Antibiotic Resistance Profile for Staphylococcus Species Recovered from Milk S.F. Gad EL-Rab 1, K.M.Osman 2 and M.A.Kamel 3 (1) General Organization of Veterinary Medicine (G.O.V.S) (2) Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt (3) Department of Microbiology, National Research Center, Dokki Received: 3/2/2016 Accepted: 2/4/2016 ABSTRACT A total of 220 milk samples from buffaloes and cows (150 from buffaloe s and 70 from cows) were investigated. Samples were cultured on Baird Parker media for isolation of Staphylococcus, especially S.aureus from apparently normal, clinical and subclinical mastitis cases. The total isolates were 42(19) from raw milk samples (27 from buffaloes and 15 from cows) 220. The aim of this study is to characterize phenotypically Staphylococcus spp. The collected samples were taken from selected areas to increase the range of information available about antibiotic resistance profile. This enhances formulating strategies to reduce the spread of this bacterium and also avoiding its health hazard on animals and human beings. INTRODUCTION Milk and milk products are home to complex microbial ecosystem; these are responsible for the broad variations in taste, aroma and texture of milk and milk byproducts.contamination of milk and milk products with pathogenic bacteria is mainly due to processing, handling and unhygienic environment. Stapylococuss was one of those bacteria that can cause minor skin infections (pimples, boils, cellulites, toxic shock syndrome, impetigo and abscesses as well as life threatening disease (pneumonia, meningitis, endocarditis and septicemia) as reported by Somaroo et al. 2003. Staphylococci are bacteria generally sensitive to the majority of antibiotics.at the same time, it is recognized that staphylococci are extremely sensitive to antibiotics pressure.it is known that the indiscriminate use of antibiotics therapy rendering the antibiotic treatment is ineffective in humans and animals.also mastitis is one of the major causes of antibiotics use in dairy cows (Mitchell et al., 1998; DANMAP, 2003). The use of antimicrobial agents is associated with the risk of inducing resistance to antimicrobial agents among bacteria, reduction of cure rates after treatment of clinical mastitis and transmission of resistance to bacteria humans via food chain (WHO, 1997; Sol et al., 2000; Ungenmuch, 1999). Over 135 different microorganisms have been isolated from bovine intra mammary infections (IMI), but the majority of infections are caused by Staphylococci,Streptococci, and other gram negative bacteria ( Bradley,2002). Materials MATERIALS AND METHODS Samples: A total number of 220 raw milk samples were collected from different areas (Giza, Cairo, Kaliobia and Elfaioum).
Studied Animals Buffaloes A total of 150 examined normal, apparently normal and clinically mastitic lactating buffaloes. Cows A total of 70 examined normal, apparently normal and clinically mastitic lactating cows. The normal milk samples were collected from a milk processing factory at the receiving point while others were collected from different farms. Clinical Inspection of the Udder Udders of the cows were examined by visual inspection and palpation for the presence of any lesion,pain,hotness and swelling.in addition,milk from each quarter was withdrawn and checked for any change in color and consistency. California Mastitis Test (CMT) California mastitis test was conducted to diagnose the presence of sub-clinical mastitis by mastitis indicator test kit (Frieso-test) obtained from Impfstoff work Friesoythe Gmbh-Germany, and it was carried out according to Schalm et al.,1971. CMT Score N (Negative) T (Trace) 1 2 3 Interpretation Healthy Quarter Subclinical Mastitis Subclinical Mastitis Serious Mastitis Infection Serious Mastitis Infection Identification Milk samples were incubated 18-24 hr at 37 C then a loop full from the samples were cultured on Baird Parker medium (selective for Staphylococci ) agar base supplemented with egg yolk tellurite emulsion and then using quadrant streaking method according to national committee for clinical laboratory standards (NCCLS 1997) and (Quinn etal;1994) then incubated at 37 C for 24-48h. Typical Colonial Appearance of Staphylococci Colonies were 0.5-3mm in diameter, typical S.aureus colonies appear gray black shiny enriched by two halo opaque zone resulting from lipolysis and other translucence due to proteolysis. A typical S.aureus colonies appear brown or white, absence or presence of just a halo irregular shape. This is according to Baird Parker 1962, Lancette and Bennett; 2001. Microscopic Examination (Quinn et al., 2002) Suspected colonies stained with Gram stain examined microscopically under oil emersion lens to find Gram positive cocci arranged single or in clusters in pairs or tetrads grape like clusters. 011
