275 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Accepted: 23.01.2020 | Available on line: 31.12.2021 1College of Veterinary Sciences and Animal Husbandry, Section Microbiology, Abdul Wali Khan University, Mardan-Khyber Pakhtunkhwa Pakistan. 2Department of Animal Health, The University of Agriculture, Peshawar-Khyber Pakhtunkhwa Pakistan. 3Center of Microbiology and biotechnology, Veterinary Research Institute, Peshawar-Khyber Pakhtunkhwa Pakistan. 4College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, PR China. 5Department of Clinical Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, P.R. China. *Corresponding author at: College of Veterinary Sciences and Animal Husbandry, Section Microbiology, Abdul Wali Khan University, Mardan-Khyber Pakhtunkhwa Pakistan. E-mail: sadeeq@awkum.edu.pk. Sadeeqe ur Rahman1*, Nazeer Muhammad2, Tariq Ali3, Umer Saddique2, Shakoor Ahmad2, Muhammad Shafiq4 and Bo Han5 Keywords ESBL-producing E. coli, NDM-1, Genotypes, Peshawar Pakistan, Poultry, OXA-48, CTXM, Poultry meat. Summary 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 bla CTXM was the most prevalent (14/36 = 38.9%) ESBL-encoding gene followed by bla SHV2 (9/36  =  25%). Strikingly, co-occurrence of multiple ESBL- and/or carbapenemase-encoding genes in a single isolate was observed, and combination of bla CTXM + bla SHV2 was the most predominant (19.4%) followed by bla CTXM + bla NDM1 + bla OXA-48 (11.1%) and bla CTXM + bla OXA-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. Genotypic characterization of multidrug resistant Escherichia coli isolates reveals co-existence of ESBL- and carbapenemase- encoding genes linked to ISCR1 2013, ur Rahman et al. 2018b). Carbapenemases on the other hands are enzymes that can inactivate carbapenem drugs-the most effective and last resort of antibiotics. ESBLs and carbapenemases are predominantly produced in Enterobacteriaceae, mainly in Escherichia coli that is utilized as crucial resistance mechanism against cephalosporins and carbapenems (Nordmann et  al. 2011, ur  Rahman et al. 2018b). Furthermore, generally, E. coli encoding for ESBL and carabepenemases are resistant to more Introduction Antimicrobial resistance (AMR) is a global emerging threat. Particularly, resistance to β-lactams and carbapenems is quite alarming as these drugs are proven safe and efficient, but are losing its effect due to emerging AMR. Bacteria achieve resistance to β-lactams through production of extended spectrum β-lactamases (ESBL) that inactivate antimicrobials including third-, fourth-generation cephalosporins and monobactams (El Salabi et  al. 276 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Genotypic characterization of ESBL-producing E. coli in Pakistan ur Rahman et al. cephalosporinases- the Ambler class C have also been described with slight expanded-spectrum activity against carbapenems (Giske et al. 2008). Infections due to β-lactamases-producing Enterobacteriaceae have serious implications both for public health and infection control strategies. Such kind of infections are difficult to treat increasing the risk of infection-related mortality by 5 fold (Kumar et al. 2009). Finally, the overwhelming ability of the β-lactamases-producing Enterobacteriaceae to persist and speedily disseminate with the help of mobile elements in different clinical- and community-settings, and their frequent association with MDR phenotypes presents a huge challenge (El Salabi et  al. 2013, Ali et  al. 2016, Nordmann and Poirel 2002). MDR Escherichia coli carrying ESBL and carbapenemases is increasingly disseminating all over the world. The situation is more alarming in the developing countries partly due to excessive use of antibiotics and absence of surveillance reports. The persistent and excessive use of antibiotics for treatment and as growth promoter in poultry industry in Pakistan is more likely providing impetus for the emergence of novel mechanisms of resistance including resistance to β-lactams in Enterobacteriaceae. Thus, this study was designed with the objectives to determine the occurrence of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing E.  coli in broiler and poultry farm environment in Peshawar-Pakistan and characterize these isolates for the types of ESBL genes and susceptibility common antimicrobials. Materials and methods Ethics Proper and prior ethical approval was obtained from the supervisory and technical committee of the University of Agriculture