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Research Article

volume 53 issue 7 (july 2019) : 938-943,   Doi: 10.18805/ijar.B-3599

Phenotypic and molecular characterization of extended spectrum b-lactamase, ampc b-lactamase and metallo b-lactamase producing Klebsiella spp. from farm animals in India

R
Rituparna Tewari
S
Susweta Mitra
N
Nimita Venugopal
S
Sangita Das
F
Feroze Ganaie
A
Arnab Sen
R
Rajeswari Shome
H
Habibur Rahman
B
Bibek Ranjan Shome
1ICAR-National Institute of Veterinary Epidemiology and Disease, Informatics, Bengaluru-560 064, Karnataka, India.
Cite article:- Tewari Rituparna, Mitra Susweta, Venugopal Nimita, Das Sangita, Ganaie Feroze, Sen Arnab, Shome Rajeswari, Rahman Habibur, Shome Ranjan Bibek (2018). Phenotypic and molecular characterization of extended spectrum b-lactamase, ampc b-lactamase and metallo b-lactamase producing Klebsiella spp. from farm animals in India. Indian Journal of Animal Research. 53(7): 938-943. doi: 10.18805/ijar.B-3599.

ABSTRACT

Animal populace has attained less attention in antimicrobial resistance research than human sector resulting in limited information available on animal origin isolates. The study aimed to investigate the occurrence of ESBL, AmpC and MBL genes, plasmids and integrons in Klebsiella spp. Fecal samples were collected from healthy livestock (cattle, pig, sheep and goat) and poultry between  2012-2015. Preliminary identification of isolates was done by conventional phenotypic methods and confirmed by genotypic methods. Antimicrobial susceptibility testing was performed by disk diffusion method. Molecular characterization by PCR was conducted for 17 antimicrobial resistance genes, 3 integrons and 18 Plasmid replicons. A total of 48 Klebsiella isolates were identified. Multidrug resistance was observed in 23% of isolates. ESBL, AmpC and MBL resistance genes were detected in 21%, 6% and 4% of isolates, respectively. Integrons [Int2 gene] and plasmids [Y and IncI genes] were identified in 6% and 8% of isolates.The study highlights the existence of ESBL, AmpC and MBL producing Klebsiella isolates with certain strains carrying mobile genetic elements  in healthy livestock and poultry as reservoirs and probable disseminators of resistance, thus imposing public health threat. Prudent use of antimicrobials and continuous intensified surveillance in animal sector is crucial to limit the spread of such emerging resistant traits.

