IndianJournals.com Isolation, Identification and

Journal of Immunology and Immunopathology Vol. 18, No. 1, January-June, 2016: 58-62 DOI: 10.5958/0973-9149.2016.00009.5

IndianJournals.com A product of Diva Enterprises Pvt. Ltd.

Research Article

Isolation, Identification and Antibiogram of Staphylococcus aureus Isolated from Bovine Mastitis Falguni M. Parth 1, H.C. Chauhan 2*, A.G. Bhagat 3, B.S. Chandel 4, Patel Bharat K. 5, Patel Kirit B.6 and H.N. Kher7 1

P.G. Scholar, 2Associate Professor, 3Assistant Professor, 4Professor and Head, 5Junior Research Fellow, 6Senior Research Fellow, 7Registrar, Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Sardar Krushi Nagar Dantiwada Agricultural University, Sardar Krushi Nagar, Palanpur-385506, Gujarat

Downloaded From IP - 14.139.121.18 on dated 11-Jul-2016

www.IndianJournals.com

Members Copy, Not for Commercial Sale

ABSTRACT Parth FM, Chauhan HC, Bhagat AG, Chandel BS, Patel BK, Patel KB and Kher HN (2016). Isolation, Identification and Antibiogram of Staphylococcus aureus Isolated from Bovine Mastitis. J. Immunol. Immunopathol. 18(1): 58-62. In the present study, out of 255 milk samples (185 from subclinical and 70 from clinical cases) screened for Staphylococcus aureus, 53 isolates were obtained giving an overall incidence of 20.78%. The incidence of S. aureus from subclinical mastitis was 8.11% (15/185) and from clinical mastitis was 54.29% (38/70). Species-wise incidence was 30.77% (16/52) in buffaloes and 18.23% (37/203) in cows. All 53 isolates were identified on the basis of colony characters on nutrient agar, staining and biochemical characters namely catalase production, oxidise test, maltose fermentation test and ohosphatase test. Out of 53 isolates, 49 (92.45%) isolates showed coagulase production in tube coagulase test. Number of isolates showing alpha, beta, gamma and alpha–beta haemolysin production were 18 (33.96%), 26 (49.06%), 4 (7.55%) and 5 (9.43%) on sheep blood agar, respectively. All the 53 S. aureus isolates were confirmed genotypically by 23S rRNA ribotyping in which a species-specific amplicon of 1,250 bp was obtained. Antibiotic sensitivity patterns of all these 53 S. aureus isolates revealed that the isolates were most sensitive to gentamicin (94.34%), whereas penicillin (94.34%) and ampicillin (86.79%) were highly resistant to S. aureus. Keywords: Bovine, Mastitis, S. aureus, Isolation, Antibiogram, Ribotyping, PCR

INTRODUCTION Bovine mastitis is the single largest problem in dairy animals, causing economic losses in the tune of millions of rupees annually in India. Among the various pathogens responsible for mastitis, Staphylococcus aureus is one of the most frequently isolated pathogen from both subclinical mastitis and chronic infections (Suleiman et al., 2012). Staphylococcus is spherical or ovoid in shape, gram positive and 0.8–1 µm in diameter arranged in irregular cluster resembling ‘bunches of grapes’. It is aerobic or facultative anaerobic, catalase-positive, oxidasenegative, non-motile, non-spore forming and

fermentative. Staphylococcus can be identified on the basis of colony morphology, biochemical and cultural characters and by molecular characterisation. Staphylococci are also characterised by production of enzymes like catalase, lecithinase, lipase, proteinase, phosphatase and others. Certain characters like production of haemolysin, coagulase, DNase and enterotoxins are taken into consideration for differentiating Staphylococcus species into pathogenic and non-pathogenic types. S. aureus can be identified by conventional methods, but as this organism shows variation in phenotypic expressions, its identification with 58

