Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Prevalence and molecular characterization of Multidrug-Resistant ESBL-Producing E. coli in commercial poultry

Sengamani Sakthikarthikeyan1,*, Mani Sivakumar2, Rajendran Manikandan1, Periyathambi Senthil Kumar1, Veerapandian Suresh Kumar2, Shanmugasamy Malmarugan1, Manimuthu Prabhu1, Vijayaragavan Ramakrishnan1
1Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Tirunelveli-627 358, Tamil Nadu, India.
2Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Theni-625 531, Tamil Nadu, India.

Background: Rural poultry farmers are gradually changing from the backyard poultry production to intensive production, which made poultry more vulnerable to diseases and lead to indiscriminate use of antimicrobials in poultry production. As per OIE, FAO and WHO, multiple spread antimicrobial resistant pathogenic bacteria are a serious global human and animal health problem. Hence the study has been carried out to know the antimicrobial resistance status of poultry in Tirunelveli, Tamil Nadu, India.

Methods: The study was aimed to find the occurrence of extended spectrum beta lactamases (ESBLs) producing E. coli from poultry origin. Caecum samples from the broiler (n=180), desi chicken (n=180) and Japanese quail (n=180) were processed for the isolation and phenotypic identification of ESBL E. coli. The isolates were tested for antimicrobial susceptibility test and characterization of ESBL and virulence genes done by PCR.

Result: On phenotypic confirmation of ESBL producing E. coli by combined disk diffusion test (CDDT), a total of 9.63% samples were positive for extended spectrum beta lactamase producer (ESBL). Multi drug resistance (MDR) was observed in 38.23% of the broiler isolates, 27.27% and 57.14% in desi chicken and Japanese quail isolates, respectively. All the phenotypically confirmed ESBL producing E. coli isolates (n=52) were positive for uspA gene by PCR confirming the isolates as E. coli. The overall presence of the blaTEM gene was 57.69%, with broiler isolates having the highest prevalence of 67.47%, followed by desi chicken 36.36% and Japanese quail 42.85%. In contrast, the blaSHV gene was found in only 17.64% of the broiler isolates, but none were positive for the blaCTXM and blaOXA-1 gene.

Poultry meat is preferred more by peoples all over the world because of its low market price, nutritional value and less space requirement for production (Kalakuntla et al., 2017). Antimicrobials are injudiciously used in poultry production for the purpose of prevention of chick mortality, treatment and as growth promoters in feed. The WHO has classified extended spectrum β-lactamase (ESBL) producing E. coli as one of the important AMR pathogens to human health and a major public health concern. The prevalence of ESBL-producing E. coli in hospitals, livestock and poultry has increased globally (Hu et al., 2019). ESBL producing organisms are more often noticed in the gram-negative bacteria of Enterobacteriaceae family. The bacteria commonly involved in production of extended spectrum β-lactamase particularly E. coli and K. pneumoniae were reported in recent year all over the world including in India (Lalzampuia et al., 2013). ESBL organism may act as a potential source for transfer of resistance to other organism, intern these organisms are transmitted to susceptible host through contaminated water, environment and food chain etc. Several investigators have reported the occurrence of ESBL E. coli in different samples of chicken meat (Sivakumar et al., 2021); chevon meat (Bhoomika et al., 2016); raw milk (Chauhan et al., 2013); cloacal swabs of broilers (Shrivastav et al., 2016) and poultry fecal samples (Lalzampuia et al., 2013) from various states of India but not many reports are available on the occurrence of ESBL E. coli in poultry in Tamil Nadu, India. Hence, the study was undertaken to find out the occurrence, antimicrobial resistance pattern and their characterization of ESBL E. coli in different poultry species (Broiler, desi chicken and Japanese quail).
Sample collection
The present study was carried out during the year 2021-2023, at Department of Veterinary Pharmacology and Toxicology, VCRI, Tirunelveli, Tamil Nadu. A total of 540 caecum samples, 180 each from broilers, desi chicken and Japanese quail were collected randomly in aseptic polythene bag at different poultry meat retail outlets of Tirunelveli. Samples were processed at the laboratory or maintained at 4°C until further processing.
Isolation and phenotypic identification of ESBL E. coli
All the collected samples were processed for isolation of E. coli on MacConkey agar, EMB agar (HiMedia, India) followed by biochemical characterization (IMViC test) as per the standard methods. Biochemically characterized isolates of E. coli were cultured on MacConkey agar, which was supplemented with cefotaxime at 2 mg/L. Any growth on the plates was considered as ESBL producer and/or AmpC resistant E. coli (Costa et al., 2009). Further, isolates shown growth on these plates was also subjected to a disk diffusion assay with commercially available antimicrobial disc (HiMedia, India): cefotaxime (CTX, 30 µg), ceftriaxone (CTR, 30 µg), ceftazidime (CAZ, 30 µg) and cefepime (CPM, 30 µg). The isolates showing resistant to any of the above antimicrobials were further subjected for combination disc method for detection of ESBL production, which is based on the principle that the ESBL producing isolate will exhibit an expanded zone of inhibition against third or fourth generation cephalosporin in the presence of beta-lactamase inhibitor like clavulanic acid (CLSI, 2015).
Antimicrobial susceptibility testing
Phenotypically confirmed ESBL producing E. coli isolates were tested for antimicrobial susceptibility using six classes of twelve different commercially available antimicrobial discs such as Cefpodoxime (CPD-10 µg), Cefepime (CPM-30 µg), Ceftazidime (CAZ-30 µg), Cefperazone (CPZ-75 µg), Cefotaxime (CTX-30 µg), Ceftriaxone (CTR-30 µg), Amoxycillin+Clavulanic acid (AMC-20/10 µg), Co-trimoxazole (COT-1.25/23.75 µg), Oxytetracycline (O-30 µg), Gentamicin (GEN-10 µg), Enrofloxacin (EX-5 µg) and Chloramphenicol (C-30 µg) by Kirby and Bauer disk diffusion method (Hudzicki, 2009). The Clinical Laboratory Standards Institute (CLSI) guidelines were followed to measure the zone of inhibition and isolates were categorized accordingly as sensitive, intermediate and resistant.
Characterization of ESBL genes and virulence genes by PCR
All the phenotypically confirmed ESBL producing E. coli isolates were further confirmed by PCR amplification of uspA gene specific primers for E. coli. The E.coli isolates confirmed by PCR were further subjected to PCR amplification of important ESBL genes viz., blaTEM, blaCTXM and blaSHV and blaOXA-1. Shiga toxin virulence genes stx1 and stx2 by using gene specific primers (Table 1). In brief, the bacterial DNA extraction from the isolates was done by Snap chill method (Mandal et al., 2017). The uniplex PCR assays were performed with reaction mixture comprised of 12.5 µL of 2X Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark), 1 µL (10 pM) of each primer, 2 µL of extracted DNA template and the final volume of 25 µL adjusted by addition of  NFW and subjected to amplification in thermocycler (Eppendorf Mastercycler® nexus X2, Germany). The PCR cyclical conditions of uspA, blaTEM, blaSHV, blaCTXM and blaOXA-1 were set at an initial denaturation of 94°C for 5 min followed by 35 cycles each of denaturation at 94°C for 30 sec, annealing at 60°C for 15 sec, extension at 72°C for 30 sec and final extension at 72°C for 5 min whereas for stx1 and stx2 initial denaturation at 94°C for 5 min followed by 35 cycles each of denaturation at 94°C for 1.5 min, annealing at 62°C for 1.5 min, extension at 72°C for 1.5 min and final extension at 72°C for 7 min. Electrophoresis was carried out in 1.5% agarose gel to visualize the PCR products and the images were captured by gel documentation system (Gelstan-1312).

