Virulence and Antibiogram Study of Escherichia coli Isolated from Faecal Sample of Buffalo

The study was undertaken for detection of virulence gene using polymerase chain reaction (PCR) and investigate their antibiotic sensitivity pattern in fecal sample of buffalo. In present study, a total of 180 samples were processed for isolation of verocytotoxic E. coli (VTEC). Out of 180 samples, 19 samples were found positive for VTEC. All VTEC strains were subjected to antibiotic sensitivity test using 16 antibiotic discs. Of these, ciprofoxacin and imipenem were found highly sensitive (100%) against VTEC strains followed by ceftriaxone (89.47%), gentamicin (84.21%), amikacin (84.21%) and chloramphenicol (78.94%). Least sensitivity was recorded for enrofloxacin (78.94%), followed by cefixime (63.15%), erythromycin (57.84%) and tetracycline (52.63%).

Diarrhea is one of the most common and multifactorial disease of man and animals mainly caused by E. coli . E. coli is the most commonly observed gastrointestinal flora of animals and environmental contaminant considered as important food borne pathogen causing serious complications in man and animals (Malik et al., 2013;Dhama et al., 2013;Ankita et al., 2014). Verocytotoxic E. coli (VTEC) was first identified as a distinct group of E. coli, which had the ability to produce toxins with profound and irreversible effect on vero cells. VTEC is also termed as shiga-like toxin producing E. coli (SLTEC) or shiga toxin producing E. coli or STEC. Acronym STEC is derived from the fact that the toxins are shiga like that is similar to those produced by Shigella dysenteriae type1 (Brien et al., 1987). The EHEC belong to the Verocytotoxin producing E. coli (VTEC). VTEC always do not induce clinical signs and are not enterohaemorrhagic until addition virulence factor are present like enterohemolysin and adherence factors (intimin). The adherence factor(s) enables the organism to attach to and colonize intestinal mucosal cells (Hiruta et al., 2001). Among VTEC, serotype O157:H7 has been closely associated with the sporadic and clinical outbreaks of hemorrhagic colitis (HC), haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in human beings (Croxen and Finlay, 2010;Gyles and Fairbrother, 2010;Sanchez et al., 2013). Healthy domestic ruminants are recognised as the main natural reservoir of STEC and large game animal maybe healthy carriers of STEC (Diaz et al., 2011;Sanchez et al., 2010).
Antimicrobial drug resistance in bacterial isolates that have potential to enter our food supply is a growing public health concern. Antibiotics are being frequently incorporated as sub therapeutic and animal food supplements to cure and prevent disease in animals. This non judicious use of antibiotics generates a selective pressure that has led to the emergence of antibiotic resistance in the microbes including E.coli (Ajayi et al., 2011). Considering the impact of pathogenic E.coli on humans, animals and its considerable public health significance, the present study was conducted to discern the virulence factors and antibiotic susceptibility profile of E.coli isolated from faeces of apparently healthy cattle as well as environment (soil) from dairy farms of Uttar Pradesh. Keeping in view the importance of this organism, the present study was planned to reveal the genotypic study of VTEC in faecal samples of domestic and wild ruminants.

Sampling and isolation of E. coli
A total of 180 buffalo fecal samples were collected from Mathura district. The samples were collected aseptically in UV sterile polythene bags and immediately transported to the laboratory under chilled conditions for microbiological analysis. For primary isolation of E. coli (VTEC), 10gm of faecal sample were enriched in 90 ml modified trypticase soya broth (mTSB) containing acriflavine (10 mg/l) to reduce the growth of gram positive organism. The method used for collection of materials, and isolation and identification techniques were made as per the lines suggested by World organization for Animal Health (OIE, 2014). These samples were incubated at 37°C for 6 h. MacConkey's Agar (MCA) was used as differential media, while Eosin methylene blue (EMB) agar (Hi-Media, India) was used as selective media. Suspected E. coli strains were subjected to morphological, cultural and biochemical characterization as per the standard method (Ewing, 1986).

Molecular characterization
Multiplex Polymerase chain reaction (PCR) was used for detection of virulent genes (stx 1 , stx 2 , eaeA and hlyA) of VTEC. All the E. coli isolates were subjected to genomic DNA isolation. The bacterial growth in TSB broth (HiMedia) was centrifuged at 3000 rpm for 15 min to make the pellet of bacterial cells. These cells were washed twice with PBS (pH 7.4) to remove any impurity of broth media. Bacterial DNA was extracted by using DNA extraction kit (Genei, Bangalore) as per the manufacturer's protocol. For the PCR reaction, PCR Master Mix solution (Genei, Bangalore) was used. To amplify DNA targeted to virulent genes (stx 1 ,stx 2 , eaeA and hlyA) of VTEC by using primers on 3µl of DNA template in 25µl reaction mixture (Paton and Paton, 1998). After an initial denaturation step at 95ºC for 4 min, 30 amplification cycles were performed, each consisting of 94 °C for 2 min., 65 °C for 2 min. and 72°C for 1.5 min and followed by a final extension step at 72ºC for 2.5 min. After the amplification, amplicons were separated in 1.5% gel in tris acetate EDTA (TAE) buffer at 60 volt for 80 min, stained with 0.5% ethidium bromide solution and visualized under ultraviolet light.

RESULTS AND DISCUSSION
Out of 180 faecal sample, a total of 180 E. coli strains were obtained. All the strains of E. coli were screened to detect the presence of VTEC genes using multiplex PCR (Fig. 1). An overall prevalence of VTEC in Buffalo faecal sample was found to be 10.56% (19/180). Out of 19 VTEC, 6 VTEC was found to be positive for stx 1 gene (180 bp) and 7 VTEC for stx 1 and stx 2 (180 bp and 255 bp), 2 VTEC were found positive for stx 1 with eaeA (384 bp) gene and 4 were positive for stx 1 , stx 2 and eaeA gene.
High level of antimicrobial sensitivity was reported to Ciprofloxacin, Imipenem, Amikacin, Ceftriaxone and Gentamicin this was in accordance to the finding of (Zinnah, 2008;Bradford et al., 1999) and (Chattopadhyay et al. 2003) also reported high sensitivity against ciprofloxacin which show similar finding as in present study. Some drugs as Table 1, shows sensitivity pattern similar to (Sharma, 2004). Present study shows high resistance to Ampicillin, Amoxicillin, Streptomycin and Tetracycline. The findings of present study are in agreement with earlier reports (Ajayi, 2011;Abdulla et al., 2013;Lim et al., 2007). Finding of drug resistance of drugs shows in Table 1, was also found similar as given (Muffling et al., 2007). However Enrofloxacin resistant was reported by (Shah, 1989;Khan et al., 2002). This increasing resistance pattern of E.coli may be attributed to the over use and non judicious use of various antimicrobials. Commensal E.coli face various selective pressures in the environments of intestine which further favours the development, persistence and dissemination of robust strains that may be resistant to antimicrobial agent.