Peshawar and Abdul Wali Khan University Mardan. All work described in here is carried out according to the local institutional and national guidelines and legislations for human and animal samples for research purpose. Prior consent was obtained from owners of the poultry farm/bird or human beings along with consent for publication of the resultant data. Study area and samples Peshawar is the capital city of Khyber Pakhtunkhwa situated in the northwest of Pakistan with 34.0150° N, 71.5805° E coordinates. Samples for study were collected in September 2016 to April 2017. A total of 200 samples including 50 samples from healthy chickens, 50 from apparently looking sick chicken, than one classes of antimicrobials and hence are multidrug resistant (MDR), and thus presenting a serious challenge in healthcare settings. In addition to implications of antimicrobials for prophylaxis and as growth promoters, recurrent bacterial infections encourages excessive and constant usage of these antimicrobials thereby resulting emergence and development of resistance against these compounds (ur Rahman et al. 2018b). Emergence of resistance against carbapenem and cephalosporins is particularly more worrisome as limited options of treatment are left for patients infected with these resistant microbes. Three major categories, bla CTXM , bla SHV and bla TEM , of ESBL-encoding genes have been described well so far with bla CTXM as the most prevailing genotype, which has been further divided into five subgroups (bla CTXM-1 , bla CTXM-2 , bla CTXM-8 , bla CTXM-9 , bla CTXM-25 ). The families of each three categories have been expanded so much that almost more than 172 variants have been described for each type of ESBL (http://www.lahey.org/studies) (Chong et  al. 2018). With obvious geographical variations among the ESBL subtypes, bla CTXM-15 genotype has been reported frequently from Asia (D'Andrea et  al. 2013), and has even frequently been isolated from food-producing-animals (Ali et  al. 2016, Ali et  al. 2017) as well as from community (Abrar et  al. 2017, Brigante et  al. 2005). E.  coli carrying bla CTXM-15 often carry many other antibiotic resistance genes and often causing community acquired infections (Chong et  al. 2018). It has been shown that ESBL carrying E.  coli spread through contaminated food chain or water thereby resulting spread of resistance elements harboring by these bacteria through the help of mobile elements on conjugative plasmids such as integron and insertion sequence common region 1 (ISCR1) (Ahmed et  al. 2016, Ali et  al. 2016, Huang et al. 2014). Carbapenemases are highly versatile family of β-lactamases recognizing almost all hydrolysable β-lactams, and are resilient against almost all available commercial β-lactamase inhibitors (Lolans et  al. 2005, Nordmann and Poirel 2002). Carbapenemase-encoding genes are not naturally found in E.  coli, however, these genes have been acquired largely from other related or environmental bacteria when found associated with mobile genetic elements such as conjugative plasmids (Queenan and Bush 2007). The first carbapenemase producing E.  coli was reported in 1993 encoded by NmcA (Naas and Nordmann 1994). Since then and particularly over the last decade, increasing number of carbapenemase encoding genes have been described in Enterobacteriaceae, enlisting them into 3 classes of β-lactamases such as the Ambler class A, B, and D β-lactamases (Queenan and Bush 2007). Moreover, rare chromosomally-encoded 277Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 ur Rahman et al. Genotypic characterization of ESBL-producing E. coli in Pakistan for E.  coli growth, (ii) MacConkey agar media plate with having cefotaxime (2 µg/mL) which was used as selective media for ESBL-producing E. coli growth and (iii) MacConkey agar media plate with having meropenem (0.5 µg/mL) which was selective for growth of E.  coli producing carbapenemase. The plates were incubated overnight at 37 °C after inoculation. Pink color colonies that grew on (ii) and (iii), and presented morphology related with E.  coli were candidates for ESBL-producing and carbapenemase-producing phenotypes and further biochemical tests were performed for E. coli identification. E.  coli species were confirmed by using API kits (bioMérieux, Marcy I’Etoile, France). Biochemically confirmed E.  coli isolates were also genotyped by specific PCR as described earlier (Tantawiwat et  al. 2005). Confirmed E.  coli isolates and suspected