Pure culture of the isolates were identified according to Cruickshank et al. 1975, Quinn.,et al 2002 and to Bergey's Manual of Systematic Bacteriology Volume 2. Biochemical Tests 1-Catalase Test: with a wooden applicator stick, inoculums from center of a suspected colony were transferred on glass side then one drop of hydrogen peroxide was added and observation of bubbles indicated a positive result. Staphylococci was catalase positive 2-Tube Coagulase Test: Aseptically 0.5ml of rabbit plasma with EDTAwas added to a bottom of a sterile tube, then 0.5 ml of 18-24hr. a pure broth culture of organism was added to tested tube and mixed by gentle rotation then, a tube placed in a37 C and formation of visible clot after 30 minutes to 4hr.considerd positive test. 3-Uresae Test: the surface of the Christensen's urea agar slant was streaked with pure culture of organism, and then incubated at 37 C for 18-24hr.a positive result was indicated by red coloration of yellow slant of medium. 4-Hemolysis on Sheep Blood Agar: Staphlococci isolates produce either alpha-hemolysin(narrow zone of clear hemolysis arroud the colony) or beta-hemolysin(broader outer zone of incomplete hemolysis). 5-Mannitol Salt Agar: inoculation of samples on mannitol salt agar and incubate 18-24hr. at 37 C.Change of pink medium to yellow color indicated a positive fermentation result. S.aureus (coagulase positive CPS) was mannitol fermenter but coagulase negative Staphylococci (CNS) non mannitol fermenter. 6-Oxidase Test: was performed to differentiate between Staphylococci and Micrococci. Oxidase test discs were applied on suspected colonies giving purple color around colonies in case of a positive test, Staphylococci positive oxidase test. 7-Nitrate Reduction Test: the isolates were inoculated into potassium nitrate medium an incubated for 96hr.immediately before equal volumes of sol. A and B were mixed to give the test reagent to the tested culture.then 0.1 ml of the test reagent was added to the test culture.a red color developing within a few min. indicated the ability of the organism to reduce nitrate. 8-Sugar Fermentation: the colonies were inoculated into 1 peptone water tubes using Bromocresol purple as indicator for acid production to which 1 solution of one of the following=g sugars was added: fructose,glucose, maltose,mannose,sucrose, trhlose and xylose the tubes were incubated at 37 C for 3days and the reactions were recorded for acid production. Antibiotic susceptibility: according to NCCL S 2007 and 2011 The following was determined by the standardized agar diffusion test on Muller Hinton agar (Oxoid) using the following disks (Oxoid): Gentamycin (10 μg)-erythromycin(15 μg)-penicillin g(10 I.U)-tetracyclines(30 μg) ampicillin+sulbectam(20 μg) oxacillin1 μg ciprofloxacin(5 μg)-vancomycin(30 μg)- chloramphenicol(30 μg).
RESULTS Table (2): the results of California mastitis test (CMT) of examined buffalos and cows milk samples Animal species examined lactating animals Apparently normal animals Normal animals (negative CMT) N= Subclinical mastitic animals(positive CMT) N= Clinically mastitic animals N= Buffaloes 150 35 23.3 90 60 25 16.6 Cows 70 45 64.3 10 14.3 15 21.4 Total 220 80 36.4 100 45.4 40 18.2 Table (3): Incidence of coagulase positive and coagulase negative Staphylococci isolates in buffalos and cows milk samples Animal species isolates coagulase positive isolates coagulase Negative isolates Buffaloes 27 7 26 20 74 cows 15 0 0 15 100 total 42 7 16.7 35 83.3 Table (4): Number and percentage of Staphylococcus spp. isolated from milk samples according to biochemical tests Staphylococcus species S.aureus S.Capitis S.saccharolyticus S.auricularis S.xylosus S.carnosus S.intermedius total isolates 11 1 10 4 1 14 1 42 26.2 2.4 23.8 9.5 2.4 33.3 2.4 100 010
Vancomycin(VA) Chloramphenicol(C) Erythromycine (E) Ampicillin+sulbectam (SAM) Tetracycline (TE) Ciprofloxacine (CIP) Oxacillin (OX) Gentamycin (CN) Penicillin G (P) Arab Journal of Nuclear Science and Applications, 50 (2), (310-318) 2017 Table (6): Antibiotic sensitivity of staphylococcus spp. Isolated From milk samples Number Strain name Susceptibility profile\diameter of inhibition zone 1 S.aureus R S S S S S I S S 2 S.aureus R S R S S S S S S 3 S.aureus S S S S R I S S S 4 S.aureus R I S I S S S S I 5 S.aureus R S S S R S I S S 6 S.aureus R S S I R S I S S 7 S.aureus R S R S R S S S S 8 S.intermedius R S R S I S S S S 9 S.carnosus R I R S S S S S S 10 S.xylosus R S R S R S R I S 11 S.carnosus R S R I R S R S S 12 S.carnosus R I R S S S S S I 13 S.carnosus R S R S S I I S S 14 S.carnosus R S R S I S S S S 15 S.xylosus R S S S R S S I S 16 S.carnosus R S S S S S S S S 17 S.carnosus R S S I S S S S S 18 S.capitis R I S S S S I S S 19 S.carnosus R S R I S S S S S 20 S.capitis R S S I S S S S S 21 S.capitis R S R S S I S S S 22 S.carnosus S S S S I S S S S 23 S.saccharolyticus S S I S S S I S S 24 S.carnosus R I S S R S I I S 25 S.capitis R S S I S S S I S 26 S.auricularis R S S S S S S I S 27 S.capitis R S S I S S S S I 28 S.aureus R S S S S S S I S
