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Detection of Multi-drug Resistant Salmonella from Milk and Meat in Bangladesh

M. A. Rahman
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh & Department of Medicine, Surgery and Obstetrics, Patuakhali Science and Technology University, Babugonj, Barisal, Bangladesh.

A. K. M. A. Rahman
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. A. Islam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Alam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

This study was conducted to investigate the prevalence of Salmonella spp. in milk, chicken meat and beef and to determine the multi-drug resistance (MDR) profile of Salmonella spp. in Mymensingh and Gazipur districts, Bangladesh. A total of 169 samples of milk (n=108), chicken meat (n=51) and beef (n=10) were collected from Bangladesh Agricultural University (BAU) dairy farm, American dairy farm, Gazipur and different small dairy farms of municipal area during July 2016 to June 2017. Salmonella spp. were isolated on various selective agar media such as: Salmonella-Shigella (SS) agar, Xylose-Lysine Deoxycholate (XLD) agar, Eosine-Methylene Blue (EMB) agar. Identification of Salmonella spp. was done by colony characteristics, Gram staining, biochemical test and Polymerase Chain Reaction (PCR). Multi-drug resistant Salmonella spp. was detected by disc diffusion test using 10 commonly used antibiotics. The overall prevalence of Salmonella spp. in all food samples was 21.89%. A total of 29 (56.86%) chicken meat, 02 (1.85%) milk, and 06 (60%) beef samples were Salmonella spp. positive. Antibiogram study showed that an overall 89.19% of Salmonella spp. was found multi-drug resistant. Specifically 100%, 66.67% and 93.10% of the Salmonella spp. isolates originated from milk, beef and chicken meat respectively were multi-drug resistant. The result of this study suggests that MDR Salmonella spp. is prevalent in the milk and meat which might cause public health hazard if proper hygienic measures are not undertaken at farm and marketing level.

Keywords: Salmonella, Milk, Beef and Chicken meat, Antibiogram, Multidrug resistance, PCR.

Location: Mymensingh and Gazipur district of Bangladesh

Start Date: 00-07-2016

End Date: 00-12-2017

Program Area: Animal Health and Management

Commodity: Diseases

Objectives:

To the presence of Salmonella including its antimicrobial-resistant profiles in foods of animal origin like chicken meat, beef and milk in Mymensingh and Gazipur districts.

Methodology:

The samples were collected randomly from farms and local markets situated in Mymensingh and Gazipur district of Bangladesh. A total of 169 samples comprised of poultry meat (n=51), beef (n=10) and milk (n=108) samples were tested from July 2016 to December 2017. Aseptically collected meat samples were placed in sterile plastic bags and transported to the Department of Medicine using an icebox. Milk samples were collected from Bangladesh Agricultural University (BAU) dairy farm, American dairy farm, Gazipur, and several small dairy farms. Aseptically 8-10 ml of milk was collected in test tube directly from teat of lactating cow and local market and transported to the Department of Medicine using an icebox. Five to ten grams of chicken (breast and cloacal muscle) meat and beef were collected aseptically in zipper bag from local municipal market and transported to the laboratory using ice box. Ten to twenty grams of meat were mixed with 10 ml of peptone (0.1%) water then homogenized suspension was prepared using sterilized pestle and mortar. The homogenized meat suspension (4 to 5 ml) was inoculated into nutrient broths (5 ml/test tube), nutrient agar, SS agar, XLD agar and EMB agar). The inoculated media were incubated at 37⁰C for 24±2 hours. Culture positive samples were subcultured several times to be pure culture. Gram staining and biochemical tests and sugar fermentation test were performed (Cheesbrough, 1985). The DNA of Salmonella spp. was extracted by heat lysis method with slight modification (Momtaz et al., 2012). The PCR assays were performed to detect Salmonella spp. using primer pairs. The nucleotide sequence (5’-3’) for the Salmonella common primer used for this study were: R- ATG TTG TCC TGC CCC TGG TAA GAG and F- ACT GGC GTT ATC CCT TTC TCT TGG with an amplicon size of 496 bp (Al-Salauddin et al., 2015). Briefly, for the detection of 16SrRNA gene PCR reaction mixture were 25 μl consisting of 5.5 μl RNAse free water, 12.5 μl PCR master mixture (Thermo Scientific, EU), 5 μl DNA template, 1 μl (20 pmol) primer. The PCR amplification was done by Initial denaturation at 95⁰C for 5 minutes followed by 30 cycle of denaturation at 95⁰ C for 30 seconds, annealing at 62⁰C for 1 minute and extension at 72⁰C for 1 minute. The final extension was at 72⁰C for 10 minutes. PCR amplify products were subjected to gel (1.5% agarose, Takara, Japan) electrophoresis with ethidium bromide fluorescence (100 v for 30 minutes) and visualized in gel documentation system via UV transilluminator (302 nm). Antimicrobial susceptibility of Salmonella spp., was performed by the disc diffusion according to the guidelines of the CLS1 (2012). Ten commercially available antibiotics (Oxoid, UK) e.g., oxytetracycline (30μg), ciprofloxacin (5μg), gentamicin (10μg), erythromycin, (15μg), azithromycin (15μg), sulphonamide-trimethoprim (25μg), neomycin (10μg), amoxicillin (10μg), doxycycline (10μg) and amikacin (30μg) were used for antibiogram study.

Source of Information: Bangl. J. Vet. Med. (2018). 16(1): 115–120

Article Link (Online Journal): DOI: 10.3329/bjvm.v16i1.37388

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The presence of Salmonella spp. in food indicates contamination with polluted water; the improper handling, processing and storage and poor personal hygiene and sanitation. Salmonella isolates of food animal and poultry origin found to be resistant to drugs commonly used in humans like azithromycin. This indicates interspecies transmission of resistance gene which might pose serious public health threats. The antibiogram profile of the isolates will help choosing the most effective antibiotics to treat the diseases conditions caused by Salmonella spp. in cattle and poultry in Bangladesh.

Knowledge Management: Journal