Analysis of biofilm formation and associated gene detection in Staphylococcus isolates from bovine mastitis

Abstract

The objective of this study was to investigate the biofilm-forming ability and distribution of biofilm associated genes in clinically isolated Staphylococcus in bovine mastitis. Silver staining, scanning electron microscopy (SEM) and crystal violet staining were conducted for the detection of biofilmforming ability in 24-well plates. The bap, icaAD, icaBC, Staphylococcal accessory regulator (sar), accessory gene regulator (agr), sigB, clumping factor A (clfA), clfB, fibronectin-binding proteins (fnbpA) and fnbpB genes were amplified by polymerase chain reaction (PCR). Formation of biofilms was found macroscopically in 120 of the 137 strains after being stained with silver (biofilm-forming rate 87.6%). Five strains did not adhere to the surface of the silica gel after being stained with crystal violet, while the remaining 132 strains did adhere. Bap was amplified in 57 isolates, and icaAD and icaBC were isolated in 43 and 54 strains, respectively. SigB, sar and agr were amplified in 73, 49 and 38 isolates, respectively, and clfA and clfB were isolated in 76 and 50 strains, respectively. FnbpA was present in 52 strains and fnbpB in 26 isolates. Our study reveals that bap, sigB, sar, icaAD and icaBC may be crucial biofilm associated genes since these genes were present more often in biofilm-positive strains than in biofilm-negative strains. There was no obvious difference between the frequencies of agr in the biofilmpositive strains and biofilm-negative strains, which indicates that the role of agr in biofilm development is still controversial. The distribution of clfA, clfB, fnbpA and fnbpB in biofilm-positive strains were not greatly different from that in biofilm-negative strains.

Key words: Bovine mastitis Staphylococcus, biofilm, silver staining, crystal violet staining.