Phenotypic and genotypic characterization of Staphylococcus aureus of mastitic milk origin from cattle and buffalo for some virulence properties
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Date
2013
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Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001
Abstract
Mastitis is one of the most important diseases of lactating animals leading to severe financial loss. Cattle and buffalo are maximum milk producing animals in India, so the occurrence of mastitis in these species is an issue of serious concern. Of the
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many causes Staphylococcus aureus is the most important infection. This organism is blessed with many virulence factors such as, toxins, enzymes and a variety of extracellular or cell wall associated factors and the ability to develop antibiotic resistance after short period of exposure. In the present investigation the overall prevalence of S. aureus in both cattle and buffalo mastitic milk was 35.95%. These isolates were studied for their phenotypic properties including colony characteristics, fermentation of various sugars, haemolysis on blood agar, production of coagulase in tube test, slime production, DNase activity, thermostable nuclease activity, beta-lactamase activity, typing as well as titration of haemolysins and antibiogram against antibiotics of different generations and production. The genotypic confirmation of S. aureus was done by polymerase chain reaction using species-specific 23S rRNA based primers. Further the isolates were characterized by PCR for some of the genes such as coa, spa X-region, clfA, hla and hlb responsible for different virulent properties. On aerobic cultivation on nutrient agar colonies obtained were round, smooth and glistening but with variable pigment colors. Of the 12 sugars eight were fermented by variable number of isolates whereas dulcitol, inositol, arabinose and raffinose were not fermented by any of the isolates. On the basis of sugar fermentation reactions the cattle isolates were divisible into six groups and buffalo isolates into seven. All the isolates produced coagulase and human plasma showed the best coagulation reaction. THE OVERALL HAEMOLYTIC REACTIONS ON SHEEP BLOOD AGAR SHOWED THAT 15.62, 62.50, 12.50 AND 9.37% ISOLATES SHOWED COMPLETE, PARTIAL, BOTH AND NO HAEMOLYSIS REACTIONS, RESPECTIVELY. DELTA (δ) HAEMOLYSIN ON HORSE BLOOD AGAR WAS SHOWN BY NINE ISOLATES (28.12%) ONLY. On toxin titration all the isolates were found to produce alpha-toxin (maximum titre being1:5120) whereas beta-toxin (maximum titre being1:1280) was produced by 68.75% cattle isolates and by 43.75% buffalo isolates. Of the total, 15.62% isolates (two from cattle and three from buffalo) detected as delta-toxin (maximum titre being 1:80) producer. In present investigation 93.7% isolates showed DNase activity and all isolates exhibited thermonuclease activity. Slime production was recorded in very high percentage (96.87%) of isolates. We developed antibiograms against 33 antibiotics belonging to different categories and generations. Seven antibiotics namely chloramphenicol, doxycycline, gentamicin, methicillin, netiline, rifampisin and tobramycin were 100% effective against all the cattle isolates and eight antibiotics namely cefdinir, doxycycline, gentamicin, linezolid, methicillin, netiline, rifampisin and
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tobramycin showed 100% efficacy against buffalo isolates. Both cattle and buffalo isolates were resistant to cephalexin, polymixin B, cefixime, cephalexin and amoxicillin. Beta-lactams production was detected only in 12.5% isolates from cattle and in 56.20% buffalo isolates. The ribotyping produced an amplicon of 1250 bp in all the isolates confirming them to be S. aureus. The cattle isolates were grouped into four coagulase types based on the size of coa gene amplicons (650, 600, 510 and 400 bp) and buffalo isolates were divisible into five groups (680, 650, 600, 510 and 400 bp). In the present investigation, six RFLP patterns of coa gene amplicon were generated using AluI restriction enzymes from five different coagulase types. The cattle isolates produced spa amplicons of 120, 150, 200, 250, 280, 300 and 330 bp with calculated number of repeats of 3, 4, 6, 8, 10, 11 and 12, respectively while buffalo isolates produced five different types and the sizes of spa amplicons were 150, 200, 250, 330 and 380 with 5, 7, 9, 12, and 14, respectively. The present study suggested a great variability in cattle and buffalo mastitic S. aureus isolates in regards to spa typing. Of the total, 75% isolates were considered pathogenic based on spa typing. The clfA gene was amplified in 84.30% isolates from both cattle and buffalo, producing two different amplicons of 900 (71.80%) and 1000 bp (12.50%) sizes showing polymorphism. In the present study 15 cattle and all the buffalo isolates (96.80%) amplified hla gene producing amplicons of 534 bp and the hlb gene was amplified by 13 cattle and 14 buffalo isolates (84.30%) producing single amplicon of 833 bp in each.
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Keywords
livestock, bacteria, genes, mastitis, antibiotics, productivity, animal husbandry, polysaccharides, enzymes, diseases