REFERENCES

  1. Abraham, S., Trott, D.J., Jordan, D., Gordon, D.M., Groves, M.D., Fairbrother, J.M., Smith, M.G., Zhang, R., and Chapman, T.A. ( 2015). Phylogenetic and molecular insights into the evolution of multidrug-resistant porcine enterotoxigenic Escherichia coli in Australia. Int J Antimicrob Agents., 44: 105-11.
  2. Alyamani, E.J., Khiyami, A.M., Booq, R.Y., Majrashi, M.A., Ahwerth, F.S., and Rechkina, E. (2017). The occurrence of ESBL-    producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia. Ann Clin Microbiol Antimicrob., 16:1.
  3. Cao, X., Xu, X., Zhang, Z., Shen, H., Chen, J., and Zhang, K. (2014). Molecular characterization of clinical multidrug-resistant Klebsiella pneumonia isolates. Ann Clin Microbiol Antimicrob., 13:16.
  4. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L, and Threlfall, E.J. (2005). Identification of plasmids by PCR-based replicon typing. J Microbiol Methods., 63: 219-228.
  5. Carattoli, A. (2008). Animal reservoirs for extended spectrum â lactamase producers. Clin Microbiol Infect., 14: 117-123.
  6. Carattoli, A. (2009). Resistant Plasmid Families in Enterobacteriaceae. Antimicrob Agents Chemother., 53: 2227-38.
  7. Cavaco, L.M.E., Abatih, F.M., Aarestrup, L., and Guardabassi. (2008). Selection and persistence of CTX-M producing Eshcherichia coli in the intestinal flora of pigs treated with amoxicillin, ceftiofur, or cefquinome. Antimicrob Agents Chemother., 52: 3612-3616.
  8. CDDEP. (2015). Resistance Map Washington DC: Center for Disease Dynamics, Economics and Policy. http://www.resistancemap.org.
  9. Chander, Y., Ramakrishnan, M.A., Jindal, N., Hanson, K., and Sagar, M.G. (2011). Differentiation of Klebsiella pneumoniae and Klebsiella oxytoca by Multiplex Polymerase Chain Reaction. InterJ Appl Res Vet Med., 9: 138-148
  10. Clinical Laboratory Standard Institute. (2014). Performance Standards for antimicrobial disk susceptibility tests approved standard. 24th ed M100-S24, CLSI; Wayne P A. 
  11. Datta, S., Wattal, C., Goel, N., Oberoi, J.K., Raveendran, R., Prasad, K.J. (2012). A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital. Indian J Med Res., 135: 907–12. 
  12. Dorina, T., Elena, M.I., Nicola, J.W., Andrew, W., and Vanessa, S. (2016). Veterinary Hospital Dissemination of CTX-M-15 Extended-    Spectrum Beta-Lactamase–Producing Escherichia coli ST410 in the United Kingdom. Microb Drug Resist., 22: 609-615.
  13. Johnson, T.J., Wannemuehler, Y.M., Johnson, S.J., Logue, C.M., White, D.G., Doetkott, C., and Nolan, L.K. (2007). Plasmid replicon typing of commensal and pathogenic Eshcherichia coli isolates. Appl Environ Microbiol., 73: 1976-83.
  14. Kar, D., Bandyopadhyay, S., Bhattacharyya, D., Samanta, I., Mahanti, A., Nanda, P.K., Mondal, B., et al (2015). Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamse producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect Genet Evol., 29: 82-90.
  15. Katiyar, R., Deorukhkar, S.C, and Siddiqui, A.U. (2016). Bacteriological profile and antibiogram of uropathogens with special reference to extended spectrum beta lactamases (ESBLs) detection in Gram negative bacilli. Indian Journal of Basic and Applied Medical Research., 5: 290-299.
  16. Khari, M.I., Karunakaran, R., Rosli, R., Tay, T.S. (2016). Genotypic and Phenotypic Detection of AmpC â-lactamases in Enterobacter spp. Isolated from a Teaching Hospital in Malaysia. PLoS ONE., 11:e0150643.