ESBL producers were stored in BHI broth containing 30% glycerol at - 80 °C. Presumptive ESBL-producing E. coli were confirmed phenotypically for ESBL production by double-disc synergy test in accordance with recommendations of the Clinical and Laboratory Standards Institute (CLSI 2014), using antimicrobial discs of cefotaxime (30 μg), cefotaxime plus clavulanic acid (30/10 μg), ceftazidime (30 μg), ceftazidime plus clavulanic acid (30/10 μg) (Becton Dickson, Sparks, MD USA) for ESBL production. The test was declared positive for ESBL production when the zone of inhibition of cefotaxime plus clavulanic acid or ceftazidime plus clavulanic acid was ≥ 5 mm larger than their respective single discs (CLSI 2014). For positive control, Klebsiella pneumoniae ATCC 700603 (ESBLs-positive strain) was used. ESBL and carbapenemase encoding genes identification Identification of ESBL and carbapenemase encoding genes was achieved by regular PCR. Total DNA was extracted by using standard boiling method (Johnson and Woodford 1998) PCR was first performed for bla CTX-M , bla SHV , and bla TEM of ESBL-producing isolates (Table I). Variants, bla CTX-M-1 and bla CTX-M-9 , of bla CTX-M were also determined through PCR using specific primers as described in Table I. All ESBL producers were also subjected to PCR for carbapenemase-encoding genes like bla OXA , and bla NDM . The primers, amplicons expected size and references are described in Table  I. The polymerase chain reaction was performed as described earlier (Ali et  al. 2016, Sartor et  al. 2014). Amplified PCR products were evaluated by 1% agarose gel electrophoresis. Antibiotic susceptibility testing Mueller-Hinton agar (DifcoTM) was used to perform 50 from poultry rearing environment within poultry farms and finally 50 samples from human beings dealing with poultry in the farms or purchase points were collected from Peshawar. Samples from sick broiler chickens were obtained from Poultry postmortem section of Veterinary Research Institute, Peshawar, where farmers from various cities including nearby peri-urban areas of Peshawar city bring sick birds for disease diagnosis or/and treatment. Samples from healthy broiler chicken were obtained from live bird market at Peshawar city at sales point, while environmental samples were obtained from open shed poultry farms of Peshawar district that provides broiler to these sales points. These samples included drinking water (n  =  20), swabs from floor (n  =  10), utensils (n  =  10) and humid walls (n  =  10). Fecal swab samples or swab samples from hands, nose and feet or blood samples of human beings (n = 50) dealing with these broiler at different spots such as butchers, live bird sellers, farmers and truck drivers who transport birds from farm to market were included in this study. Culturing, identification of Escherichia coli, screening and confirmation of ESBL production Meat samples of healthy broiler chicken at retail market and sick broiler chicken from the postmortem room, were sealed in a sterile plastic bag and transported in icebox to Microbiology Laboratory of University of Agriculture or College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University and placed at 2-8 °C for maximum of 10 hr until further processed. Cotton swab samples from the environment of the broiler farms or humans were transported in transport medium in icebox and were processed immediately for culturing and isolation. Human fecal swab samples in the transport medium or a total of 10 ml blood were aseptically obtained from people. Meat samples of broiler were processed for isolation of E. coli (Doy et al. 2010, Paterson et al. 2010). Briefly, approximately 25 mg of meat was used from each sample for culturing after mechanically mincing in 10 ml of Brain heart infusion (BHI; Sigma-Aldrich) media followed by enrichment at 37 °C overnight (Doy et  al. 2010, Paterson et  al. 2010). On the next day, 50-100 µL of this broth was streaked onto MacConkey agar for selective growth. In the case of swab samples a sterile cotton swab was streaked directly onto MacConkey agar. Screening and confirmation of ESBL was performed as defined by Clinical and Laboratory Standards Institute (CLSI) (CLSI 2014). The media plates used for each type of sample included (i) MacConkey agar plate with no antibiotics which was control 278 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Genotypic characterization of ESBL-producing E. coli in Pakistan ur Rahman et al. (XDR) if