Genotypic characterization of multidrug resistant Escherichia coli isolates reveals co-existence of ESBL- and carbapenemase- encoding genes linked to ISCR1


ESBL-producing E. coli
Peshawar Pakistan
Poultry meat

How to Cite

ur Rahman, S., Muhammad, N., Ali, T., Saddique, U., Ahmad, S., Shafiq, M., & Han, B. (2021). Genotypic characterization of multidrug resistant Escherichia coli isolates reveals co-existence of ESBL- and carbapenemase- encoding genes linked to ISCR1. Veterinaria Italiana, 57(4), 275–285. https://doi.org/10.12834/VetIt.1780.9397.4


Antimicrobial resistance in food-producing animals has not yet judiciously been reported from Pakistan. Here, we report on the isolation rate of poultry-associated multidrug resistant extended spectrum β-lactamase (ESBL) -producing Escherichia coli in Peshawar, Pakistan. A total of 200 samples, 50 from retail-poultry meat, 50 from sick birds, 50 from the boiler farm-environment, and 50 from human beings working on or exposed to poultry were analyzed for isolation of ESBL -producing E. coli, ESBL -encoding genes and antimicrobial susceptibility. A total of 81 E. coli isolates [(50.0% Phylogroup-A, 33.3% D and 16.7% phylogroup B2)], were recovered, 36 (44.4%) of them were found to be ESBL -producers. PCR revealed that blaCTXM was the most prevalent (14/36 = 38.9%) ESBL -encoding gene followed by blaSHV2 (9/36 = 25%). Strikingly, co-occurrence of multiple ESBL - and/or carbapenemase-encoding genes in a single isolate was observed, and combination of blaCTXM + blaSHV2 was the most predominant (19.4%) followed by blaCTXM + blaNDM1 + blaOXA-48 (11.1%) and blaCTXM + blaOXA-48 (8.8%). All these ESBL producers were found to be multidrug resistant (MDR) and were carrying either integron 1 (48.5%) or 2 (51.5%). Finally, 14 of the 36 isolates were also found positive for variable region and insertion sequence common region 1, which was found linked to ESBL/carbapenemase encoding genes in 5/14 isolates suggesting its role in dissemination.


Abrar S., Vajeeha A., Ul-Ain N. & Riaz S. 2017. Distribution of CTX-M group I and group III β-lactamases produced by Escherichia coli and klebsiella pneumoniae in Lahore, Pakistan. Microb Pathog, 103, 8-12. doi.org/10.1016/j.micpath.2016.12.004.

Ahmad K., Khattak F., Ali A., Rahat S., Noor S., Mahsood N. & Somayya R. 2018. Carbapenemases and Extended-Spectrum β-Lactamase–Producing Multidrug-Resistant Escherichia coli Isolated from Retail Chicken in Peshawar: First Report from Pakistan. J Food Prot, 81, 1339-1345

Ahmed H.A., El-Hofy F.I., Shafik S.M., Abdelrahman M.A. & Elsaid G.A. 2016. Characterization of Virulence-Associated Genes, Antimicrobial Resistance Genes, and Class 1 Integrons in Salmonella enterica serovar Typhimurium Isolates from Chicken Meat and Humans in Egypt. Foodborne Pathog Dis, 13(6),281-288. doi: 10.1089/fpd.2015.2097.

Ali T., Zhang L., Shahid M., Zhang S., Liu G., Gao J. & Han B. 2016. ESBL-Producing Escherichia coli from Cows Suffering Mastitis in China Contain Clinical Class 1 Integrons with CTX-M Linked to ISCR1. Front Microbiol, 7, 1931. doi: 10.3389/fmicb.2016.01931

Ali T., Ur Rahman S., Zhang L., Shahid M., Han D., Gao J., Zhang S., Ruegg P.L., Saddique U. & Han B. 2017. Characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from bovine mastitis. Oncotarget, 8(52), 90144-90163. doi: 10.18632/oncotarget.21496.

