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Molecular Identification and Antibiogram Profiles of Escherichia Coli Isolated from Apparently Healthy and Diarrheic Goats

F. Begum
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Sohidullah
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. M. L. Kabir
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. T. Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

The present study was designed for the cultural, biochemical characterization and molecular detection of E. coli from apparently healthy and diarrheic goats in and around BAU campus including their antibiogram study. A total of 50 fecal samples were collected among which 13 originated from diarrheic goat and 37 from apparently healthy goats. Out of 50 samples, 35 were found positive for E. coli i.e., overall 70% occurrence. Occurrences of E. coli from diarrheic and apparently healthy goats were 92% and 62% respectively. Occurrences were 60%, 80% and 70% in case of BAU Goat Farm, Veterinary Teaching Hospital and Boyra respectively. On age basis 93%, 54%, 66% and 54% samples originated from 6 months, 7-12 months, 13-18 months and 19 months aged goats were found positive respectively. Occurrences of E. coli on the basis of sex were 78% for male and 62% for female. In case of breed, the occurrences were 69% in Black Bengal and 100% in for Jamunapari. Molecular detection was done by PCR and 13 out of 20 isolates tested gave the bands at the 585 bp specific for E. coli 16S rRNA gene. All the isolates (100%) were found sensitive to ciprofloxacin and norfloxacin; 100% and 35% were intermediately resistant to tetracycline and gentamicin respectively and 25% isolates were resistant to streptomycin. Ciprofloxacin and norfloxacin were found to be the best choice of antibiotics for the treatment of colibacillosis in goats in the study area.

Keywords: E. coli, Goats, Occurrence, PCR, Antibiogram profile

Location: Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh

Start Date: 00-01-2016

End Date: 00-05-2016

Program Area: Animal Health and Management

Commodity: Goat

Objectives:

To isolate and molecular detection of E. coli inhabiting in feces of goats and to assess antibiogram profiles of the isolated E. coli.

Methodology:

This research work was conducted during the period of January, 2016 to May, 2016 in the Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh. A total of 50 rectal swab samples were aseptically collected from the diarrhoeic goats of BAU, Mymensingh and surrounding area. Among 50 samples, 20 originated from BAU Goat Farm (19 apparently healthy and one 1 diarrheic goat), 20 from Veterinary Teaching Hospital, BAU (10 apparently healthy and 10 diarrheic goats) and 10 from Boyra, Mymensingh (8 apparently healthy and 2 diarrheic goats). After collection, the samples were transported to the Bacteriology, Department of Microbiology and Hygiene, BAU as soon as possible for the bacteriological examinations and further characterization. The isolation and identification of E. coli were performed according to the method described by Cowan (1985).The isolated bacteria was initially identified as E. coli by observing their cultural characteristics, morphology by Gram’s stain, biochemical tests, motility test and finally confirmed by PCR through amplification of E. coli specific 16S RNA gene. Extraction of DNA from the E. coli was carried out by conventional boiling and rapid cooling method (Medici et al., 2003). In brief 200 µl deionized water was taken into an eppendorf tube, a pure colony of E. coli from nutrient agar was mixed with the deionized water. The tube was then transferred to boiling water for 10 minutes then it was immediately taken to the icebox for cold shock about 10 minutes. After cold shock centrifugation at 10,000 rpm for 10 minutes was done. The supernatant was collected which was used as template DNA. Details of the oligonucleotide primers used for the amplification of 16S rRNA gene of E. The PCR reaction mixture (25µl) for E. coli was prepared using 12.5 µl master mixture (Promega, USA), 10 pmol primer (Bioneer, South Korea) of each, 5µl DNA template and 5.5 µl nuclease free water. The cycling conditions consisted of initial denaturation for 5 minutes at 95°C, followed by 30 cycles of denaturation at 94°C for 0.5 minutes, annealing at 58°C for 1 minute and extension at 72°C for 1 minute. The final extension was conducted at 72°C for 10 minutes. Amplification was performed in a thermal cycler (Astec, Japan). The amplified products were electrophoresed into 1.5% agarose (Sigma-Aldrich, USA) gel at 80 volt for 45 minutes and then visualization was done under Gel doc/UV trans-illuminator (BioRad).100 bp DNA size marker (Promega, USA) was used. Antibiogram study was performed by employing the Kirby-Bauer disc diffusion method (Bauer et al., 1959) using five different commercially available antibiotic discs (HiMedia, India and Oxoid Ltd., England) on Mueller-Hinton agar (HiMedia, India) to assess the susceptibility and resistance pattern of the E. coli isolates. The selected antibiotics used were ciprofloxacin (5 µg/disc), gentamicin (10 µg/disc), norfloxacin (10 µg/disc), streptomycin (10 µg/disc), and tetracycline (30 µg/disc).The interpretation on susceptibility was done according to the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2012). In this study, multidrug resistant E. coli was identified by considering resistant to 2 or more drugs.

Source of Information: Bangl. J. Vet. Med. (2016). 14 (2): 203-208 ISSN: 1729-7893 (Print), 2308-0922 (Online)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

E. coli were successfully isolated from healthy and diarrheic goat and confirmed by PCR. Overall occurrence of E. coli was 70%. Based on the present study, it may be concluded that use of ciprofloxacin and norfloxacin will be of first choice of treatment against E. coli infection in goats located at the study area. Findings of this study have suggested that multidrug resistant E. coli isolated from goat might be an important concern for veterinary practitioners. Nevertheless, more studies are needed to clearly understand the genomic diversity in E. coli as well as molecular mechanisms of virulence and development of antimicrobial resistance.

Knowledge Management: Journal