found resistant to all commonly used drugs, multi-drug resistant (MDR)-if found resistant against more than two different kinds of antibiotics tested (Magiorakos et al. 2012). Determination of phylogroups of E. coli Phylogenetic analyses was performed on ESBL- producing-E.  coli isolates to place them into one of the four phylogenetic groups: phylogeneticgroup A, group B1, group B2 and group D. For this purpose, a triplex PCR assay targeted chuA, yjaA genes and the DNA fragment TspE4 was used as described previously by Clermont and colleagues (Clermont et al. 2000). antibiotic susceptibility of ESBL carrying isolates. A total of 9 different types of antibiotic discs (Becton Dickison, Sparks, MD USA) following standard Kirby-Bauer disk diffusion method according to recommendations of the CLSI (CLSI 2014) were tested. Both, β-lactam and non-β-lactam antibiotics, were tested such as ampicillin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), aztreonam (30 μg) and meropenem (10 μg), and tetracycline (30 μg), gentamicin (10 μg), tetracycline (20 μg), meropenem (10 μg) and norfloxacin (10 μg), respectively. E.  coli ATCC 25922 (ESBL-negative) and Klebsiella pneumoniae ATCC 700603 (ESBL-positive) were used as quality control strains (CLSI. 2014). The ESBL E. coli were marked declared as extensively-drug resistant Table I. Primers used in this study. Primer name Target gene Sequence (5’-3’) Size -bp Ref β -lactamases CTX-M –F bla CTXM ATGGTTAAAAAATCACTGCG 873 Paauw et al. 2006 CTX-M –R AAACCGTTGGTGACGAT CTX-M9-F bla CTXM TGGTGACAAAGAGAGTGCAACG 875 Paauw et al. 2006 CTX-M9-R TCACAGCCCTTCGGCGAT SHV –F bla SHV GGG TTA TTC TTA TTT GTC GC 567 Chang et al. 2001, Yao et al. 2007SHV -R TTAGCGTTGCCAAGTGCTC CTXM1-F1 bla CTXM-1 GCT GTT GTT AGG AAG TGT GC 490 Shibata et al. 2006 CTXM1-R CCA TTG CCC GAG GTG AAG TEM-F Bla TEM-1, -52, -71, -104 -105, -138, -151, -152 ATA AAA TTC TTG AAG ACG AAA 1086 Yao et al. 2007 TEM-R GAC AGT TAC CAA TGC TTA ATC NDM-F Bla NDM1 AGCTGAGCACCGCATTAG 720 Poirel et al. 2011 NDM-R CGGAATGGCTCATCACGATC OXA-F Bla OXA-48 GCGTGGTTAAGGATGAACAC 438 Poirel et al. 2011 OXA -R GTTGTCATCCTTGTTAGGCG Integrons and integron variable region intI1-F intI1 CCT CCC GCA CGA TGA TC 280-bp Dillon et al. 2005 intI1-R TCC ACG CAT CGT CAG GC intI2-F intI2 AAA TCT TTA ACC CGC AAA CGC 439-bp Dillon et al. 2005 intI2-R ATG TCT AAC AGT CCA TTT TTA AAT TCT A intI3-F intI3 AGT GGG TGG CGA ATG AGT G 599-bp Dillon et al. 2005 intI3-R TGT TCT TGT ATC GGC AGG TG intI1-VR-F intI1 variable region TCA TGG CTT GTT ATG ACT GT variable White et al. 2000 ntI1-VR-R GTA GGG CTT ATT ATG CAC GC Specific to E. coli UAL uidA TGG TAA TTA CCG ACG AAA ACG GC 147-bp Tantawiwat et al. 2005 UAR ACG CGT GGT TAC AGT CTT GCG E. coli phylogrouping ChuA-F ChuA GAC GAA CCA ACG GTC AGG AT 279-bp Clermont et al. 2000 ChuA-R TGC CGC CAG TAC CAA AGA CA YjaA-F YjaA TGA AGT GTC AGG AGA CGC TG 211-bp Clermont et al. 2000 YjaA-R ATG GAG AAT GCG TTC CTC AAC TspE4C2-F TspE4C2 GAG TAA TGT CGG GGC ATT CA 152-bp Clermont et al. 2000 TspE4C2-R CGC GCC AAC AAA GTA TTA CG ISCR1 ISCR1 CGC CCA CTC AAA CAA ACG 469-bp Kiiru et al. 2013 GAG GCT TTG GTG TAA CCG F = Forward; R = Reverse. 279Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 ur Rahman et al. Genotypic characterization of ESBL-producing E. coli in Pakistan the farming environment and 24% (12/50) from risk associated to humans. Chi-square test indicated a significantly (P < 0.05) higher E.  coli positivity rate from sick chicken as compared to healthy chicken and between the human and environmental samples. Screening of ESBL phenotypes by double disc synergy test indicated a total of 36 (44.4%) presumptive ESBL-producing E. coli isolates, majority (50%; 15/30) of these recovered from diseased chicks followed by healthy chicken (47.6%; 10/21), chicken rearing environment (38.8%; 7/18) and human (16.6%; 2/12) (Table II). Distribution and diversity of ESBL‑and carbapenemase‑encoding genes All ESBL- producing isolates were further assessed for the types of ESBL-encoding genes and results are presented in Table III. Our results indicated that bla CTXM was found to be the most prevalent (38.9%; 14/36) ESBL-encoding gene followed by SHV2 (25%; 9/36). Strikingly, no bla TEM and bla CTXM9 genes could be amplified from any of the isolates under study. Interestingly, these all isolates were also screened for common carbapenemase-encoding genes which showed that bla NDM1 was found to be the most prevalent gene (16.7%; 