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2017 - 201912020 - 20221
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Subject: Animal Biotechnology × Author: DUTTA, MADHUSMITA × Type: Thesis ×
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION AND GENOTYPING OF BIOFILM-PRODUCING STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2022-03) DUTTA, MADHUSMITA; Borah, Probodh
    Mastitis is an inflammatory disease of dairy animals and is considered as one of the commonest problems of the dairy industry throughout the world. Though it is a multietiological disease, Staphylococcus aureus is considered to be a common and potent cause of mastitis due to its wide array of virulence factors and ability of forming biofilm. The present study aimed at isolation and identification of biofilm forming S. aureus from mastitic and apparently normal bovine milk samples, and molecular detection of genes related to adhesion and biofilm formation, antibiotic resistance and enterotoxin production. The study also included molecular typing of representative isolates by three methods namely, multilocus sequence typing (MLST), surface protein A typing (spa typing) and accessory gene regulator typing (agr typing). The study was further extended to assessment of antibiofilm efficacy of three compounds- chitosan, EDTA and povidone iodine against selected S. aureus isolates in vitro. The study included a total of 136 milk samples comprising of 26 from mastitic and 110 from apparently healthy cows. In California Mastitis Test for detection of sub-clinical mastitis, 84 (76.40 %) of the 110 apparently normal milk samples tested positive. On bacteriological examination, 18 (69.23%) of 26 milk samples from mastitic cows and 64 (76.19%) of 84 apparently normal milk samples were to be positive for Staphylococcus with an overall positivity of 74.55%. Altogether 78 (95.12 %) of the 82 Staphylococcus isolates were found to be coagulase positive and confirmed as S. aureus based on detection of the species-specific aroA gene by polymerase chain reaction (PCR). Among the 78 S. aureus isolates, 54 (69.23 %) were identified as biofilm producers based on their characteristic growth on Congo red agar (CRA) plates. However, on PCR amplification, 58 (74.36%) of these isolates were found to carry icaA and icaD genes. All the isolates were found to have both fnbA and clfB genes, while 98.71, 61.50 and 11.53 per cent of the isolates were positive for clfA, cna and bap genes, respectively. On antimicrobial susceptibility testing, all the 78 isolates were found to be resistant to Oxacillin and Tetracycline. The isolates showed highest (25.64%) susceptibility to Cefepime followed by Ceftriaxone (23.08%) and Cefotaxime (20.51%). A very low susceptibility was shown to Gentamicin (10.26%), combination of Tricarcillin and Clavulanic acid (3.85%), Chloramphenicol (2.56%) and Co-Trimoxazole (2.56%). Among the 54 biofilm producing isolates, 48 (88.89%) and 45 (83.33%) were found to carry blaZ1 and blaZ2 genes, respectively, while not a single isolate carried the smr gene. On the other hand, among the 24 non-biofilm producing isolates, 22 (91.67%) possessed both blaZ1 and blaZ2 genes, while none carried the smr gene. Of the 78 isolates, 14 (17.94 %) were found to have at least one of the three staphylococcal enterotoxin genes (sea, seb and sed) included in the study. However, none of the isolates were found positive for see genes. Multi Locus Sequence typing (MLST) of 15 representatives biofilm-producing S. aureus isolates could detect three sequence types (STs) and one clonal complex (CC). Seven isolates belonged to ST672, five to ST1713 and three to ST2592 with slight variations in the allelic profile. ST672 had no CC while ST1713 and ST2592 belonged to CC1. Spa typing of the same isolates revealed three different spa types, t1309, t1611 and t267. Of the 15 isolates tested, two agr types were identified: agr I (60 %) and agr II (40 %). Strains belonging to agr types III and IV were not detected in this study. Antibiofilm efficacy of 5% povidone iodine (betadine®), 20 mM EDTA and 5 mg/ml of chitosan was tested in the present study based on determination of MIC of these compounds either alone or in combination against selected biofilm-producing S. aureus isolates. It was found that the combined effect of all the three compounds against biofilm-producing S. aureus was almost similar to that of the combination of chitosan and povidone iodine. Hence, the later combination was suggested as an alternative to using high concentration of an antiseptic for sanitization of the udder surface of milch cows to get rid of biofilm-producing S. aureus frequently associated with subclinical mastitis.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION AND GENOTYPING OF STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS
    (Assam Agricultural University, Khanapara,Guwahati, 2017-07) DUTTA, MADHUSMITA; Borah, P.
    Mastitis is an important disease of dairy cows and buffaloes causing huge economic losses in the form of reduced milk production. It is an inflammation of the mammary gland (udder) that causes physical and chemical changes in milk, and leads to pathological condition of the glandular tissue. It is generally associated with poor hygienic and husbandry practices. The present study was undertaken with a view to isolate and identify Staphylococcus aureus from both mastitic and apparently normal bovine milk samples. The study also included molecular typing of representative isolates and detection of important virulence-associated genes by PCR. A total of 204 milk samples comprising both clinically affected (14) and apparently normal (190) milk were used for this study. The apparently normal milk samples were subjected to California mastitis test, of which 85.79 % tested positive for sub-clinical mastitis. Bacteriological and biochemical examinations were performed to isolate and identify staphylococci associated with mastitis. A total of 60 (33.8%) out of 177 milk samples yielded Staphylococcus aureus, which were confirmed by polymerase chain reaction (PCR) amplification of conserved sequences of aroA gene. All the isolates (100 %) were found to possess three virulence-associated genes, namely surface protein A (spa), thermonuclease (nuc) and coagulase (coa) genes, while 58 (96.6%) of the isolates showed the presence of clumping factor A (clfA) gene. Antimicrobial susceptibility testing revealed that all the 60 isolates were resistant to Ampicillin and Cotrimoxazole, while the highest susceptibility (100%) was shown to Gentamicin, Kanamycin and Chloramphenicol followed by Streptomycin (80%). On the other hand, significantly lower susceptibility was shown to Ceftriaxone (13.33%), Tetracycline (8.33%) and Cefapime (1.67%). Out of the total 60 isolates, seven were subjected to PCR-RFLP of the coagulase (coa) gene. Polymorphism was shown by all the isolates (100%) with four different restriction patterns. Ten isolates were subjected to staphylococcal protein A (Spa) typing and PFGE. Spa typing revealed two different types, t165 and t1611. On the basis of phylogenetic analyses based on spa typing and PFGE, it was concluded that isolate number 9 of Spa type t165 is the ancestral strain, the clonal descendents of which are endemic in the study area causing subclinical bovine mastitis.