Isolation, Identification and Antibiogram of Staphylococcus aureus Isolated from Bovine Mastitis




genotypic methods is much more important. In addition, the pressure exercised in diverse environments with reservoirs, management and trading specific to each geographical area leads to the selection of distinct and genetically adaptable strains, which further necessitates knowledge on the epidemiological profile of a herd or region before applying mastitis control measures. Different researchers have studied the phenotypic and genetic properties to differentiate S. aureus isolates (Sanjivet al., 2008). These properties might help to understand the distribution of prevalent S. aureus clone to control infections in dairy herds. Antimicrobial therapy is one of the measures for controlling Staphylococcal mastitis. The cure rates for S. aureus mastitis are affected by several factors, including antimicrobial resistance of the isolates. The determination of antimicrobial susceptibility of clinical isolates is required not only for therapy but also for monitoring the spread of resistant strains throughout the populations. Although antimicrobial susceptibility testing is a valuable tool in determining the best therapeutic choice against a mastitis pathogen, in practice it is rarely performed and therapy decisions are usually made empirically. Therefore, susceptibility data for a large number of isolates in an area or region can be useful to veterinarians (Salmon, 2002). Therefore, the present work was undertaken with objective of isolation, identification and antibiotic sensitivity patterns of S. aureus isolated from bovine mastitis. MATERIAL AND METHODS Milk samples from suspected cases of subclinical and clinical mastitis in cows and buffaloes belonging to various places of Banaskantha district were collected aseptically in sterilised vials. A total of 185 milk samples from161 cows and 24 buffaloes were collected and screened for subclinical mastitis. Samples from subclinical cases were first processed for detection of subclinical mastitis by indirect tests namely electrical conductivity meter (Draminski 4Q MAST) and California mastitis test (CMT) using standard protocol as per the manufacturer’s instructions. A total of 70 milk samples from clinical cases of mastitis were also collected from cattle and buffaloes (42 from cattle and 28 from buffaloes). Journal of Immunology and Immunopathology

The samples which were detected positive in subclinical mastitis and the samples from clinical cases were processed for isolation of S. aureus. Milk samples were inoculated on the plates of nutrient agar by spreading heavy inoculums of thoroughly mixed milk. The plates were incubated at 37°C for 24 h. Then colonies showing golden-yellowpigmented colour indicative of S. aureus were transferred to nutrient agar slants for further identification. The isolates were identified as per the methods described by Cowan and Steel (1974) on the basis of morphological characteristics and biochemical characterisation namely catalase test, phosphatase test, oxidase test, maltose fermentation test mannitol fermentation, coagulase production and haemolysin production. For genotypic identification of the organisms, ribotyping based on 23S rRNA was carried out as per the method described by Straub et al. (1999) for which prim er-1 with sequence of 5ACGGAGTTACAAAGGACGAC-3 and primer-2 with sequence of 5-AGCTCAGCCTTAAC GAGTAC-3 were used. The Deoxyribonucleic Acid (DNA) extraction was carried out as per the protocol outlined in the manufacturer’s manual using Genpro™ 3-in-1 DNA isolation kit (GeNei, MERCK). The quality and purity of DNA were checked by agarosegel electrophoresis using 0.8% agarose and by Picodrop (Picodrop, U.K.). Polymerase Chain Reaction (PCR) was carried out in final reaction volume of 25 µl in a thin-walled 200-µl PCR tubes using a Nexus Mastercycler (Eppendorf).The PCR cycling protocol was applied as following: initial denaturation at 94°C for 5 min, followed by 37 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 60 s and extension at 72°C for 75 s. Final extension was done at 72°C for 3 min. All the S. aureus isolates obtained were subjected to in vitro antibiotic sensitivity test, as per the method described by Bauer et al. (1966). Each test strain was grown in Brain and Heart Infusion (BHI) broth overnight at 37°C. Sterile plates of Mueller and Hinton agar were seeded with about 1 ml of inoculums and were allowed to dry. Monodiscs (Hi Media, Pvt. Ltd., Mumbai) of antibiotics namely penicillin (P) G (10 mcg), ciprofloxacin (Cf) (5 mcg), gentamicin (G) 59

Falguni M. Parth et al.