6/36) followed by bla OXA-48 (13.9%; 5/36). Chi square test indicate significant association (P < 0.05) of bla CTX-M , bla SHV2 and bla OXA-48 gene with diseased chicken and healthy chicken while between human being and environment significance association was observed in bla CTX-M-1 and bla SHV-2 genes. Interestingly, there were co-occurrence of multiple ESBL-encoding genes in a single isolate, and combination of bla SHV2  + bla CTXM was found as dominant combination with a frequency of 19.4%. Of note, bla NDM1 -carrying isolates (n  =  4) were also found positive for bla CTX-M indicating an alarming combination or favorable co-occurrence. Four isolates were carrying a combination of three different genes viz., bla CTXM + bla NDM1 + bla OXA-48 , while a single isolate was carrying four different resistance genes (bla CTXM + bla SHV2 + bla NDM-1 + bla OXA-48 ) showing co-occurrence of different ESBL-encoding genes in a single isolate. Frequency of ESBL or carbapenemase encoding genes is mentioned in Table IV. Detection of integrons and variable region For identification of the integrons (class 1-3) in all ESBL-producing E.  coli isolates using integron-integrase gene specific primers, intI1, intI2 and intI3 as described (Dillon et  al. 2005). The variable regions of class 1 integrons were detected in all intI1 positive ESBL-producing E. coli using PCR amplification with primers targeting the flanking regions (White et al. 2000). Statistical analysis Data was compiled with the help of Microsoft Excel and analysed through chi-square test at P  <  0.05 probability level using SPSS 16.0 analysis software. Results Escherichia coli isolates and ESBL phenotypes A total of 81 E. coli isolates were recovered from 200 samples tested. Frequency of sample positivity of E. coli was 60% (30/50) from disease broiler chicken, 42% (21/50) from healthy chicken, 36% (18/50) from Table II. Occurrence of drug resistant Escherichia coli (n = 200). Sample nature Total no samples Sample positivity n ESBL producers n MDR n Chi- Sq Sick chicken 50 30 (60.0%) 15/30 (50.0%) 17/30 (56.7%) 52.00* Healthy chicken 50 21 (42.0%) 10/21 (47.6%) 12/21 (57.1%) Environment 50 18 (36.0%) 7/18 (38.9%) 9/18 (50.0%) 1.000* Human 50 12 (24.0%) 4/12 (33.3%) 5/12 (41.7%) Total 200 81 (40.5%) 36/81 (44.4%) 43/81 (53.1%) * Indicates statistical significance difference in the diseased chicks vs healthy chicks and Environmental vs human sample. Table III. Frequency prevalence of extended-spectrum β-lactamase encoding genes (n = 36). Name of gene Diseased chick Healthy chick Chi-sq Environment Human being Chi-sq Total n CTX-M 7/15 (46.7%) 3/12 (25.0%) 1.44* 2/7 (28.6%) 2/2 (100.0%) 1.24 14/36 (38.9%) CTX-M -1 1/15 (6.7%) 1/12 (8.3%) 1.100 2/7 (28.6%) 1/2 (50.0%) 1.110* 5/36 (13.9%) SHV-2 4/15 (26.7%) 2/12 (16.7%) 1.100* 1/7 (14.3%) 2/2 (100.0%) 1.130* 9/36 (25.0%) OXA-48 2/15 (13.3%) 1/12 (8.3%) 78.00* 1/7 (14.3%) 1/2 (50.0%) 68.00 5/36 (13.8%) NDM-1 2/15 (13.3%) 2/12 (16.7%) 1.12 1/7 (14.3%) 1/2 (50.0%) 1.22 6/36 (16.7%) TEM 0/15 (0.0%) 0/12 (0.0%) 1.22 0/7 (0.0%) 0/2 (0.0%) 1.32 0/36 (0.0%) * Indicates statistical significance difference in the diseased chicks vs healty chicks and Environmental vs human sample. 280 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Genotypic characterization of ESBL-producing E. coli in Pakistan ur Rahman et al. present in a total of 17 isolates (51.5%) followed by integron 1, which was found in 16 (48.5%) isolates. Notably, integron 3 could not be amplified from any of the isolates. Interestingly, a combination of integron 1 and 2 was also observed in a total of 7  isolates (21.1%) that was found mainly in isolates recovered from sick chicken. Only 3 isolates recovered from healthy chicken carrying class 1  integron, while 2 isolates of them were carrying class 2. Of the 36 isolates, 14 were also found positive for the presence of variable region with amplicon size ranging from ~  0.25 kb to 2  kb. Furthermore, insertion sequence ISCR1 was also PCR-amplified from a total of 14 isolates (42.4%). Finally, association of the ESBL genes with the ISCR1 was assessed by primer combination targeting ISCR1 and an ESBL gene, indicating a total of 7 isolates produced an amplicon of the expected size showing association with the insertion sequence and more likely role in dissemination of these resistance encoding genes. Overall, our results showed ESBL producing multidrug resistant E. coli carrying diverse ESBL and carbapenemase encoding