  17. Kojima, A., Ishii, Y., Ishihara, K., Esaki, H., Asai, T., Oda, C., Tamura, Y., Takahashi, T., and Yamaguchi, K. (2005). Extended-spectrum-    beta-lactamsase-producing Eshcherichia coli strains isolated from farm animals from 1999 to 2002: report from the Japanese Vaterinary Antimicrobial Resitance Monitoring Program. Antimicrob Agents Chemother., 49: 3533-7.
  18. Mendes, R.E., Kiyota, K.A., Monteiro, J., Castanheira, M., Andrade, S.S., Gales, A.C., Pignatari, A., and Tufik, S. (2007). Rapid detection and Identification of metallo-â-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol., 45: 544-547.
  19. Perez-Perez, F.J., and Hanson, N.D. (2002). Detection of Plasmid-Mediated AmpC-â-Lactamase genes in Clinical Isolates by using Multiplex PCR. J Clin Microbiol., 40: 2153-2162.
  20. Pitout, J.D., Hossain, A., and Hanson, N.D. (2004). Phenotypic and molecular detection of CTX-M-â-Lactamases produced by Eshcheria coli and Klebsiella spp. J Clin Microbiol., 42: 5715-5721.
  21. Pitout, J.D. (2012). Extraintestinal pathogenic Escherichia coli: an update on antimicrobial resistance, laboratory diagnosis and treatment. Expert Rev Anti Infect Ther., 10: 1165-76.
  22. Ravi, S.G., Namratha, W.N., Krishna, B.V.S., and Chandrashekar, M.R. (2011). Comparison of Disc Diffusion Methods for the detection of extended-spectrum Beta lactamase producing enterobacteriaceae. J Lab Physicians., 3: 33-06.
  23. Sachdeva, R., Sharma, B., Sharma, R. (2017). Evaluation of different phenotypic tests for detection of metallo-â-lactamases in imipenem-    resistant Pseudomonas aeruginosa. J Lab Physicians., 9:249-53.
  24. Shah, M.D., Zahurul, H.A., Akhter, S., Rahman, M.M., Mohammad, N., and Hafez, M.A. (2014). ESBL positive organisms: method of routine reporting and prevalence in health care settings. Bangladesh J Med Microbiol., 8: 23-27.
  25. Sharma, C., Rokana, N., Chandra, M., Singh, B.P., Gulhane, R.D., Gill, J.P., Ray, P., Puniya, A.K., and Panwar, H. (2018). Antimicrobial Resistance: its surveillance, impact, and alternative management strategies in dairy animals. Front Vet Sci., 4:237.
  26. Timofte, D., Maciuca, I.E., Evans, N.J., Williams, H., Wattret, B.A, Fick, J.C., and Williams, N. (2014). Detection and molecular characterization of Eshcherichia coli CTX-M-15 and Klebsiella pneumonia SHV-12 â-lactamases from bovine mastitis isolates in the United Kingdom. Antimicrob Agents Chemother., 58: 789-94.
  27. Valentina, D., Fabiola, F.N., Rene, S.H., Christina, A.S., Gessica, C., Aurora, G.F., Serena, L., et al (2014). Extended-spectrum-Beta-    Lactamases, AmpC Beta-Lactamases and Plasmid Mediated Quinolone Resistance in Klebsiella spp. from Companion Animals in Italy. PLoS ONE 9: e90564. 
  28. Veena, K., Vijaykumar, G.S., Sudeepa, K.M., Prashanth, H.V., Prakash, R., and Nagaraj, E.R. (2013). Phenotypic & Genotypic methods for detection of ESBL producing E.coli & Klebsiella pneumoniae isolated from Ventilator associated pneumonia. J Clin Diagn Res., 7: 1975-1978.
  29. World Health Organization. (2010). Prevention and containment of antimicrobial resistance. Report of a Regional Meeting Chiang Mai, Thailand, 8th to 11th of June. http://www.searo.who.int/entity/ antimicrobial_resistance/BCT_Reports_SEA-HLM-    408.pdf?ua=1 
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Indian Journal of Animal Research
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    Research Article