(10 mcg), enrofloxacin (EX) (10 mcg), chloramphenicol (C) (30 mcg), cephalexin (Cp) (30 mcg), amoxycillin/sulbactam (30 mcg), ampicillin (AM) (10 mcg), amoxycillin/clavulanic acid (AC) (30 mcg) were then placed in the plate and were incubated aerobically at 37°C overnight. Zones of inhibition were measured and compared with zone size interpretative table furnished by the manufacturer and graded as sensitive and resistant.




RESULT AND DISCUSSION Mastitis is an infectious disease of dairy ruminants that affects milk production and quality. This disease has been singled out as the most significant cause of economic loss to the dairy industry. Although several bacterial pathogens can cause the disease, S. aureus has emerged as one of the most prevalent ones, and once it is established in the mammary gland of the milking animal, it is very difficult to eradicate. A total of 53 isolates were obtained from subclinical and clinical cases of mastitis, which included 15 from Sub Clinical Mastitis (SCM) and 38 from clinical cases of mastitis. Of subclinical cases, 11 were from cows and 4 from buffaloes. Similarly, 38 isolates of clinical cases, 26 from cows and 12 from buffaloes. This way, 37 isolates were from cows and 16 isolates from buffaloes were found positive for clinical and subclinical mastitis. Overall incidence of S. aureus in clinical and subclinical mastitis was found to be 20.78% (53/255). Considering specieswise incidence, 30.77% (16/52) buffaloes were found positive for S. aureus from the clinical and subclinical mastitic milk, whereas 18.23% (37/203) cows were detected positive. Higher incidence of S. aureus may be attributed to the fact that the principal reservoirs of S. aureus are the skin of the udder and milk of the infected gland (Davidson, 1961), and S. aureus is a contagious organism, with capacity to penetrate into the tissue producing deep seated foci. The isolation of predominantly S. aureus from SCM was in accordance with the study of Suleiman et al. (2012). In contrast to the present study, Bhanderi et al. (2009) observed higher rate of incidence for S. aureus mastitis. The possible reasons for the lower incidence of S. aureus responsible for clinical and subclinical mastitis compared with other reports may be 60

attributed to the etiology of mastitis that the Staphylococcus may not be responsible for the mastitis or some other reasons like breed of the animal, seasons of the sampling, mastitic status of the animals and others. All the 53 isolates were found catalase positive, oxidase negative, positive for maltose fermentation and positive for phosphatase test. In the present study, all the 53 (100%) isolates were able to ferment mannitol on MSA. Similar findings were reported by Makwana et al. (2012) and Thaker et al. (2013), who observed cent percent mannitolfermentator S. aureus, whereas Bhanderi (2007) found 74.41%mannitol fermentation by S. aureus isolates. Out of 53 isolates, 49 (92.45%) isolates were found positive for coagulase production and 4 isolates were found negative. These coagulase negative isolates in further study were confirmed not to possess coa gene but were S. aureus as confirmed by 23S rRNA gene ribotyping using species specific primers. Similar findings were reported by Pandya (1991) using rabbit plasma. Turutoglu et al. (2005) reported 89.77% coagulase S. aureus from cows with subclinical mastitis. Makwana et al. (2012) reported that among 100 Staphylococcal isolates, 94 isolates (94.00%) were positive for tube coagulase test. Haemolysins are extra cellular enzymes that can be detected by their ability to lyse red blood cells. Haemolysins are produced by various pathogenic bacteria and are believed to play an important role in the virulence of the organism. In the present study, incidence of S. aureus producing different haemolysins was detected on sheep blood agar. The incidence of S. aureus producing alpha 18 (33.96%), beta 26 (49.06%), gamma 4 (7.55%) and 5 alpha– beta (9.43%) haemolysin on sheep blood agar observed in the present study is more or less similar to the findings of Pandya (1991), who observed alpha (11.71%), beta (54.68%) and alpha–beta (19.53%) haemolysin and Patel (2008) observed alpha (27.5%), beta (48.75%), gamma (13.75%) and alpha–beta (11.25%) haemolysin among 80 isolates of Staph. aureus. S. aureus can be identified by conventional methods, but as this organism shows variation in phenotypic expressions, its identification with genotypic methods is much more important. In Vol. 18, No. 1, January-June, 2016




