mrsa

Short Communication Indian J. Compo Microbiol. Immunol. Infect. Dis. Vol. 34 No.2 (July-Dec.), 2013 : 56-59

BACTERIOLOGICAL INVESTIGATION AND ANTIBIOGRAM ON METHICILLIN-RESISTANT STAPHYLOCOCCUSAUREUS (MRSA) CAUSING SUBCLINICAL MASTITIS IN DAIRY CATTLE POPULATION OF WEST BENGAL I. Paul*, D.P. Isore, S.N. Joardar, S.K. Mukhopadhayayl, S. Ganguly and S. Pal Department of Veterinary Microbiology, Faculty of Veterinary & Animal Sciences West Bengal University of Animal and Fishery Sciences 37, Kshudiram Bose Sarani, Belgachia, Kolkata -700037, WB, India

Keywords : Sub clenical mastitis, MRSA, Staphylococcus Aureus, Antibiogram

Downloaded From IP - 122.180.105.90 on dated 20-Feb-2015

www.IndianJournals.com

Members Copy, Not for Commercial Sale

Received: 18 Dec., 2013 Accepted: 03 Jan., 2014 quarters of cross-breed milch cows from different farms and clinics of different districts of West Bengal and stored at 4°C for further study. These milk samples were collected from acute, sub acute and chronic cases of mastitis.

INTRODUCTION Mastitis is a multietiopathogenic condition ofmammary gland affecting dairy cows and remains the most economically important disease of dairy industries around the world. It is characterized by physical, chemical and microbiological changes in the milk and pathological changes in the glandular tissues of the udder. Keeping this in priority, the present study was carried out for the isolation, identification and antibiogram of pathogenic agents responsible for causing the infection. Out of 60 samples, 38 samples (63.33%) were positive for staphylococci and 10 samples (16.66%) were positive for streptococci. Coagulase positive staphylococci organisms were identified by reacting the isolates with rabbit plasma in the ratio of 1: 10 dilution. It was observed that 84.21 % isolates were coagulase positive. 89.5% staphylococci isolates were resistant to sulphamethizole, 84.2% to ampicillin, 79% to colistin, 60.5% to penicillin G, 52.63% to methicillin, 42.1 % to ciprofloxacin, 26.3% to tetracycline, 23.7% to ceftizoxime and enrofloxacin and 13.2% to ceftriaxone by disc diffusion method. All the streptococci isolates were resistant to penicillin G.

Altogether, 38 strains of Staphylococci and 10 strains of Streptococci isolated from cattle were examined to determine their biochemical properties. For this purpose, all the isolates were subjected to various biochemical tests which included catalase test, oxidase test, urease test, indole test, methyl-red test, Voges-Proskauer test and sugar fermentation test.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

For the present study, milk samples were collected aseptically into screw capped bottles from the affected

All Staphylococci isolates showed positive results to catalase, urease, Voges-Proskauer test and negative to

Biochemical and sugar fermentation tests were carried out for characterization and identification of the bacterial isolates as per the method of Buxton and Fraser (1977). Antibiotic sensitivity tests of Staphylococci and Streptococci isolates were carried out with commonly used antibiotic discs on Mueller Hinton agar by disc diffusion method (Bauer et aI., 1966). Fermentation reactions were carried out with all the isolates. Six different sugars viz., glucose, lactose, maltose, mannitol, sucrose and salicin were included in this purpose.

*Corresponding author:Ernail: indirapaul45 @grnail.com IDepartrnent of Fish Processing Technology, Faculty of Fishery Sciences West Bengal Univ. of Animal and Fishery Sciences, Kolkata, WB, India

56

Bacteriological investigation and antibiogram on MRSA causing subclinical mastitis

oxidase and indole test. 97.37% strains were MR positive. Out of 10 Streptococcal isolates 100% showed catalase, oxidase, urease, indole, MR and VP negative (Table 1).

were positive to glucose and lactose fermentation, 80% positive to maltose, 60% positive to sucrose, 70% positive to salicin fermentation. No strains were found to be positive to mannitol (Table 3).

