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Multi-drug Resistant Staphylococcus Aureus Isolated from Milk, Chicken Meat, Beef and Egg in Bangladesh

Mohammad Anisur Rahman
Department of Medicine, Surgery and Obstetrics, Patuakhali Science and Technology University, Babugonj, Barisal, Bangladesh

A. K. M. Anisur Rahman
Department of Medicine, Surgery and Obstetrics, Patuakhali Science and Technology University, Babugonj, Barisal, Bangladesh

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

Md. Mahbub Alam
Department of Medicine, Surgery and Obstetrics, Patuakhali Science and Technology University, Babugonj, Barisal, Bangladesh

Abstract/ Executive Summary:

Staphylococcal infection is one of the most common food-borne diseases in the world. Moreover antimicrobial resistance of pathogenic bacteria, including Staphylococcus (S.) aureus is an emerging problem of food safety. This study was conducted to investigate the prevalence of S. aureus in milk, chicken meat, egg and beef; and to determine the multi-drug resistance (MDR) profile of S. aureus in Mymensingh and Gazipur districts, Bangladesh. A total of 189 samples of milk (n=108), chicken meat (n=51), egg (n=20) and beef (n=10) were collected from Bangladesh Agricultural University dairy farm, American dairy farm, Gazipur and different dairy farms of municipal area and retail shops during July 2016 to June 2018. S. aureus were isolated and identified by conventional methods and polymerase chain reaction (PCR). Antimicrobial susceptibility tests were done through disc diffusion test using 10 commonly used antibiotics. The overall prevalence of S. aureus in all food samples was 43.39%. A total of 39 (76.47%) chicken meat, 25 (23.15%) milk, 11(55%) egg and 07 (70%) beef samples were S. aureus positive through conventional method. Among 82 culture positive samples only 39 samples (47.56%) were confirmed by PCR. Antibiogram study showed that S. aureus isolated from chicken meat were mostly resistant to oxytetracycline (71.79%); and highly sensitive to amikacin (100%) and neomycin (100%). S. aureus isolated from milk samples were highly sensitive to neomycin (100%), and resistant to amikacin (56%). Only 28.57% isolates of S. aureus originated from beef samples were resistant to oxytetracycline and 100 % isolates were sensitive to ciprofloxacin, gentamicin, erythromycin, azithromycin, doxycycline. Similarly, S. aureus isolated from egg samples were resistant to erythromycin (81.82%) and 100% sensitive to amikacin. Out of 41.46% MDR isolates 12%, 53.85%, 90.91% and 0% of the S. aureus originated from milk, chicken meat, egg and beef respectively. The higher prevalence of S. aureus in chicken meat, beef, egg and milk indicates unhygienic production, marketing and processing of these foods. Presence of MDR S. aureus in these foods might pose serious public health threats. Rational use of antibiotics with higher sensitivity should be prescribed in managing poultry diseases to reduce re-emerging MDR in Bangladesh

Keywords: S. aureus, Beef, Chicken meat, MDR, PCR

Location: Mymensingh and Gazipur districts, Bangladesh

Start Date: 00-07-2016

End Date: 00-06-2018

Program Area: Animal Health and Management

Commodity: Beef, Chicken, Milk

Objectives:

To determine the prevalence of S. aureus along with multiple-drug resistance profile in in chicken meat, egg, beef and milk in Bangladesh.

Methodology:

The samples were collected randomly from farms and local markets situated in Mymensingh and Gazipur district of Bangladesh. A total of 189 (51 poultry meat, 20 egg, 10 beef and 108 milk) samples have been tested from July 2016 to June 2018. Milk samples have been collected from BAU dairy farm, American dairy farm, Gazipur, surrounding other local small dairy farm. Aseptically 8-10 ml of milk was collected in test tube directly from teat of lactating cow and local market and send to the Medicine laboratory using icebox. Ten to twenty grams of chicken breast meat or beef were collected aseptically in sterile zipper bag and send to the Medicine laboratory using ice box. Egg aseptically collected with sterilize zipper bag from different retail shop of municipal market, Mymensingh. 10 to 20 grams of meat were mixed with 45 ml of peptone (0.1%) water then homogenized suspension was prepared using sterilized pestle and mortar. Egg surface were washed with peptone water (0.1%) within zipper bag. The homogenized suspensions (4 to 5ml) and egg washing water were then transferred into nutrient broths (5 ml/test tube) separately, nutrient agar and other selective media (MSA). In every step, samples were incubated at 37⁰C for 22-24 hours. The positive samples were then subculture several times to obtain pure S. aureus. Gram staining and Biochemical test (five sugar fermentation test) were done to be confirmed (Cheesbrough, 1985). Antibiotic disc diffusion test were done for S. aureus in Muller- Hinton agar (Hi-Media, India) according to CLSI, 2012. DNA extractions were performed through boiling method (Goering et al., 2008) with some modification. One or two single colonies of Staphylococcus spp. were suspended in 200 μl of distilled water and boiled for 10 minutes in a heat block, then placed on ice for 10 minutes. After centrifugation at 10,000 rpm for 10 minutes at 4⁰ C, the supernatant (150 μl) was placed in an eppendorf tube and kept at -20⁰ C until used as a DNA template. PCR assay were applied in all the 82 isolates to confirm the Staphylococcus sp. and S. aureus based on 16S rRNA and nuc gene respectively. PCR reaction mixture were 25 μl consisting of RNAse free water 5.5 μl, PCR master mixture (Thermo Scientific, EU) 12.5 μl, genomic DNA 5 μl and primer (Reverse and Forward) 2 μl. The PCR protocol consisted of the following steps : i) Denaturation- 94⁰C for 1 min, ii) 30 cycles of 94⁰ C for 30 second, 55⁰C for 30 second, 72⁰C for 1 min, iii) Final Elongation 72⁰C for 05 minute. 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 (Biosciences, Germany) via UV transilluminator (302 nm). Antimicrobial susceptibility of S. aureus, was performed by the disc diffusion test applied on Muller-Hinton agar (Hi-media, India) in vitro using 10 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) according to the guidelines of the CLS1 (2012).

Source of Information: Res. Agric. Livest. Fish. Vol. 5, No. 2, August 2018 : 175-183

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The higher prevalence of S. aureus in milk, chicken meat, egg and beef indicates unhygienic production and processing of these foods. Presence of multi-drug resistant S. aureus in these foods may pose serious public health threats and this study may help the clinician as well as physician to take proper therapeutic decision to treat the cattle and poultry affected by Staphylococcus spp. in Bangladesh.

Knowledge Management: Journal