Phenotypic Characterization and bla Gene Detection in Antibiotic Resistant Escherichia coli Obtained from Poultry Fecal Samples

Extended spectrum-b-Lactamase (ESBL) producing bacteriaare a major threat to public health threat worldwide. The emergence of ESBL is due to improper use of anti biotics both in veterinary and human medicine (Geidam et al., 2014; Alisadi et al., 2015.). Antibiotics are widely used inpoultry and livestock breeding to improve growth and feed efficiency and reduce the disease outbreaks. Most of the bacterial pathogens associated with human diseases derived from animals and occurs indirectly through eggs, chicken meat and contaminated water, etc., (Donoghue, 2003; Kamini et al., 2012; Boamah et al., 2016; Newel et al., 2010.). b-Lactamase encoded by plasmids and transmitted between different bacterial species, which promotess increase emergence of ESBL strains. The proper methods for ESBL detection raise serious concern due to treatment failure of 3^rd generation cephalosporins and Aztreonam Nordman et al., (2011). Currently, there is paucity of information on ESBL producing E. coli from poultry droppings and the possible contribution of these resistant species to the ever growing antimicrobial resistance observed in humans. Therefore, the present study demonstrates to detect ESBL producing E.coli from healthy poultry of small-scale farmers.

Study materials
 
A total of 60 poultry farms with the age groups of 1-5 months was included in the study. The study was conducted from July 2019 to March 2020 by the Veterinary University Training and Research Centre, Melmaruvathur. Random sampling techniques were adopted to choose 60 poultry farms.
 
Sample collection
 
The faecal samples were collected by inserting a swab into the vent and gently swabbing the mucosal wall than the swab was removed gently from the cloaca and placed the swab into sterile tubes containing enough enrichment medium (Mueller Hinton broth) to moisten and cover the end of the swabs and incubated at 37°C for 18-24 h.
 
Identification of pathogens
 
For identification of E. coli a loop-full culture from enrichment broth was taken and streaked onto MacConkey’s lactose agar (HiMedia, India) and incubated at 37°C for 24 h. The suspected E. coli colonies, pink to red were picked up and further streaked on eosin methylene blue agar (HiMedia, India) and incubated at 37°C for 24 h. Dark-centered and flat colonies with metallic sheen were considered as E. coli. On microscopic examination motile long slender Gram negative bacilli on the gram staining were suggestive of E. coli. All the samples were subjected to biochemical charectrization as Catalase, Indole and Motility test to confirm as E. coli.
 
Antimicrobial susceptibility test
 
All the confirmed E. coli isolates were tested for their antimicrobial drug susceptibility test on Mueller-Hinton agar (MHA) (HiMedia, India) by the disc diffusion method (CLSI 2012). The antibiotics used were oxytetracycline (30 µg), cefpodoxime (30 µg), enrofloxacin (30 µg), gentamicin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg) with Clavulanicacid (10 µg) and ceftazidime (30 µg) with Clavulanicacid (10 µg) (HiMedia, India). The diameter of the zones of complete inhibition was measured and compared with the zone size interpretation chart and was graded as sensitive, intermediate and resistant.
 
Detection of ESBL isolates by combined disc diffusion methods
 
All confirmed E. coli isolates were tested for their antibiotic sensitivity pattern on Mueller-Hinton agar (MHA) (HiMedia, India) by the disc diffusion method. Prepared the inoculums of the suspected test isolate and streak in the MHA plates and kept the plates for not more than 15 min for evaporation of excess media. The antibiotics used were oxytetracycline (30 µg), cefpodoxime (30 µg), Enrofloxacin (30 µg), gentamicin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg) with Clavunic acid (10 µg) andceftazidime (30 µg) with Clavunic acid (10 µg) (HiMedia, India). The diameter of the zones of complete inhibition was measured and compared with the zone size interpretation chart and was graded as sensitive, intermediate and resistant.

