Md. Kamrul Islam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
S. M. Lutful Kabir
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
A. K. M. Ziaul Haque
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Y. A. Sarker
Department of Pharmacology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
M. H. Sikder
Department of Pharmacology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
The study was conducted to isolate, identify and characterize bacterial samples from broiler meat collected from 20 different upazilla markets of Jamalpur, Tangail, Netrokona and Kishoreganj districts of Bangladesh. A total of 20 samples were subjected to bacteriological isolation and identification, and the isolated bacteria were subjected to antimicrobial susceptibility testing using disc diffusion method. Among the samples, 75% (n=15) were contaminated with Campylobacter spp., 70% (n=14) were with Salmonella species and 85% (n=17) were contaminated with Escherichia coli. The Campylobacter spp., Salmonella spp. and E. coli were isolated and identified by culturing on Blood agar, Xylose-Lysine Deoxycholate (XLD) agar, and MacConky and eosin methylene blue (EMB) agar respectively. Isolates of Campylobacter spp., Salmonella spp. and E. coli were biochemically analyzed. Campylobacter specific 16S rRNA genes were amplified from the isolates. Campylobacter spp. and E. coli isolates were positive to 16S rRNA gene based polymerase chain reaction (PCR). Almost all isolates of Campylobacter spp., Salmonella spp. and E. coli showed highest susceptibility to ciprofloxacin, norfloxacin and gentamicin. However, most isolates were resistant to amoxicillin and erythromycin. Some isolates showed susceptibility to tetracycline, streptomycin and azithromycin. The findings of this study revealed that there is presence of multidrug resistant isolates of Campylobacter spp., Salmonella spp. and E. coli in broiler meat. Results of this research project demonstrated the high level of microbial contamination and occurrence of pathogenic bacteria in broiler meat sold in markets of Bangladesh.
Broiler, Escherichia coli, Salmonella, Campylobacter, Molecular detection
Jamalpur, Tangail, Kishoreganj and Netrokona district of Bangladesh.
Animal Health and Management
Broiler, Diseases
A total of 20 apparently healthy dressed broilers were collected randomly from 20 different upazilla live bird markets of Jamalpur, Tangail, Kishoreganj and Netrokona district of Bangladesh. Five upazilla were selected randomly from each district. After collection, samples were immediately brought to Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh through maintaining cool chain using ice box. Solid and liquid culture media were used to isolate the bacteria. Blood agar (BA), MacConkey (MC), Salmonella-Shigella (SS), Eosin Methylene Blue (EMB), Xylose-Lysine Deoxycholate (XLD), Mueller Hinton agars were used as solid culture media for this study. The liquid media used in the study were nutrient broth (NB), peptone broth, methyl-red and voges-proskauer broth (MR-VP), and sugar media, 1% hippurate solution, 3.5% ninhydrin solution, oxidase solution and sugar media. Pure culture of E. coli and Salmonella spp. were obtained as per the methods described by Krieg et al. (1994). Briefly; 10 g of samples were homogenized with 90 ml of 0.1% peptone water and 50 µl of homogenized sample was poured on to selective agar media and spread with glass spreader and incubated at 37°C for 24 h. Isolation of Campylobacter spp. was carried out by filtration method (0.45 μm filter) according to Shiramaru et al. (2012). The collected samples were allowed to prepare meat homogenates and then 50 μl of meat homogenates were spread on the filter papers that were placed on the surface of Blood base agar no.2 and allowed to stand for 30 min at room temperature, after 30 min filter papers were removed from the BA and the plates were incubated at 37°C for 48 h in microaerobic condition (5% O2, 10% CO2 and 85% N2). The colonies of primary cultures were repeatedly sub-cultured by streak plate method (Cheesbrough, 1985) until the pure cultures with homogenous colonies appeared. The representative bacterial colonies were characterized morphologically using Gram's stain according to the method describe by Merchant and Packer (1967). Biochemical characterizations of the E. coli and Salmonella isolates were performed with Sugar fermentation test, Methyl Red test (MR), Voges-Proskauer test (V-P) and indole test (Cheesbrough, 1985). Differentiation of isolated Campylobacter spp. with supporting growth characteristics were subjected to various biochemical tests such as catalase, oxidase and hippurate hydrolysis test according to the procedures followed by Nachamkin (2003) and Foster et al. (2004). DNA template was prepared by boiling method as described by Rawool et al. (2007). All the samples were examined by two pairs of primers to detect 16S rRNA gene of Campylobacter spp., E. coli and Histidine transport operon gene of Salmonella spp. For Campylobacter spp. the PCR reactions were carried out using a thermocycler (ASTEC, Japan) with the following programme: initial denaturation for 5 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 47°C for 30 s and extension at 72°C for 1 min and 30 s. The final extension was conducted at 72°C for 10 min. For E. coli, the PCR reactions were carried out using a thermocycler (ASTEC, Japan) with the following programme: Initial denaturation for 5 min at 95°C followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 1 min. The final extension was conducted at 72°C for 5 min. For Histidine transport operon gene identification in Salmonella spp. initial denaturation for 5 min at 94°C, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 45 s. The final extension was conducted at 72°C for 5 min. 1 and 2% agarose (Invitrogen, USA) gel was used for electrophoresis of the PCR products. All isolates randomly selected from the three genera were tested for antimicrobial drug susceptibility against eight commonly used antibiotics by disc diffusion method according to the guidelines of Clinical and Laboratory Standard Institute (CLSI), 2012. The selected antibiotics used were ciprofloxacin (5 μg/disc), azithromycin (30 μg/disc), amoxicillin (30 μg/disc), gentamicin (10 μg/disc), Norfloxacin (10 μg/disc), erythromycin (30 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 Standard Institute (CLSI, 2007)
African Journal of Microbiology Research
Journal