Isolation and identification of Staphylococcus aureus from milk and milk products and their drug resistance patterns in Anand, Gujarat

Aim: The study was carried out with aim to isolate Staphylococcus aureus from milk and milk products (pedha and curd) and determine antibiogram pattern of S. aureus isolates. Materials and Methods: During 9 months duration of study a total of 160 milk and milk product samples (pedha and curd) were collected from different places in and around Anand city such as milk collection centre of Co-operative milk dairies, cattle farms, individual household, milk vendors and sweet shops. The samples were collected under aseptic precautions and were enriched in Peptone Water (PW) followed by direct plating on selective media viz. Baird-Parker Agar. The presumptive S. aureus isolates were identified by biochemical tests. Antibiogram pattern of S. aureus to antimicrobial agents were evaluated by disk diffusion method. Results: Analysis of result revealed that out of total 160 samples of milk (100) and milk products i.e. curd (30) and pedha (30) resulted in the isolation of 10 isolates (6.25 %) of S. aureus. In the present study S. aureus isolates were found variably resistant to the antibiotics tested. The S. aureus isolates showed highest sensitivity towards cephalothin (100.00 %), co-trimoxazole (100.00 %), cephalexin (100.00 %) and methicillin (100.00 %) followed by gentamicin (90.00 %), ciprofloxacin (80.00 %), oxacillin (70.00 %), streptomycin (60.00 %) and ampicillin (60.00 %). The pattern clearly indicated that the overall high percent of S. aureus isolates were resistant to Penicillin-G (100.00 %) followed by ampicillin (40.00 %), oxytetracycline and oxacillin (20.00 %) and streptomycin and gentamicin (10.00 %) Conclusions: Results clearly suggested a possibility of potential public health threat of S. aureus resulting from contamination of milk and milk products with pathogenic bacteria is mainly due to unhygienic processing, handling and unhygienic environment.


Introduction
milk products can cause severe health hazards to people as they are highly susceptible to variety of Food-borne diseases (FBD) are defined by the microorganism because of high nutritive value and World Health Organization as diseases of infectious or complex chemical composition [2].toxic nature caused by, or thought to be caused by the Many contaminants find their way to raw milk, consumption of food or water.The pathogenesis of from which they gain access to dairy products [3,4].bacteria causing food-borne poisoning depends on Chapaval [5] found production of staphylococcal their capacity to produce toxins after ingestion (in the enterotoxins in milk when milk was stored at tempdigestive tract) or intoxication (ingestion of preformed eratures of 37 ºC to 42 ºC or when exposed to variations toxins in foodstuff).Among the bacteria predominantly in temperature.involved in these diseases, Staphylococcus aureus is a Staphylococcal food poisoning include symptoms leading cause of gastroenteritis resulting from the such as sudden onset of nausea, vomiting, abdominal consumption of contaminated food.Staphylococcal cramps and diarrhea [6].food poisoning is due to the absorption of Staphylo-On heating at normal cooking temperature, the coccal enterotoxins preformed in the food [1].
bacteria may be killed but the toxins remains active Milk and milk products are the prime habitat to [7].Staphylococcal enterotoxins are highly heat complex microbial ecosystems; these are responsible resistant and are thought to be more heat resistant in for the broad variations in taste, aroma and texture of foodstuffs than in a laboratory culture medium [8].milk and milk products.Contamination of milk and Besides these, enterotoxins producing S. aureus milk products with pathogenic bacteria is mainly due to are most dangerous and harmful for the human health.processing, handling and unhygienic environment.The About 50 % strain of this organism are able to produce occurrence of these pathogenic bacteria in milk and enterotoxins associated with food poisoning [9].Illness through S. aureus range from minor skin infection such The selective medium used for isolation of S. as pimples, boils, cellulites, toxic shock syndrome, aureus was Baird Parker Agar (BPA) (HiMedia Pvt.impetigo, and abscesses to life threatening disease such Ltd.).A loopful of inoculum from enrichment were as pneumonia, meningitis, endocarditis, and septicaemia streaked on BP agar and incubated for 48 hours at 37°C.[2].Especially in India, rate of infection is still higher Characteristic appearance of jet black colonies because of warm and humid climate [10].For many surrounded by a white halo were considered to be years, S. aureus was the only staphylococcal species presumptive S. aureus (Fig- 1).The pure cultures were known to produce enterotoxins [11].
streaked on Nutrient agar (HiMedia Pvt.Ltd.) and Thus, the objective of this study was to investigate incubated for 24 hours at 37°C and were further the occurrence of S. aureus in milk and milk products characterized by biochemical tests.(pedha and curd) and determine antibiogram pattern of Morphological characteristics: The smear was prepared S. aureus isolates.
from the isolated culture on clean grease free microscopic glass slide and stained with Gram's method of staining.