Table 1: Details of primers used in this study.

DNA sequencing and phylogenetic analysis
Among the ESBL genes, only the blaTEM gene from broiler, desi chicken and Japanese quail was subjected for gene sequencing and phylogenetic analysis. GeneJET gel extraction kit (Thermo Fisher scientific, USA) was used for purification of the amplified products from the excised gel as per manufacturer’s directions. The purified DNA was sequenced using the same set of PCR primers at Eurofins Genomics India Pvt Ltd., Bangalore. blaTEM gene sequences were submitted to NCBI database and obtained accession numbers. The present study sequences were compared with 11 distinct isolates of the E. coli blaTEM gene from GenBank database. Multiple alignment and comparison of the study sequences with GenBank references were performed using clustal W. Further, phylogenetic tree construction and molecular evolutionary analysis were conducted using MEGA (Molecular Evolutionary Genetics Analysis) version 11.0 by neighbour joining method with maximum likelihood substitution model at 1000 boot straps replicates (Tamura et al., 2021).
Statistical analysis
All the statistical analyses were performed using SPSS computer software version 22. Chi-square analysis was performed to compare the isolation of E. coli and phenotypic confirmation of ESBL E. coli by CDDT for broiler, desi chicken and Japanese quail.
Isolation and phenotypic identification of ESBL E. coli
In the present study, a total of 89.44% (483/540) E. coli isolates were isolated from caecum samples. Among them highest isolation rate was obtained in broiler 91.6% (165/180), followed by Japanese quail 89.4% (161/180) and desi birds 87.2% (157/180). Chi-square analysis showed that no significant association (P≥0.05) between broiler, Desi chicken and Japanese quail for the isolation of E. coli (Table 2). The prevalence of E. coli in this study also indicates that poultry meat is significant sources of E. coli and potential risk factors for E. coli infection. Further, phenotypic confirmation of ESBL producing E. coli done by CDDT. A total of 9.63% (52/540) of isolates were ESBL producer in which broiler, desi chicken and Japanese quail were positive for 18.89% (34/180), 6.12% (11/180) and 3.88% (07/180), respectively. Chi-square analysis showed that the highly significant association (P<0.01) between broiler, desi chicken and Japanese quail for the phenotypic confirmation of ESBL E. coli by CDDT method (Table 3).