It was observed that all Staphylococci strains were positive to glucose, lactose, maltose, mannitol, sucrose fermentation. 100% strains were negative to salicin fermentation (Table 2). Sugar fermentation test revealed that 100% strains

Table 1. Biochemical tests of Staphylococci isolates collected from bovine milk. Total no. of

Catalase test

Oxidase test

Urease test

VP test

MR test

Indole test

Staphylococci isolates Downloaded From IP - 122.180.105.90 on dated 20-Feb-2015

+ve



38

-ve

+ve

-ve

+ve

-ve

+ve

-ve

+ve

-ve

+ve

-ve

0

0

38

38

0

0

38

37

1

38

0

0%

100%

100%

0%

0%

-100%

97.37%

2.63%

100% 0%

100% 0%

Table 2. Showing the number of Staphylococci strains fermenting various sugars. Name of the sugar used

No. of strains tested

Positive No.

%

Glucose

38

38

100

Lactose

38

38

100

Maltose

38

38

100

Mannitol

38

38

100

Sucrose

38

38

100

Salicin

38

0

0

Table 3. Showing the number of Staphylococci strains fermenting various sugars. Name of the sugar used

No. of strains tested

Positive No.

%

Glucose

10

10

100

Lactose

10

10

100

Maltose

10

8

80

Mannitol

10

0

0

Sucrose

10

6

60

Salicin

10

7

70

57

Pauletal.

All 38 strains of Staphylococci were subjected to pathogenicity test by coagulase test.

observed that the 32 isolates (84.21 %) were coagulase positive and 6 isolates (15.79%) were coagulase negative out of 38 staphylococci isolates (Table 4).

Coagulase test was performed for identification of isolated Staphylococci and determine the pathogenicity. It was

Table 4. Showing percent positivity of coagulase test on sample collected from cattle. Total no. of staphylococci

No. of coagulase positive strains

No. of coagulase negative strains

32 (84.21 %)

6 (15.79%)

isolated 38

Altogether, 38 staphylococci isolates and 10 streptococci isolates were tested for their sensitivity to antimicrobial drugs used in veterinary practice by disc diffusion techniques. The analysis reflected in the Table 5 indicated that 89.5%,84.2%,79.0%,60.5%,52.63%,42.1 %,26.3%,

23.7%, 23.7% and 13.2% were resistant against sulphamethizole, ampicillin, colistin, penicillin G; methicillin, ciprofloxacin, tetracycline, ceftizoxime, enrofloxacin and ceftriaxone respectively. All the streptococci strains were resistant to penicillin G (Table 5).




Table 5. Showing degree of sensitivity of Staphylococci isolated from bovine milk against various antibiotics.

Antimicrobial

Resistant strains(%)

Intermediate strains(%)

Sensitive strains(%)

agents

Methicillin

52.63

Tetracycline

26.3

34.2

39.5

Ciprofloxacin

42.1

34.2

23.7

Penicillin G

60.5

39.5

Ampicillin

84.2

15.8

Ceftizoxime

23.7

76.3

Ceftriaxone

13.2

79.0

7.8

Enrofloxacin

23.7

13.2

63.1

Sulphamethizole

89.5

10.5

Cnli~tin

79,0

47.37

21.0

In the present study, the rate of incidence of staphylococci infection in affected milch CO'~TS 'Nas found to be 63.33% and Streptococcal infection was 16.66%. This finding corroborates with the findings of NagaI et a1. (1s)s)Y) who observed the incidence of Staphylococcous aureus to be 61.76%, Streptococcus pyogenes 12.03%, Streptococcus epidermis 2.4% and findings ofBalakrishnan et a1. (2004) who repo1ted the incidence of Streptococcus agalactiae to be 17.5%.

were positive for Voges-Proskauer, catalase test" urease test and negative for inrlol~ :;Jno oxirla~e test These observations support the findings ofVarshney et a1. (1993) and Bhanderi et a1. (2009). Sugar fermentation test revealed that all the Staphylococci isolates produced acid with glucose, lactose, maltose, mannitol and sucrose but not with salicin. These findings were in agreement with the findings of Saitoh et a1. (1994). Varshney et a1. (1993) reported that 100% glucose and mannitol fermented by Staphylococcus aureus.