Blaak H., van Hoek A. H., Hamidjaja R. A., van der Plaats R. Q., Kerkhof-de Heer L., de Roda Husman A. M. & Schets F. M. 2015. Distribution, Numbers, and Diversity of ESBL-Producing E. coli in the Poultry Farm Environment. PloS one, 10(8), e0135402.doi.org/10.1371/journal.pone.0135402

Brigante G., Luzzaro F., Perilli M., Lombardi G., Colì A., Rossolini G.M., Amicosante G. & Toniolo A. 2005. Evolution of CTX-M-type β-lactamases in isolates of Escherichia coli infecting hospital and community patients. Int J Antimicrob Agents, 25, 157-162.

Chong Y., Shimoda S. & Shimono N. 2018. Current epidemiology, genetic evolution and clinical impact of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Infect Genet Evol, 61, 185-188.

Clermont O., Bonacorsi S. & Bingen E. 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol, 66(10), 4555–4558. doi.org/10.1128/AEM.66.10.4555-4558.2000.

Clinical and Laboratory Standards Institute (CLSI). (2014). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI Document M100-S24, Wayne, 34.

D'Andrea M.M., Arena F., Pallecchi L. & Rossolini G.M. 2013. Ctx-m-type beta-lactamases: A successful story of antibiotic resistance. Int J Med Microbiol, 303(6-7), 305-317. DOI 10.1016/j.ijmm.2013.02.008.

Dillon B., Thomas L., Mohmand G., Zelynski A. & Iredell J. 2005. Multiplex PCRr for screening of integrons in bacterial lysates. J Microbiol Methods, 62(2), 221-232. DOI 10.1016/j.mimet.2005.02.007.

Doi Y., Paterson D.L., Egea P., Pascual A., López-Cerero L., Navarro M.D., Adams-Haduch J.M., Qureshi Z.A., Sidjabat H. E. & Rodríguez-Baño J. 2010. Extended-spectrum and CMY-type beta-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain. Clin Microbiol Infect, 16(1), 33-38. doi:10.1111/j.1469-0691.2009.03001.x

Egea P., López-Cerero L., Torres E., Gómez-Sánchez M.d.C., Serrano L., Navarro Sánchez-Ortiz M.D., Rodriguez-Baño J. & Pascual A. 2012. Increased raw poultry meat colonization by extended spectrum beta-lactamase-producing Escherichia coli in the South of Spain. Int J Food Microbiol, 159(2), 69-73. https://doi.org/10.1016/j.ijfoodmicro.2012.08.002.

El Salabi A., Walsh T.R. & Chouchani C. 2013. Extended spectrum beta-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in gram-negative bacteria. Critical Reviews Microbiol, 39(2), 113-122. DOI 10.3109/1040841x.2012.691870.

Girlich D., Poirel L., Carattoli A., Kempf I., Lartigue M.F., Bertini A. & Nordmann P. 2007. Extended-spectrum beta-lactamase ctx-m-1 in Escherichia coli isolates from healthy poultry in France. Appl Environ Microbiol, 73(14), 4681-4685. DOI 10.1128/aem.02491-06.

Giske C.G., Sundsfjord A.S., Kahlmeter G., Woodford N., Nordmann P., Paterson D.L., Canton R. & Walsh T.R. 2008. Redefining extended-spectrum β-lactamases: Balancing science and clinical need. J Antimicrob Chemother, 63(1), 1-4.

Gu B., Pan S., Wang T., Zhao W., Mei Y., Huang P. & Tong M. 2008. Novel cassette arrays of integrons in clinical strains of enterobacteriaceae in China. Int J Antimicrob Agents, 32(6), 529-533. DOI 10.1016/j.ijantimicag.2008.06.019.

Habeeb M.A., Sarwar Y., Ali A., Salman M. & Haque A. 2013. Rapid emergence of ESBL producers in E. Coli causing urinary and wound infections in Pakistan. PakJ Med Sci, 29(2), 540.

Hasan B., Sandegren L., Melhus Å., Drobni M., Hernandez J., Waldenström J., Alam M. & Olsen B. 2012. Antimicrobial drug–resistant Escherichia coli in wild birds and free-range poultry, Bangladesh. Emerg Infect Dis, 18(12), 2055-2058. DOI 10.3201/eid1812.120513.