    volume 53 issue 7 (july 2019) : 938-943,   Doi: 10.18805/ijar.B-3599

    Phenotypic and molecular characterization of extended spectrum b-lactamase, ampc b-lactamase and metallo b-lactamase producing Klebsiella spp. from farm animals in India

    R
    Rituparna Tewari
    S
    Susweta Mitra
    N
    Nimita Venugopal
    S
    Sangita Das
    F
    Feroze Ganaie
    A
    Arnab Sen
    R
    Rajeswari Shome
    H
    Habibur Rahman
    B
    Bibek Ranjan Shome
    1ICAR-National Institute of Veterinary Epidemiology and Disease, Informatics, Bengaluru-560 064, Karnataka, India.
    Cite article:- Tewari Rituparna, Mitra Susweta, Venugopal Nimita, Das Sangita, Ganaie Feroze, Sen Arnab, Shome Rajeswari, Rahman Habibur, Shome Ranjan Bibek (2018). Phenotypic and molecular characterization of extended spectrum b-lactamase, ampc b-lactamase and metallo b-lactamase producing Klebsiella spp. from farm animals in India. Indian Journal of Animal Research. 53(7): 938-943. doi: 10.18805/ijar.B-3599.

    ABSTRACT

    Animal populace has attained less attention in antimicrobial resistance research than human sector resulting in limited information available on animal origin isolates. The study aimed to investigate the occurrence of ESBL, AmpC and MBL genes, plasmids and integrons in Klebsiella spp. Fecal samples were collected from healthy livestock (cattle, pig, sheep and goat) and poultry between  2012-2015. Preliminary identification of isolates was done by conventional phenotypic methods and confirmed by genotypic methods. Antimicrobial susceptibility testing was performed by disk diffusion method. Molecular characterization by PCR was conducted for 17 antimicrobial resistance genes, 3 integrons and 18 Plasmid replicons. A total of 48 Klebsiella isolates were identified. Multidrug resistance was observed in 23% of isolates. ESBL, AmpC and MBL resistance genes were detected in 21%, 6% and 4% of isolates, respectively. Integrons [Int2 gene] and plasmids [Y and IncI genes] were identified in 6% and 8% of isolates.The study highlights the existence of ESBL, AmpC and MBL producing Klebsiella isolates with certain strains carrying mobile genetic elements  in healthy livestock and poultry as reservoirs and probable disseminators of resistance, thus imposing public health threat. Prudent use of antimicrobials and continuous intensified surveillance in animal sector is crucial to limit the spread of such emerging resistant traits.