Materials and Methods
The stained smear was observed under microscope.

(60%) and ampicillin (60%). The pattern clearly indicated
This study presents the sensitivity of the S. aureus that the overall high percent of S. aureus isolates were isolates towards life saving drugs, viz., cephalothin, resistant to Penicillin-G (100%) followed by ampicillin cephalexin and co-trimoxazole followed by gentamicin (40%), oxytetracycline and oxacillin (20%) and and ciprofloxacin thus indicating the safety of food streptomycin and gentamicin (10 %).
bacteria poses a problem of concern.Effectiveness of Discussion current treatments and ability to control infectious diseases in both animals and humans may become Milk is normally sterile in the udder of the cow hazardous.and buffalo provided they do not suffer from mastitis (udder infection).If they have mastitis, a large number

Conclusion of generally Gram positive bacteria such as
Staphylococcal food poisoning is of major concern Streptococcus and Staphylococcus spp.may be present in public health programs worldwide.S. aureus may be in milk when it leaves the udder [14].
present in milk and milk products as a result of milk Negligence of hygienic condition such as improper collected from the animal suffering from disease cleaning of bulk tank, dirty udder, milking equipments, condition and excreting S. aureus in milk or due to milk handling technique and improper storage will unhygienic conditions during production, processing, increase the proportion of Gram-positive and Gramstorage and handling of milk products, which are the negative bacteria in the bulk tank milk [15,16].main causes of food borne diseases.Results clearly Food products serve not only as a source of indicated that milk and milk based products available nutrition but also as substrates for the growth of in the market were contaminated with S. aureus, posing microorganisms.The growth of microorganisms causes a high risk of food poisoning.Thus more hygienic food spoilage.It may result in food-borne illness.In preventive measures are required to reduce the bacterial tropical countries raw milk and milk products are contamination, so as to increase the wholesomeness of responsible for many outbreaks of gastrointestinal these milk and milk based products.tract.It is also reported that immunocompromised The indiscriminate use of antibiotics/antiindividuals are prone to food-borne infection [17].microbials agents for prophylactic as well as other Of the 100 milk samples, 30 pedha and 30 curd therapeutic purpose could be the reasons for increased samples, 6 (6%), 3 (10%) and 1 (3.33 %) respectively antimicrobial resistance of S. aureus.A relatively high were found to be positive for S. aureus.The highest number of strains are also resistant to the antibiotics incidence of S. aureus was from pedha (10%), followed commonly used in the therapeutic protocols of many by milk samples (6%) and curd (3.33 %).
human and animal infections.This study highlights the The present overall isolation rate in milk and milk products was 6.25% which seems to be similar to the need for continuous surveillance of antibiotic findings of 7.3% by Fagundes [18], 8.3 % by Shah [19] sensitivity pattern of Staphylococcus aureus with a and 6.6% by Kumar and Prasad [20].As compared to view to selecting appropriate therapy.present findings higher level of incidence of S. aureus Region.Acta Agri.Slov., 92(1): 83-88.manuscript for publication.All authors read and 13.Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck,M.approved the final manuscript.(1966).Antibiotic susceptibility testing by a standardized single disk method.Amer.J. Clin.Pathol., 45: 493-496.

Sample collection: From
February 2011 to October Smear revealed Gram positive, spherical cells arranged 2011, a total of 160 milk and milk product samples in irregular clusters resembling to bunch of grapes.which includes raw milk (100 samples), curd (30 Biochemical examination: Biochemical tests were samples) and pedha (30 samples) were collected from performed to confirm S. aureus using Catalase test, different places in and around Anand city such as milk Coagulase test, DNase test, Acetoin production, collection centres of Co-operative milk dairies, cattle Oxidase test and D-mannitol fermentation.farms, individual household, milk vendors and sweet shops.The samples were collected in sterilized milk

Table - 1
. Morphological and culture characteristics of S. aureus

Isolated bacteria Gram staining Culture characteristics on selective media Staphylococcus
aureus Gram positive cocci (in clusters)BPA: Typical jet black colonies surrounded by halo zone