firsov AA, Strukova EN, Portnov YA, Shlykova DS, Zinner SH. Bacterial antibiotic resistance studies using in vitro dynamic models: population analysis vs. susceptibility testing as endpoints of mutant enrichment. Int J Antimicrob Agents 2015; 46: 313-318.
Sharma M, Pathak S, Srivastava P. Prevalence and antibiogram of extended spectrum β-lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. J Clin Diagn Res 2013; 7: 2173-2177.
Sleigh JD, Duguid JP. Enterobacteriaceae : Escherichia , Klebsiella , Proteus and other enterobacteria. In: Collee JG, Duguid JP, Fraser AG, Marmion BP, editors. Mackie and McCartney Practical Medical Microbiology. 13th ed. Edinburgh, UK: Churchill Livingstone; 1989. pp. 432-455.
Clinical and Laboratory Standards Institute. Methods for Detection of Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard. 9th ed. Wayne, PA, USA: CLSI; 2012.
Sharma J, Sharma M, Ray P. Detection of TEM and SHV genes in Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital from India. Indian J Med Res 2010; 132: 332-336.
Yasmin T. Prevalence of ESBL among Escherichia coli and Klebsiella spp. in a tertiary care hospital and molecular detection of important ESBL producing genes by multiplex PCR. MD, Dhaka University, Mymensingh, Bangladesh, 2012.
Mushi MF, Mshana SE, Imirzalioglu C, Bwanga F. Carbapenemase genes among multidrug resistant gram negative clinical isolates from a tertiary hospital in Mwanza, Tanzania. Biomed Res Int 2014; 2014: 303104.
Damborg P, Gaustad IB, Olsen JE, Guardabassi L. Selection of CMY-2 producing Escherichia coli in the faecal flora of dogs treated with cephalexin. Vet Microbiol 2011; 151: 404-408.
Dutil L, Irwin R, Finley R, Ng LK, Avery B, Boerlin, P, Bourgault AM, Cole L, Daignault D, Desruisseau A et al. Ceftiofur resistance in Salmonella enterica serovar Heidelberg from chicken meat and humans, Canada. Emerg Infect Dis 2010; 16: 48-54.
Madec JY, Lazizzera C, Chatre P, Meunier D, Martin S, Lepage G, Menard MF, Lebreton P, Rambaud T. Prevalence of fecal carriage of acquired expanded-spectrum cephalosporin resistance in Enterobacteriaceae strains from cattle in France. J Clin Microbiol 2008; 46: 1566-1567.
Geser N, Stephan R, Kuhnert P, Zbinden R, Kaeppeli U, Cernela N, Haechler H. Fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in swine and cattle at slaughter in Switzerland. J Food Prot 2011; 74: 446-449.
Reist M, Geser N, Hachler H, Scharrer S, Stephan R. ESBL- producing Enterobacteriaceae : occurrence, risk factors for fecal carriage and strain traits in the Swiss slaughter cattle population younger than 2 years sampled at abattoir level. PLoS One 2013; 8: e71725.
Hordijk J, Wagenaar JA, van de Giessen A, Dierikx C, van Essen-Zandbergen A, Veldman K, Kant A, Mevius D. Increasing prevalence and diversity of ESBL/AmpC-type β-lactamase genes in Escherichia coli isolated from veal calves from 1997 to 2010. J Antimicrob Chemother 2013; 68: 1970- 1973.
Dahms C, Hubner AO, Kossow A, Mellmann A, Dittmann K, Kramer A. Occurrence of ESBL-producing Escherichia coli in livestock and farm workers in Mecklenburg-western Pomerania, Germany. PLoS One 2015; 10: e0143326.
Olowe OA, Adewumi A, Odewale G, Ojurongbe O, Adefioye OJ. Phenotypic and molecular characterisation of extended- spectrum beta-lactamase producing Escherichia coli obtained from animal fecal samples in Ado Ekiti, Nigeria. J Env Pub Health 2015; 2015: 497980.
Mahanti A, Samanta I, Bandopaddhay S, Joardar SN, Dutta TK, Batabyal S, Sar TK, Isore DP. Isolation, molecular characterization and antibiotic resistance of shiga toxin- producing Escherichia coli (STEC) from buffalo in India. Lett Appl Microbiol 2013; 56: 291-298.
Mandakini R, Dutta TK, Chingtham S, Roychoudhury P, Samanta I, Joardar SN, Pachauau AR, Chandra R. ESBL- producing Shiga-toxigenic E. coli (STEC) associated with piglet diarrhoea in India. Trop Anim Health Prod 2015; 47: 377-381.
Cavaco LM, Abatih E, Aarestrup FM, Guardabassi L. Selection and persistence of CTX-M-producing Escherichia coli in the intestinal flora of pigs treated with amoxicillin, ceftiofur, or cefquinome. Antimicrob Agents Chemother 2008; 52: 3612- 3616.
Singh F, Hirpurkar SD, Shakya S, Rawat N, Devangan P, Khan FF, Bhandekar SK. Presence of enterobacteria producing extended-spectrum β-lactamases and/or carbapenemases in animals, humans and environment in India. Thai J Vet Med 2017; 47: 35-43.
Carattoli A. Resistance plasmid families in Enterobacteriaceae . Antimicrob Agents Chemother 2009; 53: 2227-2238.
Malkawi HI, Youssef MT. Antibiotic susceptibility testing and plasmid profiles of Escherichia coli isolated from diarrhoeal patients. J Trop Pediatr 1998; 44: 128-132.
Jacoby GA, Medeiros AA. More extended-spectrum beta- lactamases. Antimicrob Agents Chemother 1991; 35: 1697- 1704.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests; Twenty- fifth Informational Supplement: Approved Standard. 12th ed. Wayne, PA, USA: CLSI; 2015.
Mroczkowska JE, Barlow M. Fitness trade-offs in bla TEM evolution. Antimicrob Agents Chemother 2008; 52: 2340- 2345.
Endimiani A, Hujer AM, Perez F, Bethel CR, Hujer KM, Kroeger J, Oethinger M, Paterson DL, Adams MD, Jacobs MR et al. Characterization of bla KPC -containing Klebsiella pneumoniae isolates detected in different institutions in the eastern USA. J Antimicrob Chemother 2009; 63: 427-437.