    REFERENCES

    1. Abraham, S., Trott, D.J., Jordan, D., Gordon, D.M., Groves, M.D., Fairbrother, J.M., Smith, M.G., Zhang, R., and Chapman, T.A. ( 2015). Phylogenetic and molecular insights into the evolution of multidrug-resistant porcine enterotoxigenic Escherichia coli in Australia. Int J Antimicrob Agents., 44: 105-11.
    2. Alyamani, E.J., Khiyami, A.M., Booq, R.Y., Majrashi, M.A., Ahwerth, F.S., and Rechkina, E. (2017). The occurrence of ESBL-    producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia. Ann Clin Microbiol Antimicrob., 16:1.
    3. Cao, X., Xu, X., Zhang, Z., Shen, H., Chen, J., and Zhang, K. (2014). Molecular characterization of clinical multidrug-resistant Klebsiella pneumonia isolates. Ann Clin Microbiol Antimicrob., 13:16.
    4. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L, and Threlfall, E.J. (2005). Identification of plasmids by PCR-based replicon typing. J Microbiol Methods., 63: 219-228.
    5. Carattoli, A. (2008). Animal reservoirs for extended spectrum â lactamase producers. Clin Microbiol Infect., 14: 117-123.
    6. Carattoli, A. (2009). Resistant Plasmid Families in Enterobacteriaceae. Antimicrob Agents Chemother., 53: 2227-38.
    7. Cavaco, L.M.E., Abatih, F.M., Aarestrup, L., and Guardabassi. (2008). Selection and persistence of CTX-M producing Eshcherichia coli in the intestinal flora of pigs treated with amoxicillin, ceftiofur, or cefquinome. Antimicrob Agents Chemother., 52: 3612-3616.
    8. CDDEP. (2015). Resistance Map Washington DC: Center for Disease Dynamics, Economics and Policy. http://www.resistancemap.org.
    9. Chander, Y., Ramakrishnan, M.A., Jindal, N., Hanson, K., and Sagar, M.G. (2011). Differentiation of Klebsiella pneumoniae and Klebsiella oxytoca by Multiplex Polymerase Chain Reaction. InterJ Appl Res Vet Med., 9: 138-148
    10. Clinical Laboratory Standard Institute. (2014). Performance Standards for antimicrobial disk susceptibility tests approved standard. 24th ed M100-S24, CLSI; Wayne P A. 
    11. Datta, S., Wattal, C., Goel, N., Oberoi, J.K., Raveendran, R., Prasad, K.J. (2012). A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital. Indian J Med Res., 135: 907–12. 
    12. Dorina, T., Elena, M.I., Nicola, J.W., Andrew, W., and Vanessa, S. (2016). Veterinary Hospital Dissemination of CTX-M-15 Extended-    Spectrum Beta-Lactamase–Producing Escherichia coli ST410 in the United Kingdom. Microb Drug Resist., 22: 609-615.
    13. Johnson, T.J., Wannemuehler, Y.M., Johnson, S.J., Logue, C.M., White, D.G., Doetkott, C., and Nolan, L.K. (2007). Plasmid replicon typing of commensal and pathogenic Eshcherichia coli isolates. Appl Environ Microbiol., 73: 1976-83.
    14. Kar, D., Bandyopadhyay, S., Bhattacharyya, D., Samanta, I., Mahanti, A., Nanda, P.K., Mondal, B., et al (2015). Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamse producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect Genet Evol., 29: 82-90.
    15. Katiyar, R., Deorukhkar, S.C, and Siddiqui, A.U. (2016). Bacteriological profile and antibiogram of uropathogens with special reference to extended spectrum beta lactamases (ESBLs) detection in Gram negative bacilli. Indian Journal of Basic and Applied Medical Research., 5: 290-299.
    16. Khari, M.I., Karunakaran, R., Rosli, R., Tay, T.S. (2016). Genotypic and Phenotypic Detection of AmpC â-lactamases in Enterobacter spp. Isolated from a Teaching Hospital in Malaysia. PLoS ONE., 11:e0150643.
    17. Kojima, A., Ishii, Y., Ishihara, K., Esaki, H., Asai, T., Oda, C., Tamura, Y., Takahashi, T., and Yamaguchi, K. (2005). Extended-spectrum-    beta-lactamsase-producing Eshcherichia coli strains isolated from farm animals from 1999 to 2002: report from the Japanese Vaterinary Antimicrobial Resitance Monitoring Program. Antimicrob Agents Chemother., 49: 3533-7.
    18. Mendes, R.E., Kiyota, K.A., Monteiro, J., Castanheira, M., Andrade, S.S., Gales, A.C., Pignatari, A., and Tufik, S. (2007). Rapid detection and Identification of metallo-â-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol., 45: 544-547.
    19. Perez-Perez, F.J., and Hanson, N.D. (2002). Detection of Plasmid-Mediated AmpC-â-Lactamase genes in Clinical Isolates by using Multiplex PCR. J Clin Microbiol., 40: 2153-2162.
    20. Pitout, J.D., Hossain, A., and Hanson, N.D. (2004). Phenotypic and molecular detection of CTX-M-â-Lactamases produced by Eshcheria coli and Klebsiella spp. J Clin Microbiol., 42: 5715-5721.
    21. Pitout, J.D. (2012). Extraintestinal pathogenic Escherichia coli: an update on antimicrobial resistance, laboratory diagnosis and treatment. Expert Rev Anti Infect Ther., 10: 1165-76.
    22. Ravi, S.G., Namratha, W.N., Krishna, B.V.S., and Chandrashekar, M.R. (2011). Comparison of Disc Diffusion Methods for the detection of extended-spectrum Beta lactamase producing enterobacteriaceae. J Lab Physicians., 3: 33-06.
    23. Sachdeva, R., Sharma, B., Sharma, R. (2017). Evaluation of different phenotypic tests for detection of metallo-â-lactamases in imipenem-    resistant Pseudomonas aeruginosa. J Lab Physicians., 9:249-53.
    24. Shah, M.D., Zahurul, H.A., Akhter, S., Rahman, M.M., Mohammad, N., and Hafez, M.A. (2014). ESBL positive organisms: method of routine reporting and prevalence in health care settings. Bangladesh J Med Microbiol., 8: 23-27.
    25. Sharma, C., Rokana, N., Chandra, M., Singh, B.P., Gulhane, R.D., Gill, J.P., Ray, P., Puniya, A.K., and Panwar, H. (2018). Antimicrobial Resistance: its surveillance, impact, and alternative management strategies in dairy animals. Front Vet Sci., 4:237.
    26. Timofte, D., Maciuca, I.E., Evans, N.J., Williams, H., Wattret, B.A, Fick, J.C., and Williams, N. (2014). Detection and molecular characterization of Eshcherichia coli CTX-M-15 and Klebsiella pneumonia SHV-12 â-lactamases from bovine mastitis isolates in the United Kingdom. Antimicrob Agents Chemother., 58: 789-94.
    27. Valentina, D., Fabiola, F.N., Rene, S.H., Christina, A.S., Gessica, C., Aurora, G.F., Serena, L., et al (2014). Extended-spectrum-Beta-    Lactamases, AmpC Beta-Lactamases and Plasmid Mediated Quinolone Resistance in Klebsiella spp. from Companion Animals in Italy. PLoS ONE 9: e90564. 
    28. Veena, K., Vijaykumar, G.S., Sudeepa, K.M., Prashanth, H.V., Prakash, R., and Nagaraj, E.R. (2013). Phenotypic & Genotypic methods for detection of ESBL producing E.coli & Klebsiella pneumoniae isolated from Ventilator associated pneumonia. J Clin Diagn Res., 7: 1975-1978.
    29. World Health Organization. (2010). Prevention and containment of antimicrobial resistance. Report of a Regional Meeting Chiang Mai, Thailand, 8th to 11th of June. http://www.searo.who.int/entity/ antimicrobial_resistance/BCT_Reports_SEA-HLM-    408.pdf?ua=1 
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