DETECTION OF VIRULENCE FACTORS GENES AND INVESTIGATION OF ANTIBIOTIC RESISTANCE PATTERN IN Staphylococcus aureus ISOLATED FROM MASTITIC MILK SAMPLES OF BUFFALOES
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Date
2013-04
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA
Abstract
ABSTRACT:
Mastitis in buffaloes is responsible for heavy economic losses to Indian dairy industry. Staphylococcus species are the most common pathogens responsible for mastitis in cows and buffaloes. Staphylococcus aureus is implicated in the sub-clinical mastitis. Rapid and accurate detection of S. aureus is important for controlling mastitis in a herd. Many virulence factors responsible for pathogenicity are reported in S. aureus. Detection of the virulence factors helps to identify the strains of S. aureus circulating in the area. Among the important virulence factors coagulase and enterotoxins assume significance. Genes coding for these virulence factors in S. aureus can be detected by polymerase chain reaction (PCR) test. Reports on antibiotic resistance phenomenon also prompt to test the S. aureus isolates from bubaline mastitis cases for antibiotic sensitivity. Based on isolation on selective media, haemolysis on blood agar, catalase test, oxidase test and coagulase test 57.33% of the mastitic milk samples collected in the present study were provisionally confirmed for Staphylococcus species, which were later subjected to confirmation by PCR test. On screening by California Mastitis Test (CMT), 69.76% of these mastitic milk samples were found to be from sub-clinical mastitis cases of buffaloes. For PCR test, bacterial DNA from the enriched bacterial cultures was extracted by inexpensive high salt method, which yielded required quality and concentration of DNA. All the 43 mastitic milk samples that were provisionally confirmed for Staphylococcus species were tested with Staphylococcus genus specific oligonucleotide primers (Sta I and Sta II) and S. aureus specific oligonucleotide primers (Staur 4 and Staur 6) in PCR test. All the 43 samples were confirmed as Staphylococcus species (PCR products of 100 to 200 bp), whereas 69.76% (n=30) of them were confirmed as S. aureus (PCR product of 1250 bp). Bacterial cultures that were confirmed as S. aureus by PCR test were further tested for coagulase gene (coa) in PCR test using coa gene specific primers. Out of the 30 S. aureus isolates from mastitic milk samples 8 samples (26.66%) did not yield any PCR products on reactivity with coa gene specific primers. The remaining 22 S. aureus isolates (73.33%) yielded PCR products of different sizes ranging from 610 bp to 960 bp. The S. aureus isolates TVCC 18 and GV 6 yielded a PCR product of 610 bp. Six S. aureus isolates viz. TVCC 23, GDV 1, KSP 3, KSP 4, KSP 6 and KSP 7 yielded PCR product of 740 bp. Twelve isolates of S. aureus viz. TVCC 2, TVCC 4, TVCC 6, TVCC 7, TVCC 8, TVCC 9, TVCC 10, GDV 2, GDV 3, GV 1, GV2 and KSP 21 yielded a PCR product of 870 bp. Two S. aureus isolates KSP 17 and KSP 25 yielded PCR product of 960 bp. The present study confirms coa gene polymorphism in S. aureus isolates from bubaline mastitis. It is interesting to note that the S. aureus isolates GDV 5, GV 7, KSP 14, KSP 15 and KSP 16 were negative for coagulase activity in tube coagulase test and also didn’t yield any PCR product with coa gene specific primers in PCR test. These S. aureus isolates are surmised to be to be coagulase negative variants.
All the 30 S. aureus isolates were tested for staphylococcal enterotoxin genes seg, seh, sei in PCR test using oligonucleotide primers specific to these three enterotoxin genes. In PCR test 30% of S. aureus isolates (n=9) viz. TVCC 10, TVCC 14, TVCC 18, TVCC 23, GDV 1, GDV 3, GV 6, KSP 15 and KSP 21 were positive for seg gene; 20% of S. aureus isolates (n=6) viz. TVCC 10, TVCC 18, TVCC 23, GDV 1, KSP 3 and KSP 4 were positive for seh gene; 40% of S. aureus isolates (n=12) viz. TVCC 10, TVCC 14, TVCC 18, TVCC 23, GDV 1, GDV 3, GV 2, GV 6, GV 7, KSP 3, KSP 15 and KSP 21 were positive for sei gene. Four isolates TVCC 10, TVCC 18, TVCC 23 and GDV 1 were positive for all the three enterotoxin genes seg, seh and sei. Five isolates TVCC 14, GDV 3, GV 6, KSP 15 and KSP 21 were positive for two enterotoxin genes seg and sei. One isolate KSP 3 was positive for seh and sei genes, whereas only one isolate KSP 4 is positive for one enterotoxin gene seh. Therefore in the present study just 13.33% of S. aureus isolates were
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