Table 2: Sample-wise isolation rate of E. coli.


Table 3: Phenotypic characterization of ESBL E. coli by combined disc diffusion test (CDDT).

In agreement with this study, the phenotypic results of ESBL E. coli in poultry isolates were 5.22% (Lalzampuia et al., 2014); foods of animal origin and human clinical samples were 10.99% (Bhoomika et al., 2016). In contrast, existing investigations reported a higher prevalence rate of 30% in poultry droppings, (Durairajan et al., 2021) and 33.35% in healthy broilers (Shrivastav et al., 2016).
Antimicrobial susceptibility testing
All the ESBL isolates further subjected for antimicrobial testing of which highest resistance was observed to oxytetracycline, cefotaxime and cefperazone (100%) followed by ceftazidime (94.23%), cefpodoxime (88.46%), ceftriaxone (82.69%), chloramphenicol (76.92%) and low resistance in gentamicin (11.53) and enrofloxacin (9.61%). However, no resistance was observed in cefepime and amoxycillin+clavulanic acid. Resistance of antimicrobials obtained in this study in field conditions were also are reported by the different researchers (Bhoomika et al., 2016; Durairajan et al., 2021).  Of the 52 isolates 38.46 % (20/52) of them were MDR isolates, including 38.23% (13/34), 27.27% (3/11) and 57.14% (4/7) isolates from broilers, desi chicken and Japanese quail, respectively. Rahman et al., (2020) observed the prevalence of MDR E. coli in broiler 78.17% and layer is 73.71% with overall prevalence of 75.06% in Bangladesh. The transmission of MDR E. coli in healthcare and food chain influenced by factors such as the incorrect usage of antimicrobials and inadequate hygiene practices. Thereby, consuming meat and meat products that contain MDR E. coli increases the risk of human infection (Adzitey et al., 2021).
Characterization of ESBL genes and virulence genes by PCR
All the phenotypically confirmed 52 isolates of broiler (n=34), desi chicken (n=11) and Japanese quail (n=7) were subjected to PCR for genotypic detection of uspA and ESBL genes viz. blaTEM, blaSHV, blaCTXM, blaOXA-1, stx1 and stx2. The presence of uspA gene were 100% (52/52), of which 100% in broiler (34/34), desi chicken (11/11) and Japanese quail (7/7) respectively (Fig 1). The overall presence of blaTEM gene were 57.69% (30/52), of which predominant in broiler isolates 67.47% (23/34) followed by 42.85% (3/7) in Japanese quail and 36.36% (4/11) in desi chicken (Fig 2). Whereas blaSHV gene detected in 17.64% (6/34) only from broiler isolates (Fig 3) and none of the broiler, desi chicken and Japanese quail isolates were positive for blaCTXM gene and blaOXA-1 gene. Samanta et al., (2015) in poultry observed that blaTEM, blaCTXM and blaSHV detected in 21.7%, 34.7% and 43.4% of the isolates respectively. In contrast, Durairajan et al., (2021) observed in poultry droppings that 50% of the isolates pose the blaCTXM gene and blaTEM genes, but blaSHV genes were detected in none of the isolates. Screening of stx1 and stx2 gene specific PCR revealed none of the isolates were found positive for shiga toxin which was similar to the study conducted by (Sivakumar et al., 2021).

Fig 1: PCR amplification of Escherichia coli specific uspA gene.


Fig 2: PCR amplification of ESBL producing Escherichia coli blaTEM gene.


Fig 3: PCR amplification of ESBL producing Escherichia coli blaSHV gene.

DNA Sequencing and phylogenetic analysis
In this study, blaTEM genes were submitted to the NCBI database and received accession numbers OP870148, OQ355037and Q355038 respectively. Phylogenetic analysis revealed that the aforesaid accession numbered genes shared an evolutionary relationship with other blaTEM genes from different E. coli serogroups reported. Even though all these isolates were descended from a common ancestor, isolates OP870148, OQ355038 are closely related and clustered in the same clade (Fig 4). Furthermore, Isolate OQ355038 was clustered with CP116911.1 sequence from an E. coli that showed resistance against third-generation cephalosporin (Pankok et al., 2022).

Fig 4: The phylogenetic tree is based on partial nucleotide sequences of E. coli blaTEM gene.

The overall isolation rate of ESBL E. coli in broiler, desi chicken and Japanese quail is 9.63%. The MDR- ESBL E. coli strains in poultry meat may transmit to humans by means of direct as well as indirect contact. This study highlighted the emergence of multidrug resistant ESBL E. coli in healthy poultry has potential threats to public. The continuous monitoring and surveillance of poultry, poultry products and their environment for the important food borne pathogens, antimicrobial resistance and virulence factors are necessary.
The authors thank The Dean, VCRI, Tirunelveli, TANUVAS, for providing the facilities to carry out the research work successfully.
On behalf of all authors there is no conflict of interest in publishing this research article.

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