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Advisor: A.K. Kataria
Publisher: Rajasthan University of Veterinary and Animal Sciences Bikaner-334001
Language: en
Type: Thesis
Agrotags: null
Abstract: The present study was aimed to find genetic variability among Staphylococcus aureus isolates from various clinical and non-clinical animal and human settings in relation to some virulence factors and antibiotic resistance pattern including their associated genes. Of the 517 samples processed from, various clinical and non-clinical sources viz. clinical infections of human, meat pieces, horse wounds, pig nasal cavity, camel wounds, dog skin infections, clinical infections of sheep and mastitic milk of buffalos, goats and cattle, overall prevalence of S. aureus was 30.3% (157/517) with highest prevalence in human 43.75% (35/80) samples using species-specific 23S rRNA based primers with 1250bp size amplicon. In phenotypic characterization, four types of colonies viz. pale yellow (46.5%), whitish (31.8%), golden yellow (15.9%), mustard yellow (5.7%) were obtained on nutrient agar, 97.5% isolates were mannitol fermenter while 2.5% isolates were non-fermenters, 92.4% isolates produced slime while 7.6% did not. Free coagulase was produced by 94.2% isolates produced while 5.7% isolates did not produce it. Human plasma showed the best coagulation reaction. Haemolysis was shown by 94.90% isolates of which 68.8% exhibited complete haemolysis, 24.8% incomplete / partial haemolysis and 1.3% isolates showed both complete and partial hemolysis while 5.1% isolates were ahaemolytic. In qualitative toxin assay, 75 (47.8%) isolates were beta-toxin produce and 121 (77.1%) isolates were delta-toxin producers. The β toxin titres were recorded between 1:5 and 1:320 and delta () toxin titres between 1:5 and 1:40. In antibiogram studies more than 95.0% isolates were recorded resistant to ampicillin and penicillin-G while approximately 100% isolates were sensitive to chloramphenicol, meropenem and nitrofurantoin, more than 85% isolates were sensitive to ampicillin+ sulbactum, cefalothin, ceftazidime+ clavulanic acid, doxycycline hydrochloride, imipenem, oxacillin, piperacillin+ tazobactam, polymxin-B and tobramycin. Isolates were detected with highly significant (p≤0.01) variation in their resistance patterns for 39 antibiotics, significant variation (p≤0.05) for levofloxacin and nitrofurantoin, and no significant variation (p˃0.05) for clindamycin. The isolates were grouped in to three hierarchical ascendant clusters on the basis of antibiogram, one cluster comprising of buffalo, cattle, sheep, dog and goat, second cluster included horse, camel, meat piece and pig while third cluster included human isolates. Human isolates had highest (0.40) MAR index. Sixty six (42%) isolates had 0.2 or more than 0.2 MAR index while 91 (58%) isolates had less than 0.2 MAR index. The MIC for azithromycin was 73.40 mcg/ml, for vancomycin 1.22 mcg/ml, for oxacillin 4.92 mcg/ml, for gentamicin 12.35 mcg/ml, for ciprofloxacin 18.38 mcg/ml, for chloramphenicol 5.85 mcg/ml, for ceftriaxone 18.55 mcg/ml and for penicillin 16.35 mcg/ml, with highly significant (p≤0.01) variation for three antibiotics namely azithromycin, ciprofloxacin and penicillin, significant variation (p≤0.05) for vancomycin, and no significant variation (p>0.05) for ceftriaxone, chloramphenicol, gentamicin and oxacillin in all isolates. Phenotypically, 54.1% and 12.1% isolates were identified as MRSA by MeReSa agar base method and methicillin disk methods, respectively. The Beta-lactamase production, ESBL and VRSA activity was detected in 90.4%, 68.8% and 33.1% isolates, respectively. The mecA and blaZ genes were found in 23.6% and 83.3% isolates with single amplicon of 533bp and 517bp, respectively. Non-significant variations were recorded in the sequences analysis of mecA gene. Thirty six different rep patterns were obtained which, comprising of 300 to 1400bp band sizes with different arrangements and 0.8892 discriminatory index. The patterns were, grouped into five clusters at 80% genetic similarity level and most of the animal isolates were separately clustered in Ist and Vth clusters in contrast to human and meat piece isolates. In adherence genes, clfA (1000bp) and clfB (205bp) were found in 94.3% isolates, icaA (1315bp) in 95.5%, icaD (381bp) in 91.7% and trap (504bp) was found in 98.0% isolates. Except one isolate (H24) all were typeable with agr typing system, in which 40.1%, 26.8%, 17.2% and 15.3% were typed as agrI, agrII, agrIII and agrIV with 441bp, 575bp, 323bp and 659bp of single amplicon in multiplex PCR, respectively. In capsule genes, 46.5% 37.6% and 14.0% isolates carried cap5K (361bp), cap8K (173bp) and both genes while three isolates (D9, B55 and C34) were negative for any of these genes. In exoenzyme genes, coa gene was detected in 100% isolates with nine amplicon sizes varying from 500 to 900bp. Thirty three 33 different coa-RFLP patterns were obtained with 0.9301 discriminatory index. All coa-RFLP types were grouped into three clusters at 80% genetic similarity. The aur gene (1526bp) was found in 96.8% which, comprised of three different aur-RFLP types (A1, A2 and A3). In sequence analysis of above 12 aur gene samples, overall more than 200 nucleotide and their corresponding amino acid variations were recorded at various positions. Significant (p≤0.05) variations were recorded in sequence analysis between aur-RFLP types with nucleotide variations at more than 200 positions and aur-RFLP types were classified in separate clusters in phylogenetic tree analysis of aur gene sequences. The immune invasion genes, 72.6% and 25.5% isolates carried chp (404bp) and scn (320bp) genes, respectively. The spa-IgG gene was found in all isolates with three polymorphic (600bp, 750bp and 950bp) band patterns and spa-X gene was detected in 100% isolates with nine polymorphic bands (D.I.- 0.7407) ranging from 150bp (4 repeats) to 380bp (14 repeats). The 85.3% isolates had more than 7 repeats while only 14.6% isolates had less than seven repeats. Most of spa-X sequences were significantly (p≤0.05) different from each other with large overhanging flanks, 24bp big insertion and big nucleotide gaps. Five separate clusters were detected in spa-X gene sequences in phylogenetic tree analysis. The sak (403bp) gene was found in 50.3% isolates. Twenty one non-significant (p>0.05) nucleotide variations were recorded in sak gene sequence analysis which, revealed three distinct clusters in phylogenetic tree analysis. Most of isolates of animal origin were deficient in sak and scn genes. In genes for toxins, 98.7%, 59.9%, 95.5% and 12.1% isolates carried hla (534bp), hlb (833bp), hld (111bp) and tst (350bp) genes respectively. The tst gene sequence analysis revealed 12 non-significant (p>0.05) nucleotide variations in isolates with two distinct clusters in phylogenetic tree analysis. Total 83 virulotypes were detected among 157 isolates on the basis of presence or absence of studied 19 various virulence associated genes. All 19 genes were present in virulotype1 (V1) which included one human (H3) isolate while three virulotypes V81, V82 and V83 had 11, nine and eight genes which included J4, C15 and G16 isolate, respectively.
Subject: Veterinary Microbiology
These Type: M.V.Sc.
Issue Date: 2016
Appears in Collections:Thesis

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