Basana Sarker
Department of Microbiology, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh
Mohammad Arif
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Broiler chickens, Aeromonas hydrophila, Molecular identification, Antibiogram profile
Mymensingh Sadar: KR market, Bangladesh Agricultural University (BAU) and Shesh mor bazar, Mymensingh, Bangladesh
Animal Health and Management
Sample collection and processing:
This study was conducted during the period from July to November, 2019 to isolate Aeromonas hydrophila from different broiler samples in the laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh. A total of 60 samples from 20 broiler chickens were collected from two different locations of Mymensingh Sadar: KR market, BAU and Shesh mor bazar (10 birds from each location). The samples included 20 skins, 20 legs and 20 breast samples from 20 broiler chickens. Collected samples were immediately brought to the laboratory maintaining proper cool chain and processed as early as possible with 1% alkaline peptone water (HiMedia).
Cultural and biochemical characterization:
Isolation of Aeromonas hydrophila from boiler samples was performed following the procedures described by Koca and Sarimehmetoglu (2009) with some modifications. 25 g of each samples were taken, placed in sterile zipper bags and homogenized with 225 ml of 1% alkaline peptone water (HiMedia) and incubated at 30ºC for 24 hours. After incubation, enrichment fluid was streaked on Aeromonas selective agar (HiMedia) with ampicillin supplement and incubated at 30ºC for 24 hours. Following the incubation, dark green centered green translucent colonies were further sub-cultured until pure culture of bacteria was obtained. Presumptive Aeromonas hydrophila isolates were stored in 20% glycerol at –800C until further use. The isolated bacteria were identified according to their biochemical characteristics (Ahammed et al., 2016; Samal et al., 2014).
Molecular identification of Aeromonas hydrophila by PCR:
Template DNA preparation was carried out by boiling method. Cultures were grown in nutrient broth at 37ºC for 24 hrs and 1 ml of the overnight culture was centrifuged at 5,000 rpm for 3 minutes using eppendorf tubes. Supernatant was carefully removed and the pellet was resuspended with 200 μl of sterile TE buffer, boiled at 100ºC for 15 minutes and immediately incubated on ice for 10 minutes. The mixture was then centrifuged at 12,000 rpm for 10 minutes and the supernatant with template DNA were then transferred into sterile tubes and stored at -80oC for PCR amplification.
Antimicrobial susceptibility testing:
Eight different antimicrobial discs: ampicillin (10 μg), chloramphenicol (30 μg), co-trimoxazole (25 μg), ciprofloxacin (5 μg), erythromycin (15 μg), gentamycin (10 μg), imipenem (10 μg) and tetracycline (30 μg) were selected for the antimicrobial susceptibility test against 12 isolated Aeromonas hydrophila. All the antimicrobial discs were purchased from HiMedia, India. Antibiotic susceptibility of the isolates was determined using the disc diffusion or Kirby–Bauer method (Bauer et al., 1966). Stock cultures of the bacterial strains were grown on TSA for 24 h at 37oC. Then colonies of each of the isolates were adjusted to 0.5 McFarland’s turbidity standard (equivalent to 1x108 colony-forming unit/ml) in sterile phosphate-buffered saline (PBS) and the bacterial suspension was spread onto Mueller–Hinton agar (Oxoid). Antibiotic-impregnated discs were kept on the solid medium and the plates were incubated at 37oC for 24 h. Zone of inhibition formed around the discs was measured and antibiotic sensitivity was assayed from the length of the diameter of the zones (in mm). The zone radius was actually scaled from the centre of the antibiotic disc to the end of the clear zone where bacteria could be seen growing. Tested bacterial strains were classified into three categories: sensitive, intermediate, and resistant depending on the diameters of inhibition zones and standards supplied by HiMedia Laboratories and comparing with other related references.
Asian Australas. J. Food Saf. Secur. 2020, 4 (1), 22-30; ISSN 2523-1073 (Print) 2523-2983(Online)
Journal