In the present study, 97.37% staphylococci isolates were methyl-red positive. All of the staphylococci isolates 58

Bacteriological investigation and antibiogram on MRSA causing subclinical mastitis

REFERENCES




Lafi et al. (1994) reported that the most common organism isolated from clinical cases of mastitis was coagulase positive Staphylococcus spp. Junda et al. (1994) found that out of 130 Staphylococcus aureus strains isolated, 94 (91.3%) showed tube coagulase test positive. In this study 32 isolates (84.21 %) were coagulase positive and 6 isolates (15.79%) were coagulase negative out of 38 staphylccocci isolates.

Balakrishnan, G., Dnny, M., Dorairanjan, D. and Subramanian, M. (2004) Studies on bovine mastitis at Namakkal. Indian Vet. J. 81: 1166-7. Bauer, A.W., Kirby, W.M.M.; Sherris, J.C. and Turk, M. (1966) Antibiotic susceptibility testing by a standard disc method. Am. J. Anim. Path., 45: 493-6.

Bhanderi, B.B., Roy, A., Yadav, M.M. and Joshi, C.G. (2009) PCR

In tht-, present study, 89.5% isolates of Staphylococci were resistant to sulphamethizole, followed by 84.2% to ampicillin, 79.0% to colistin, 60.5% to penicillin G; 52.63% to methicillin, 42.1 % to ciprofloxacin, 26.3% to tetracycline, 23.7% to ceftizoxime and enrofloxacin, 13.2% to ceftriaxone. Ceftriaxone was highly effective to most of isolates. The result was similar to Ramprabhu et al. (2004). The isolates were more sensitive to enrofloxacin and least sensitive to ampicillin. This finding was similar like Sahay et al. (2007). Moon et al. (2007) isolated the Staphylococci strains and observed that 2.5% S. aureus and 2.4% CNS strains were resistant to methicillin.

based detection of virulence associated genes of Staphylococcus aureus from bovine clinical and subclinical mastitis. The Royal Vet. J. India, 5: 20-6.

BuxtonAandFraserG. (1977) Animal Microbiology. 1st Edn. Blackwell Scientific Publication Ltd. Victoria, Australia. Moon, J. -S., Lee, A-R., Kang, H.M., Lee, E.-S., Kim, M.N., Paik, Y.H., Park, Y.H., Joo, Y.S. and Koo, H.C. (2007) Phenotypic and genetic antibiogram of methicillin-resistant Staphylococci isolated from bovine mastitis in Korea. J. Dairy Sci., 90: 1176-85. NagaI, K.B., Sharma, M. and Katoch, R.C. (1999) Etiology of bovine mastitis in and around Palampur in Himachal Pradesh. Indian J. Anim. Sci., 63: 150-2.

ACKNOWLEDGEMENT

Sahay, S., Sinha, B.P., Verma, S.P. (2007) Incidence of sub clinical

The authors are thankful to the Vice-Chancellor, West Bengal University of Animal and Fishery Sciences for providing the necessary facilities to carry out this original research work.

mastitis in dairy animals and sensitivity of isolated organisms. Indian J. Vet. Med., 27: 29-30.

Varshney, J.P., Kapur, M.P. and Sharma, A. (1993). Studies on some biochemical characteristics of Staphylococcus aureus of buffalo mammary origin. Indian J. Compo Immunol. Microbiol. In! Dis.,

16: 317-21.

59