MULTI-DRUG RESISTANT Staphylococcus aureus ISOLATED FROM MILK , CHICKEN MEAT , BEEF AND EGG IN BANGLADESH

PCR 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.


INTRODUCTION
S. aureus (S. aureus) is an opportunistic pathogen in human and other different animal species.The pathogen is mainly related to food poisoning and is the third largest cause of food related illness throughout the world (Sasidharan et al., 2011;Achi and. Madubuike 2007).A number of infectious diseases such as dermatitis, pneumonia, meningitis, osteomyelitis in human, bovine mastitis in cattle and bumble foot disease in poultry (Ali et al., 2017) are produced by enterotoxigenic strains of coagulase-positive staphylococci, mainly S. aureus (Hennekinne et al., 2012).Among the food borne outbreaks bacterial toxin were 9.8% of which 55.4% cases were due to staphylococcal enterotoxins (Anonymous, 2010).There are some reports on the prevalence of Staphylococcus organism in poultry (Rahman andSamad, 2003, Ali et al, 2017), in milk (Mueena et al., 2015), in restaurant (49.3%) and household (Islam et al., 2016) cockroachs (26.7%), in pet (Habibullah et al., 2017) animal (40.86%) , and in frozen meat (95.83%) rinse ( Islam et al.2014) in Bangladesh.
The use of antimicrobial compounds in food animal production provides improved animal health and reduces foodborne diseases in humans.However, use of antibiotics has been shown to contribute to the increased prevalence of antibiotic-resistant bacteria of human significance (Mathew et al., 2007).Multidrug resistant S. aureus in poultry meat have been reported in the USA (Andrew et al., 2011) and very limited research on antibacterial resistant in foods of animal origin (Ashrafudoulla et al. 2017) and no report was found on staphylococcal infection in beef in Bangladesh.Hence, this study was carried out to determine the prevalence of S. aureus along with multiple-drug resistance profile in in chicken meat, egg, beef and milk in Bangladesh.

Study area and Collection of samples
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.

Sample preparation
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.

Isolation and identification of S. aureus
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 37 0 C 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.

Polymerase chain reaction (PCR)
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 4 0 C, the supernatant (150 μl) was placed in an eppendorf tube and kept at -20 0 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 (Table 1) 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-94 0 C for 1 min, ii) 30 cycles of 94 0 C for 30 second, 55 0 C for 30 second, 72 0 C for 1 min, iii) Final Elongation 72 0 C 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).

Cultural, staining and biochemical characteristics
The cultural characteristics showed that S. aureus produce turbid growth on nutrient broth and smooth white to grayish white colony on nutrient agar, golden-yellow color on manitol salt agar.On Gram staining, S. aureus, were found as Gram positive coccus and arranged as grape like.All five basic sugars like dextrose, maltose, lactose, sucrose and mannitol were fermented with the production of acid without gas.Coagulase positive Staphylococcus was detected by observing clamp or precipitation in coagulase test.The positive catalase test of Staphylococcus spp was determined by producing gas bubbles.

Molecular detection
The result of PCR is presented in Figure 1 and Figure 2. The amplicon size of PCR product was 756 bp (16SrRNA gene) and 297 bp (nuc gene) reconfirmed as Staphylococcus sp. and S. aureus, respectively.