29 S.aureus R S S S R S R S S 30 S.aureus R I S S R S S S S 31 S.aureus R S S S S S S S S 32 S.carnosus R S S I S S I I S 33 S.capitis R S S S S I S I S 34 S.xylosus S S R S R S S S S 35 S.carnosus R I S S S S S S S 36 S.capitis R S R S S S R S S 37 S.carnosus R S S I S S I I S 38 S.capitis R S S S S S S I S 39 S.capitis R S S I R S S I S 40 S.capitis R S S S S S S S S 41 S.carnosus R S I S S S S S S 42 S.xylosus S S R S R S I S S Key: R= resistant, I= intermediate, S= sensitive Table (7): Antibiotics discs zone diameter interpretation (NCCLS 2007 and 2011) Antibiotics CN CIP C TE SAM Ox VA E P G S (sensitive) 15 21 18 19 15 13 17 23 29 I (intermediate) 13-14 16-20 13-17 15-18 12-14 11-12 15-16 14-22 ------- R (resistant) Table (8): Incidence of antimicrobial sensitivity test on the recovered Staphylococcus species (42) from milk samples Susceptibility profiles Antimicrobial agent R (resistant) I (intermediate) S (sensitive) n= n= n= 1 Penicillin G P 37 88 0 0 5 11.90 2 Chloramphenicol C 0 0 11 26.19 31 73.80 3 Gentamycin CN 0 0 12 28.57 30 71.42 4 Ciprofloxacin CIP 0 0 11 26.19 31 11.9 5 Tetracycline T 13 30.95 3 7.14 26 61.90 6 Vancomycin VA 0 0 4 9.5 38 90.47 7 Erythromycin E 4 9.5 7 16.66 31 73.8 8 Ampicilin+sulbectam SAM 0 0 7 16.6 35 83.33 9 Oxacillin OX 14 33.33 2 4.76 26 61.90 12 15 12 14 11 10 14 13 28
DISCUSSION Negligence of hygienic condition such as improper cleaning of bulk tank, dirty udders, milking equipment, cooking temperature, milk handling technique and improper storage increases the proportion of staphylococci in the bulk tank milk (Vasavada, 1988; Bonfoh et al., 2003). These parameters play an important role in determining characteristics of milk and milk products. If these foods were contaminated with S.aureus.they indicate serious health problems. This study revealed that milk may be contaminated with multiply resistant S.aureus. the high frequency of resistance with penicillin (88), oxacillin (33) and tetracycline (30) could be attributed to their use in treatment of diseases in animals and humans. Resistant bacteria may transfer resistant genes to other bacteria and become important in the spread of antibiotic resistance.the indiscriminate use of antimicrobial agents and antibiotic misuse (for example,sale of antibiotics without prescription,sale of under dose and substituting brands ) enhance the development of drug resistance ( Indalo, A.A. 1997). Isolation of oxacillin or methicillin resistant S.aureus conducted in this study was of great significant as this resistant strain is considered the cause of most nosocomial cases. These S.aureus are known to be resistant to beta- lactams including penicillins, cephalosporins,carbepenes, and disinfectant. Currently,penicillin, erythromycin and teracyclines are frequently used for the treatment of mastitis and several reports indicate that S.aureus isolates had high level of such antibiotics resistance. The resistance of S.aureus to penicillin was closely associated with blaz gene Which encode β lactamase. (Olsen, Christensen.2006 ) Zscheck,Murray.1993) S.aureus resistance to erythromycin was predominately mediated by a methylase encoded by erm genes (Eady,Ross,Tipper. et al.,1993). Resistance to tetracyclines in Staphylococci was conferred by two different mechanisms: active efflux of the drug and protection of the tetracyclines sites which were the most common in conferring tetracyclines resistance in S.aureus (Schmitz,Krey.,et al.2001) This study revealed that milk may be contaminated with multiple resistant Staphylococcus species with S.aureus.The high frequency of resistance with penicillin (88), oxacillin (33), and tetracycline (30) could be attributed to their use in treatment of diseases in animals and humans. CONCLUSION High incidence of S.aureus in cows and buffalos milk may cause a risk on public health which leads to food poisoning and transmitting other zoonotic diseases. Thus, more hygienic preventive measures are required to reduce the bacterial contamination,so as to increase the wholesomeness of milk. High prevalence of CNS(coagulase negative Staphylococcus) isolates (83.3) resulting from the present study indicates bad hygiene during milking process. This is considered a significant result because emerging data indicate that they are associated with several sub-clinical and clinical infections (Ontario Ministry of Agriculture 1999) (Pyörälä S.1995) Results of hemolytic assay showed that most Staphylococci isolates which revealed a positive hemolysis(62) increase the virulence of the isolates and show the ability of Staphylococci to attach to contact surface at milk containers in milk plants. REFERENCES (1) Soomoro., etal.2003.isolation of Staphylococcus aureus from milk products sold at sweet meat shops of Hedrabad.online journal of biological sciences 91-94.
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