Huang L., Yao L., He Z., Zhou C., Li G., Yang B. & Deng X. 2014. Roxarsone and its metabolites in chicken manure significantly enhance the uptake of as species by vegetables. Chemosphere, 100, 57-62. DOI 10.1016/j.chemosphere.2013.12.074.

Johnson A. P. & Woodford N. 1998. Plasmid analysis. In Molecular Bacteriology: protocols and clinical applications (Woodford N. & Johnson A.P., Eds).Humana Press Inc., Totowa, NJ, 51–62.

Kar D., Bandyopadhyay S., Bhattacharyya D., Samanta I., Mahanti A., Nanda P.K., Mondal B., Dandapat P., Das A.K., Dutta T.K., Bandyopadhyay S. & Singh R.K. 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. DOI 10.1016/j.meegid.2014.11.003.

Khan E., Schneiders T., Zafar A., Aziz E., Parekh A. & Hasan R. 2010. Emergence of CTX-M Group 1-ESBL producing Klebsiella pneumonia from a tertiary care centre in Karachi, Pakistan. J Infect Dev Ctries, 4(8), 472-476. doi:10.3855/jidc.674

Kiiru J., Butaye P., Goddeeris B.M. & Kariuki S. 2013. Analysis for prevalence and physical linkages amongst integrons, ISEcp1, ISCR1, Tn21 and Tn7 encountered in Escherichia coli strains from hospitalized and non-hospitalized patients in Kenya during a 19-year period (1992-2011). BMC Microbiol,13, 109. doi:10.1186/1471-2180-13-109

Kumar A., Ellis P., Arabi Y., Roberts D., Light B., Parrillo J. E., Dodek P., Wood G., Kumar A., Simon D., Peters C., Ahsan M., Chateau D. & Cooperative Antimicrobial Therapy of Septic Shock Database Research Group 2009. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest, 136(5), 1237–1248. https://doi.org/10.1378/chest.09-0087

Lolans K., Queenan A.M., Bush K., Sahud A. & Quinn J.P. 2005. First nosocomial outbreak of Pseudomonas aeruginosa producing an integron-borne metallo-beta-lactamase (VIM-2) in the United States. Antimicrob Agents Chemother, 49(8), 3538-3540. doi:10.1128/AAC.49.8.3538-3540.2005

Magiorakos A.P., Srinivasan A., Carey R.B. Carmeli Y., Falagas M. E., Giske C. G., Harbarth S., Hindler J. F., Kahlmeter G., Olsson-Liljequist B., Paterson D. L., Rice L. B., Stelling J., Struelens M. J., Vatopoulos A., Weber J. T. & Monnet D. L. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect, 18(3), 268-281. doi:10.1111/j.1469-0691.2011.03570.x

Mirza S.H., Salman M., Khurshid U. & Wiqar M.A. 2006. CTX-M ESBL enzyme in Escherichia coli from urology patients in Rawalpindi, Pakistan. J Pak Med Assoc, 56(12), 576-578.

Mitema E.S. 2010. The role of unregulated sale and dispensing of antimicrobial agents on the development of antimicrobial resistance in developing countries. In Antimicrobial resistance in developing countries. (Sosa A., Byarugaba D.K., Amabile-Cuevas C., Hsueh, P.-R., Kariuk, S. & Okeke I.N., Eds). Springer, New York, 403-411.

Naas T. & Nordmann P. 1994. Analysis of a carbapenem-hydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein. Proc Natl Acad Sci U S A, 91(16), 7693-7697. doi:10.1073/pnas.91.16.7693.

Naeem, M., Khan K. & Rafiq S. 2006. Determination of residues of quinolones in poultry products by high pressure liquid chromatography. J Appl Sci, 6(2), 373-379.

Nordmann P. & Poirel L. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect, 8(6), 321-331. doi:10.1046/j.1469-0691.2002.00401.x

Nordmann P., Naas T. & Poirel L. 2011. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis, 17(10), 1791-1798. doi:10.3201/eid1710.110655.