genes with CTX-M in dominance followed by SHV-2. Discussion Over the last couple of years, ESBL- and carbapenemase-producing E.  coli have been frequently reported from food-producing animals presenting a global challenge for public health and food security (Ali et al. 2016, Ali et al. 2017, Ali et al. 2018, Seiffert et al. 2013). Quite often, these isolates exhibiting MDR phenotypes further complicating its eradication during a disease condition by reducing the number of choices of drugs available against them (ur  Rahman et  al. 2018, Nordmann and Poirel 2002). Antimicrobial resistance (AMR) is even more serious in developing countries like Pakistan where usage of antimicrobials are not strictly regulated, Antimicrobial susceptibility patterns ESBL- producing isolates were subjected to drug susceptibility testing by standard disc diffusion method and results are presented in Table V. Results indicated that almost all isolates under study were found resistant to first generation cephalosporins (Cefalexin), while resistance to third generation cephalosporins, cefotaxime and ceftazidime was noted to be 100% and 79.4%, respectively. Of note, 82.4% isolates were resistant against aztreonam, 58.8% against gentamicin and 11.8% against meropenum. Moreover, 29.4% were found resistant to norfloxacin and 52.9% were resistant against tetracycline. Altogether, all of the isolates were found MDR by showing resistance against at least two classes of antimicrobials tested. Phylogenetic analysis A triplex PCR based phylogenetic analysis of the ESBL-producing E.  coli was carried out and results are depicted in Table VI. Our results showed that the commensal phylogroup A was found to be the most prevalent (50.0%), followed by virulent extra-intestinal group D (33.3%) and phylogroup B2 (16.7%), respectively. Integron, variable region and insertion sequence ISCR1 All ESBL-producing isolates were further characterized for the type of integrons, presence of variable regions and insertion sequence common region 1 (ISCR1) and results are presented in Table VI. Overall, our results showed that integron  2 was Table IV. Distribution of extended-spectrum β-lactamase-encoding genes (n = 36) β-lactamase gene Total number of isolates Frequency (%) CTXM 14 38.9 CTXM-1 5 13.9 SHV2 9 25.0 TEM 0 0.0 NDM-1 6 16.7 OXA-48 5 13.9 CTXM + SHV2 7 19.4 CTXM + NDM-1 4 11.1 SHV2 + OXA48 2 5.6 SHV2 + NDM-1 3 8.3 OXA-48 + NDM-1 3 8.3 OXA-48 + CTXM 4 11.1 CTXM + SHV2 + NDM-1 2 5.6 CTXM+ NDM+ OXA-48 4 11.1 CTXM + SHV2 + OXA-48 + NDM-1 1 2.8 Table V. Antibiotic sensitivity profile of E. coli isolates (n =36). Drug Concentration (µg) Resistant % Intermediates % Susceptible % CLR 30 94.1 5.9 0.0 AM 10 88.2 11.8 0.0 CTX 30 100.0 0.0 0.0 CAZ 30 79.4 8.8 11.8 AZT 30 82.4 11.8 5.9 GM 10 58.8 29.4 11.8 TE 20 52.9 14.7 32.4 MPN 10 11.8 14.7 73.5 NOR 10 29.4 32.4 38.2 CLR = Cefalexin; AM = Ampicillin; CTX = Cefotaxime; CAZ = Ceftazidime; AZT = Aztreonam; GM = Gentamicin; TE = Tetracycline; MPN = Meropenem; NOR = Norfloxacin. 281Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 ur Rahman et al. Genotypic characterization of ESBL-producing E. coli in Pakistan provides a lion share of 28% of total meat produced in the country (Pakistan 2016). Unfortunately, the use of antibiotics in poultry industry is not strictly regulated (Mitema 2010), raising concerns about the possible emergence of antimicrobial-resistant microorganisms (Naeem et  al. 2006, Shahid et  al. 2007). We designed this study to identify the prevalence of ESBL- and carbapenemase-producing E. coli isolated from poultry meat and risk-associated human beings. The results showed a prevalence of 41.9% of ESBL- particularly in animals, triggering emergence of AMR. This phenomenon corroborates with bacterial response of toxin production in response to signals in the surroundings of microbes (ur  Rahman and van Ulsen 2013, ur  Rahman et  al. 2014, van Ulsen et  al. 2014, Piet et  al. 2016). Furthermore, lack of surveillance and monitoring data regarding usage of antimicrobials and emergence of drug resistance further complicates the scenario in developing countries. Poultry industry in Pakistan is one of the biggest and dynamic industry with over 200 billion Pak. Rs. (2 Bill. $) investment, and this sector Table VI. Frequency prevalence of extended-spectrum β-lactamase encoding genes (n = 36). ID Source P.G ESBL genotype Carbapenemase Integron typing VR ISCR1 ISR1 + ESBL R/I phenotypes CTXM CTXM1 SHV2 TEM Oxa-48 