MOLECULAR CHARACTERIZATION AND GENOTYPING OF BIOFILM-PRODUCING STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS
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Date
2022-03
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College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
Abstract
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.