Studies on Phenotypic and Molecular Characterization of Coagulase Negative Staphylococci Isolated from Clinical and Subclinical Cases of Bovine Mastitis
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Date
2012-05-15
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Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
Abstract
The present study was carried out with the objective of isolation, identification
and study of antimicrobial resistance patterns of coagulase negative staphylococci
(CoNS) associated with bovine mastitis. In all, 313 bovine milk samples collected from
different organized/ unorganized sectors were screened for subclinical mastitis (SCM) by
somatic cell count, electrical conductivity test, California mastitis test and bromo thymol
blue strip tests. These tests revealed SCM status at 42.8% and 48.2%, 21.4% and 26.8%,
respectively. On processing all the samples for isolation, a total of 152 CoNS isolates
including 14 isolates from clinical mastitis samples (n=42) were obtained. Biochemical
characterization of the isolates by HiStaph™ Identification Kit revealed S. arlettae, S.
fleurettii, S. equorum and S. epidermidis as predominant CoNS species in SCM cases.
Further, these isolates were subjected to two-tube multiplex PCR targeting
gap/rpoB/sodA genes for species level identification, subsequent to the confirmation at
genus level by the partial amplification of tuf gene. Of the 10 species targeted by m-
PCR, S. epidermidis, S. chromogenes and S. sciuri were the major CoNS detected.
Coagulase negative variants of S. aureus were also detected by S. aureus specific triplex
PCR. The overall agreement between phenotype- and genotype-based speciation of
CoNS was only 11%. Antibiogram studies of CoNS isolates revealed highest resistance
to oxacillin/ methicillin, followed by ceftriaxone with sulbactum/ tazobactum, penicillin
G and aminopenicillins. Highest sensitivity was recorded for chloramphenicol followed
by enrofloxacin and gentamicin. Another multiplex PCR to detect antibiotic resistance
genes and coa gene revealed mecA, aacA-D and aph3'-IIIa genes in 10, 15 and 5 isolates,
respectively. Coa gene was detected only in 15 of the 19 coagulase negative S. aureus
isolates. The two-tube multiplex PCR assay developed in the present study is an easy and
rapid method to simultaneously detect the ten major CoNS prevailing in bovine mastitis.
Description
Ph.D. Thesis