Isolation, Identification and Antibiogram of Staphylococcus aureus Isolated from Bovine Mastitis

addition, the pressure exercised in diverse environments with reservoirs, management and trading specific to each geographical area leads to the selection of distinct and genetically adaptable strains, which further necessitates knowledge on the epidemiological profile of a herd or region before applying mastitis control measures. Different researchers have studied the phenotypic and genetic properties to differentiate S. aureus isolates (Sanjiv et al., 2008). In the present investigation, all the 53 isolates after their identification by conventional microbiological procedures were subjected to 23S rRNA-based ribotyping developed by Straub et al. (1999) for confirmation. The ribotyping produced an amplicon of 1,250 bp in all the isolates confirming them to be S. aureus (Plate 1). The same method of genotypic confirmation using the similar primers has been reported by Khichar et al. (2014).

Plate 1: 23S Rrna ribotyping of Staphylococcus aureus isolates obtained from bovines with clinical and subclinical mastitis Lane Marker: 100 bp DNA Marker Lane1: Negative Control Lane 2–7: 1,250 bp PCR products

Over the last few decades, there was a sudden increase in the use of antibiotics in veterinary as well as medical science to circumvent various infections. Most of the time, the dosage of antibiotics is not accurately measured during the treatment. This indiscriminate use of antibiotics has contributed immensely to the development of antimicrobial resistance. These resistant microorganisms become part of the environment and are transmitted from animals to humans or vice-versa. Presently, there is growing concern among scientists in regards to Journal of Immunology and Immunopathology

Plate 2: Antibiotic sensitivity Staphylococcus aureus isolate

pattern

of

increasing resistance in pathogens. Out of 53 isolates of S. aureus, 50 (94.34%), 48 (90.57%), 44 (83.02%), 34 (64.15%), 28 (52.83%), 17 (32.08%), 12 (22.64%), 7 (13.21%) and 3 (5.66%) isolates were sensitive to gentamicin (G), ampicillin/sulbactam (A/S), chloramphenicol (C), enrofloxacin (EX), ciprofloxacin (Cf), amoxycillin/clavulanic acid (AC), cephalexin (Cp), ampicillin (AM), penicillin (P), respectively (Plate 2). The result of the present study shows that S. aureus isolates were highly resistant to penicillin (94.34%) and ampicillin (86.79%). Resistant to penicillin of 87, 94.4 and 100% was reported by Abera et al. (2013) and Thaker et al. (2013). In contrast to these findings, Corti et al. (2003) recorded low resistance of isolates towards penicillin. Higher resistant to ampicillin of 83% was reported by Mekonnen et al. (2005). The resistant of S. aureus to penicillin and ampicillin may be attributed to the production of beta lactamase, an enzyme that inactivates penicillin and closely related antibiotics. It is believed that around 50% of mastitis causing S. aureus strains produce beta-lactamase (Green and Bradley, 2004). In the present study, gentamicin was found highly effective against 94.34% of the isolates. The highest (100%) sensitivity to gentamicin was reported by Abera et al. (2013). REFERENCES Abera M, Demie B, Aragaw K, Regassa F and Regassa A (2013). Isolation and identification of Staphylococcus aureus from bovine mastitic milk

61

Falguni M. Parth et al.

and their drug resistance patterns in Adama town, Ethiopia. Afr. J. Dairy Farming Milk Prod., 1: 019– 023. Bauer AW, Kirby WMM, Sherris JC and Toun M (1966). Antibiotic susceptibility testing using standard single disc diffusion method. Am. J. Clin. Pathol., 45: 493–496. Bhanderi B (2007). Isolation, identification, biochemical characterization, antibiogram patternand molecular characterization of Staphylococcus aureus from clinical and subclinical mastitic milk. M.V.Sc. Thesis. Submitted to Anand Agricultural University, Anand.