Pakistan Government. 2016. Economic survey of Pakistan 2016-2017 (Economic advisor’s wing, Finance division, Islamabad, Pakistan). https://www.finance.gov.pk/survey/chapters_17/Pakistan_ES_2016_17_pdf.pdf

Piet J.R., van Ulsen P., Ur Rahman S., Bovenkerk S., Bentley S.D., van de Beek D. & van der Ende A. 2016. Meningococcal Two-Partner Secretion Systems and Their Association with Outcome in Patients with Meningitis. Infect Immun, 19, 2534-2540

Queenan A.M. & K. Bush 2007. Carbapenemases: The versatile β-lactamases. Clin Microbiol Rev, 20(3), 440-458. DOI 10.1128/cmr.00001-07.

Rahman H., Naeem M., Khan I., Khan J., Haroon M., Bari F., Ullah R. & Qasim M. 2016. Molecular prevalence and antibiotics resistance pattern of class A bla CTX-M-1 and bla TEM-1 beta lactamases in uropathogenic Escherichia coli isolates from Pakistan. Turk J Med Sci, 46(3), 897-902. doi:10.3906/sag-1502-14.

Sartor A.L., Raza M.W., Abbasi S.A., Day K.M., Perry J.D., Paterson D.L. & Sidjabat H.E. 2014. Molecular epidemiology of NDM-1 producing Enterobacteriaceae and Acinetobacter baumannii isolates from Pakistan. Antimicrob Agents Chemother, 58(9), 5589-5593. doi:10.1128/AAC.02425-14

Seiffert S.N., Hilty M., Perreten V. & Endimiani A. 2013. Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health?. Drug Resist Updat, 16(1-2), 22-45. doi:10.1016/j.drup.2012.12.001

Shahid M.A., Siddique M., Abubakar M., Arshed M.J., Asif M. & Ahmad A. 2007. Status of oxytetracycline residues in chicken meat in rawalpindi/islamabad area of pakistan. Asian J Poultry Sci, 1, 8-15.

Tantawiwat S., Tansuphasiri U., Wongwit W., Wongchotigul V. & Kitayaporn D. 2005. Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water. Southeast Asian J Trop Med Public Health, 36(1), 162-169.

Ullah F., Malik S.A. & Ahmed J. 2009. Antimicrobial susceptibility pattern and esbl prevalence in klebsiella pneumoniae from urinary tract infections in the north-west of Pakistan. African J Microbiol Res, 3(11), 676-680.

Ullah W., Qasim M., Rahman H., Khan S., Rehman Z. U., Ali N. & Muhammad N. 2017. CTX-M-15 and OXA-10 beta lactamases in multidrug resistant Pseudomonas aeruginosa: First report from Pakistan. Microbial Pathog, 105, 240–244. https://doi.org/10.1016/j.micpath.2017.02.039.

Upadhyay S., Hussain A. , Mishra S., Maurya A.P., Bhattacharjee A. & Joshi S.R. 2015. Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India. PLoS One, 10(9), e0138056. DOI 10.1371/journal.pone.0138056.

Ur Rahman S., Ahmad S., Khan I. & Pakistan P. 2018a. Incidence of ESBL-Producing-Escherichia coli in Poultry Farm Environment and Retail Poultry Meat. Pakistan Vet J, 39(1), 116-120

Ur Rahman S., Ali T., Ali I., Khan N.A., Han B. & Gao J. 2018. The Growing Genetic and Functional Diversity of Extended Spectrum Beta-Lactamases. Biomed Res Int, 9519718. doi:10.1155/2018/9519718

White P.A., McIver C.J., Deng Y. & Rawlinson W.D. 2000. Characterisation of two new gene cassettes, aadA5 and dfrA17. FEMS Microbiol Lett, 182(2), 265-269. doi:10.1111/j.1574-6968.2000.tb08906.x

Xu G., An W., Wang H. & Zhang X. 2015. Prevalence and characteristics of extended-spectrum β-lactamase genes in Escherichia coli isolated from piglets with post-weaning diarrhea in Heilongjiang province, China. Front Microbiol, 6, 1103. doi:10.3389/fmicb.2015.01103.

Younas M., ur Rahman S., Shams S., Salman M.M. & Khan I. 2019. Multidrug Resistant Carbapenemase-Producing Escherichia coli from Chicken Meat Reveals Diversity and Co-Existence of Carbapenemase Encoding Genes. Pakistan Vet J, 39(2), 241-245. DOI: 10.29261/pakvetj/2018.091

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