NDM-1 Int.1 Int.2 Int.3 1 PD 20 DC D - - + - + - + + - + + + CLR, AM,GM,TE, NOR 2 PD 30 DC D + + - - + - - + - - - - CLR, AM,GM, C,TE 3 PH 3 HC A + + + - - + + - - + - - CLR,GM,TE, C, AM, NOR 4 O HC D - - + - - - + - - + - - CLR,TE, AM 5 PE 32 E A - + - - - - + + - - + - CLR, AM 6 PD 21 DC D + - + - - - - - - - + + CLR, AM, GM,TE, NOR 7 PD 46 DC A + - - - - - - + - + - - CLR, AM,TE 8 B HC A - - - - - - - - - - - - CLR 9 PD 33 DC D + - + - - - + + - + - - CLR, AM,GM,TE 10 PH 4 HC A + - - - + - - - - - - - CLR, GM, C, 11 PD 32 DC D + - - - - + + - - + + - CLR, AM, C,TE 12 PD 28 DC D - - - - - - + + - + - - - 13 PE 33 E A + + + - - - - - - - - - CLR,TE,C 14 PD 25 DC D - - - - - - + + - - + + CLR 15 PE 43 E A - - - - - - - + - - - - - 16 PD 24 DC D - - - - - - + - - + - - CLR, AM 17 PD 34 DC D - - - - - - + - - + + + CLR 18 PD 1 DC A - - - - - - - + - - - - CLR ,TE 19 PD 31 HC A - - - - - - - + - - - - CLR 20 PE 31 E B2 + - - - + - + + - + + + CLR,GM,TE 21 PD 41 DC A - - + - - - - - - - + - CLR,TE 22 PD 5 DC A - - - - - - - + - - - - CLR, AM 23 Phh 44 HC A + - - - - - - + - - + - CLR, AM 24 HL 13 HC A - - - - - + + - - + + - CLR, AM, GM,TE, C 25 PE 47 E A - - - - - - - - - + + - CLR 26 PE 5 E D - - - - - - - + - - - - CLR 27 PD 39 DC A + - - - - + + - - + + + CLR, MPN,AM,GM, NOR, C,TE 28 PH 45 HC A - - - - - - - - - - - - - 29 PD 29 DC A - - - - - - - + - - - - CLR, AM 30 Phb 3 Human B2 + - + - + + - + - - + - CLR,TE, AM,NOR,C 31 Phb 17 E B2 + - - - - + + - - - - - CLR,TE, NOR,C 32 Phh 40 HC B2 - - - - - - + - - + + - CLR 33 Phb 11 Human B2 + + + - - - + + - + - - CLR,TE,GM, MPN,NOR,C 34 Phb 37 HC A - - - - - - - + - - - CLR,TE 35 Phb 25 Human B2 - - - - - - - - - - - - CLR,TE 36 Phb 20 Human D - - - - - - - - - - - - CLR,TE, NOR, C DC = Diseased chicken; HC = Healthy chicken; E = Environment; + = Present; - = Absent; Int.1 = Integron class 1; Int.2 = Integron class 2; Int.3 = Integron class 3; VR = Variable region; P.G = Phylogenetic groups; CLR = Cephalexin; C = Chloramphenicol; GM = Gentamicin; TE = Tetracycline. 282 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Genotypic characterization of ESBL-producing E. coli in Pakistan ur Rahman et al. first generation cephalosporins and cefotaxime, while almost 50% of isolates are found resistant to other antibiotics tested, except carbapenems. This observed lower susceptibility of these isolates to majority of antibiotics tested is most probably indicative of consistent use of these antibiotics that might provide selective pressure for the resistant bacteria. Finally, the majority of these isolates were found to be MDR that belonged to commensal phylogroup A. Although resistance determinants were also found to be carried by virulent groups of E.  coli (D/B2), however, acquiring these elements by commensal groups is highly worrisome given the increasing chances of dissemination of these resistant elements. This is suggesting the need for an improvement of the rationale use of antibiotics in poultry production. Mobile elements such as integron and insertion sequences are considered crucial for the dissemination of resistance conferring elements and emergence of MDR bacteria. Previous reports suggested an overwhelming presence of clinical class  1 integron in ESBL-positive E.  coli (Dillon et  al. 2005, Gu et  al. 2008). Integron of class 1 generally carries a variable region comprising a gene cassette arrays encoding different resistance elements. Our results of identification of class 1 integron in the majority of isolates along with variable regions (Table  V) suggesting its involvement in dissemination of resistance elements. Strikingly, we determined that majority of bla CTX-M genes were associated with ISCR1. These results corroborate with previous findings of our lab (Ali et  al. 2016) and other studies from different countries of the world (Kar et al. 2015, Kiiru et al. 2013, Xu et al. 2015). Notably, our findings of the most pre-dominant ESBL genotype (CTX-M) and its strong association with the ISCR1 indicated that they are more likely mobilized by ISCR1 elements. Conversely, only one carbapenemase encoding gene (OXA-48) was found linked to ISCR1. Conclusions Our study concludes a high prevalence of ESBL- and carbapenemase-producing E.  coli in the region. Taken together, the current high occurrence of multidrug resistant ESBL-producing E.  coli carrying clinical class 1 integron and its association with ISCR1 is quite worrisome. This calls for an efficient