Bhanderi BB, Roy A, Yadav MM and Joshi CG (2009). PCR based detection of virulence associated genes of Staphylococcus aureus from bovine clinical and subclinical mastitis. RVJI., 5: 20–26. Corti S, Sicher D, Regli W and Stephan R (2003). Current data on antibiotics resistance of the most important bovine mastitis pathogens in Switzerland. Schweiz. Arch. Tierheilkd., 145: 571–575. Cowan ST and Steel KJ (1974). Cowan and Steel’s manual for the identification of Medical bacteria. 2 nd Edition, Cambridge University Press, Cambridge. Davidson I (1961). Observations on the pathogenic Staphylococci in dairy herd during a period of six years. Res. Vet. Sci., 2: 22. Green M and Bradley A (2004). Clinical forum: Staphylococcus aureus mastitis in cattle. U.K. Vet., 9: 1–9. Khichar V, Kataria AK and Sharma R (2014). Characterization of Staphylococcus aureus of cattle mastitis origin for two virulence associated genes (coa and spa). Comp. Clin. Pathol., 23: 603-611 DOI: 10.1007/s00580-012-1675-5. Makwana GE, Gadhavi H and Sinha M (2012). Comparison of tube coagulase test with mannitol fermentation test for diagnosis of Staphylococcus aureus. Natl. J. Integr. Res. Med., 3: 73–75.

62

Mekonnen H, Workineh S, Bayleyegn M, Moges A and Taddele K (2005). Antimicrobial susceptibility profile of mastitis isolates from cows in three major Ethiopian dairies. Med. Vet., 176: 391–394. Pandya K (1991). Incidence of Staphylococcus aureus in cow milk and assessment of characteristics associated with its virulence. M.Sc. Thesis. Submitted to Gujarat Agricultural University, Sardar Krushi Nagar. Patel SS (2008). Virulence determinants in Staphylococcus aureus isolated from pus and mastitic milk of bovines. M.V.Sc. Thesis, Submitted to Sardar Krushi Nagar Dantiwada Agricultural University, Sardar Krushi Nagar. Salmon SA (2002). Use of antimicrobial susceptibility data to assist in determining the best therapy for clinical mastitis. In: Proc. NMC 41 st Annu. Mtg., Orlando, FL. National Mastitis Council, Madison, WI. 36p. Sanjiv K, Kataria AK, Sharma R and Singh G (2008). Epidemiological typing of Staphylococcus aureus by DNA restriction fragment length polymorphism of coa gene. Veterinarski Arh., 78: 31–38. Straub JA, Hertel C and Hammes WP (1999). A 23S rRNA target polymerase chain reaction based system for detection of Staphylococcus aureus in meat starter cultures and dairy products. J. Food Prot., 62: 1150–1156. Suleiman AB, Kwaga JKP, Umoh VJ, et al. (2012). Macrorestriction analysis of Staphylococcus aureus isolated from subclinical bovine mastitis in Nigeria. Afr. J. Microbiol. Res., 6: 6270–6274. Thaker HC, Brahmbhatt MN and Nayak JB (2013). Isolation and identification of Staphylococcus aureus from milk and milk products and their drug resistance patterns in Anand, Gujarat. Vet. World, 6:10–13. DOI: 10.5455/vetworld.2013.10-13. Turutoglu H, Tasci F and Ercelik S (2005). Detection of Staphylococcus aureus in milk by tubecoagulase test. Bull. Vet. Inst. Pulawy, 49: 419–422.

Vol. 18, No. 1, January-June, 2016