DISCUSSION
The overall prevalence of S. aureus in all food samples was 43.39%.We isolated S. aureus from 76.47% of chicken meat.Previous studies conducted in Bangladesh reported a wide range of S. aureus prevalence in chicken meat which varied from 24.56-95.83%(Islam et al., 2014;Datta et al., 2012;Sultana et al., 2014 andAndrew et al, 2011).This variation in prevalence with our findings might be due to the variation in the type of sample (Andrew et al, 2011) like raw and frozen sample.About 23% of our milk samples contained S. aureus.Mueena et al. (2015) also reported similar prevalence in milk sample (25.57%) sold from different open markets in Bangladesh.However, some authors reported higher prevalence (35.29-41.03%) of S. aureus in raw milk samples in Bangladesh (Tanzin et al., 2016); and in Tanzania (Jibril et al., 2018).We collected sample directly from teat which might reduce the chance of contamination in our samples.Around 70% of our beef samples were found to be contaminated with S. aureus.No inland literature was found about the prevalence of S. aureus in beef samples in Bangladesh.However, 39.7%, 9.4 % and 3% of the beef samples were reported to be contaminated with S. aureus in Sudan (Goja et al. 2013) , Ethiopia (Adugna et al. 2018) and Egypt (Osman et al. 2017) respectively.We detected S. aureus in 55% egg shell.However, other authors from Bangladesh reported higher prevalence of S. aureus from hen egg (66.67%) and duck egg (75%) in open market (Fardows et al. 2016;Fateha et al. 2018).These variations might be due to source of sample collection, hygienic condition of the farm and personal hygiene of the processors and handlers.Among the 82 culture positive samples only 39 (47.56%) samples were Staphylococcus spp.and 16 (19.51%)samples were S. aureus confirmed through PCR technique (Table 2).However, only 5.83% S. aureus in chicken and 15% S. aureus in beef were PCR positive out of conventional positive samples reported by Islam et  S. aureus isolated from our chicken meat samples were resistant to oxytetracycline (71.79%), azithromycin (64.10%) and erythromycin (58.97%); and sensitive to amikacin (100%), neomycin (100%), amoxicillin (94.87%), gentamicin (82.05%) and ciprofloxacin (71.79%).Similar findings also reported by other authors (Islam et al. 2016;Islam et al. 2014;Ali et al. 2017).S. aureus isolated from our milk sample were sensitive to neomycin (100%), gentamicin (92%) ciprofloxacin (92%), amoxicillin (88%), doxycycline (88%) azithromycin (84%) and oxytetracycline(80%), and resistant to amikacin (56%) and erythromycin (44%).Our results were supported by the findings of other authors (Islam et

CONCLUSION
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.

Table 3 .
Antibiogram study of Staphylococcus aureus isolates from different food samples

Table 4 . Detection of multi-drug resistant S. aureus Antibiogram applied Milk Chicken meat Beef Egg Total
al. 2016; Tanzin et al., 2016; Hoque et al. 2018) from Bangladesh. S. aureus isolated from our beef sample were resistant to oxytetracycline (28.57%) and 100 % sensitive to ciprofloxacin, gentamicin, erythromycin, azithromycin and doxycycline; and 85.71 % sensitive to sulphonamide-trimethoprim, neomycin, amoxicillin and amikacin.These results were in contrast with the report of Sultana et al. (2014) who reported S. aureus as resistant to ciprofloxacin.However, Ashrafudoulla et al. (2017) reported S. aureus as sensitive to gentamicin and ciprofloxacin in human patients.S. aureus isolated from our egg samples were 81.82% resistant to erythromycin, 72.73% resistant to ciprofloxacin, amoxicillin and sulphonamide-trimethoprim; and 100% sensitive to amikacin; 90.91% sensitive to gentamicin, azithromycin and neomycin.Similar to our report S. aureus were reported to be sensitive to gentamicin (Fateha et al. 2018; Ferdows et al., 2016).Ferdows et al. (2016) reported S. aureus as sensitive to ciprofloxacin which contradicts with our result.Similar to our result, Sangeda et al. (2017) reported S. aureus as resistant to ciprofloxacin and sulphonamide-trimethoprim in humans.We observed 41.46% of S. aureus isolates as MDR (includes 3 or more antibiotic resistant) in which 12%, 53.85%, 90.91% and 0% of the S. aureus isolates originated from milk, chicken meat, egg and beef respectively.Similar to our findings, Andrew et al. (2011) reported 52 % MDR in poultry meat.Other authors also reported 8.3 to 63% of S. aureus isolates as MDR in different food samples (Datta et al. 2012; Sultana et al. 2014; Sangeda et al. 2017; Hoque et al. 2018; Fateha et al. 2018).Ashrafudoulla et al. (2017) also found MDR S. aureus in human clinical samples.Indiscriminate and recurrent use of different antibiotic agents in veterinary clinical practices could be the cause of high level of MDR.