control policy with restriction on the consumption of extended-spectrum cephalosporins for long term use in poultry industry in the area. Funding This study was partially supported by Natural producing E.  coli. This is a higher incidence rate of ESBL producing E.  coli recovered from poultry as compared to previously published report of 32% in Peshawar Pakistan (Ahmad et  al. 2018), 30% in poultry in Bangladesh (Hasan et al. 2012) and 10.7% in France (Girlich et  al. 2007). The higher incidence rate of ESBL-producing E. coli is possibly due to over use or consistent usage of antibiotics during poultry production. Contrary to our findings, however, even higher incidence rate of ESBL-producing E. coli have been reported from other parts of the world such as 93% in Spain (Egea et al. 2012) and in 81% in the Netherlands (Blaak et al. 2015). ESBL-producing E. coli has been widely reported from human patients that were hospitalized (Abrar et  al. 2017, Rahman et  al. 2016, Ullah et  al. 2017) as well as from community and environment in Pakistan (Ullah et  al. 2009). However, results of our study cannot be generalized as we analyzed a limited number of samples from a single district, i.e. Peshawar of Khyber Pakhtunkhwa province, Pakistan. Due to limited resources, we did not extend our study further to investigate the occurrence and trend of ESBL-producing E.  coli in different mechanized and open shed poultry farms in the whole district. Also, we did not investigate the occurrence of ESBL- and carbapenemase-production in bacteria other than E. coli. Our study indicates that bla CTX-M remained the predominant genotype in ESBL-producing E.  coli. These results are in agreement with other contemporary findings from Pakistan (Khan et  al. 2010, Mirza et  al. 2006), China (Ali et  al. 2016, Ali et  al. 2017) and India (Upadhyay et  al. 2015), etc. This strongly suggests that bla CTX-M is the most dominant genotype of ESBL. This goes along with higher occurrence of SHV genotype in our study and as reported by others (Habeeb et  al. 2013). More worrisome is the combination of ESBL with carbapenemase encoding genes such bla OXA-48 and bla NDM-1 (Table III). Co-occurrence of ESBL and carbapenemase encoding genes have also been reported earlier from Pakistan (Ullah et  al. 2017, ur Rahman et al. 2018a, Younas et al. 2019) from other districts from human patients and poultry. Isolation of E.  coli harboring resistance elements encoding for ESBL and carbapenemase enzymes from poultry meat and its environment is in fact highly alarming posing threats to public health as these resistance elements can finally be acquired by ham pathogens. Combination of bla CTX-M + bla SHV + bla OXA-48 and bla NDM-1 is quite challenging. Such combinations of different resistance conferring-genetic elements would certainly have an impact on the severity of resistance to different antibiotics and play a role in the development of MDR. This is partly reflected by phenotypic resistance to most of the antibiotics tested in this study (Table V). Our results show that most of the isolates are found resistant to 283Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 ur Rahman et al. Genotypic characterization of ESBL-producing E. coli in Pakistan Acknowledgments The authors acknowledge the support from technical staff of Khyber Teaching Hospital during collection of samples from targeted human patients. National Science Foundation of China under the project “International Young Scientist Award” awarded to Dr Sadeeq ur  Rahman (-Project No. 31550110200) and Relief International under the umbrella of USAID program “fighting zoonosis” awarded to Dr Sadeeq. The authors specially thanking staff members of Khyber teaching hospital for help during collection of human samples. 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. References 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. 284 Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 Genotypic characterization of ESBL-producing E. coli in Pakistan ur Rahman et al. 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. 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. Pak J 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 285Veterinaria Italiana 2021, 57 (4), 275-285. doi: 10.12834/VetIt.1780.9397.4 ur Rahman et al. Genotypic characterization of ESBL-producing E. coli in Pakistan 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. & 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.