For detection of ESBL producing islolatesthe disks containing cefotaxime (30 µg) or ceftazidime (30 µg) alone and with clavulanic acid (10 µg) were placed diagonally with a distance of 25 mm, center to center. Incubate at 35°C for 16-18 hour. An increase of 5 mm (50%) or more in the zone of inhibition around the combined disk containing clavulanic acid than the corresponding disk with cefotaxime or ceftazidime is considered positive for ESBL production. Phenotypically confirmed ESBL-producing E. coli isolates were analyzed for the presence of genes encoding bla TEM, bla SHV and bla CTX-M by multiplex polymerase chain reaction (m-PCR) described by Monstein et al (2007). Primers used in this study for ESBL resistance and cyclic conditions for the polymerase reaction to detect ESBL resistance genesis given in Table 1 and 2.



​

In the present study, 40 isloates of E. coli were isolated from 60 faecal samples. Among 40 isolates, 12 isolates were resistant to two or more than two antibiotics. The resistance in isolates was as follow (Table 3): Oxytetracycline (80%), Gentamicin (37.5%), Enrofloxacin (75%), Cefotoxime (62.5%), Ceftazidime (50%), Cefpodoxime (47.5%), Aztreonam (45%), Cefotoxime +Clavulanicacid (CEC) (25%) and Ceftazidime+Clavulanicacid (CAC) (25%). Among 40 isolates, 12 isolates were resistant to two or more than two antibiotics. The E. coli isolates were also reported by Jaulkar et al., (2011) and Durairajan et al., (2021). The indiscriminate use of antibiotics in mass production of poultry has promoted the emergence of antimicrobial resistance (AMR) E. coli in poultry. Tame et al., (2019) reported 46.9% of ESBL producers from fecal dropping of poultry. The molecular methods helped to confirm the presence of genes in E. coli which is regard to ESBL production. Out of 12 E.coli presumptive ESBL producers; 6 isolates have CTX-m genes in PCR (Fig 1) and the prevalence of CTX-m gene is 50%. There was no presence of Bla-SHV and Bla-TEM in the confirmed E. coli isolates. Though AMR is hot title for concerning in human and animal health (Schwarz et al., 2001; Schink et al., 2013; WHO 2014). The failure is in accordance with WHO survey (WHO 2015B) where the majority of responsibilities indicated the need for a more rationale antimicrobial use (AMU) in veterinary medicines as the first priorities of actions. Incidence of beta lactamase producing E. coli is due to frequent administration of drug such as penicillin, cephalosporin, monobactum and carbapenam (Cheaito and Matar, 2014), which is associated with resistance to other type of antibiotics leading to multidrug resistance. Haldorsen (2011) reported plasmid mediated gene transfer is responsible for AMR and ESBL. The contamination of several resistant genes causes bacteria to be resistant most classes of antibiotics in both farm and hospital set up Allocati et al., (2013). From the study it was observed that E.coli isolates were more susceptible to Gentamicin, Aztreonarm, Cefrtrazindime and Cefotoxime. This report are similar with finding of Unal et al., (2017) and Tame et al., (2019). The high susceptibility of antibiotics mentioned above may be due to the fact that the drugs not like abused and not affordable by farmers. Also, Gentamicin and cefotoxime are available in injectable form only and because of pain and laborious to administration such antibiotics not likely to be used indiscriminately or substandard antibiotics in animal husbandry especially in poultry. Parent stocks used combination of antibiotics which simultaneously transmitted to progeny also. The genotypic charecterization are same as to Olowe et al., (2015) and Apka et al., (2010) who reported that none of the isolates were expressed Bla (TEM) genes for resistance to antibiotics. The percentage of ESBL genes observed in this study suggestive of the gene may be responsible for the production of ESBL enzymes that is resistant to most Beta lactam antibiotics. Sometimes multiples genes are responsible for production of ESBL enzymes than single gene alone. This implies that the antibiotics are useful in the treatment of infection caused by E. coli in particular area. Laboratory monitoring and detection of E. coli of ESBL producing bacteria important steps in the appropriate treatment for a farm based poultry industry and infection control efforts.

The explosions of Indian population enhance the demand of poultry. The high human population needs higher polutry production for satisfying the animal nutritive nutritive protein requirements. The continuous use of antibiotics for treatment and antibiotics free feed and feed materials has also reduced the problem of multidrug resistence Indian soils and protect to poultry health. Screening and surveillance of E. coli for ESBL producing bacteria important steps in the appropriate treatment in farm based poultry industry and infection control efforts.

The authors are grateful to the TANUVAS, Chennai, Tamil Nadu, India for providing necessary financial assistance to carry out this research programme.