S. aureus is the most significant causative agent of Bovine Mastitis. Adhesion proteins on cell wall of bacteria are an essential tool for establishment and continuation of chronic infection in mammary gland. The aim of present study was to screen isolates of S. aureus from mastitic milk via amplification of fnb protein and fib protein genes. PCR amplification of both genes revealed 404 bp product of fib gene and 524 bp product of fnbB gene.
S. aureus is extremely versatile pathogen and most significant bacterial pathogen associated with bovine mastitis. It has been reported that nearly 19% of the intramammary infections are caused by S. aureus (Wilson et al., 1997). S. aureus expresses several adhesion proteins collectively called as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which endorse bacterial interaction with components of extra cellular matrix (ECM). Its binding to ECM allows bacteria to adhere, colonize in host tissue follows establishment of infection. These proteins recognize fibronectin, fibrinogen, collagen, vitronectin, laminin, elastin, von Willebrand factor etc. Staphylococcal surface adhesion molecules like fibronectin, fibrinogen and collagen binding proteins have been shown to contribute to persistence of bacteria by adhering to sub epithelial tissue components after epithelial damage or after bacterial invasion through epithelia (Kot et al., 2016). Adhesion genes are the essential virulence genes of organism for establishment of infection in bovine mammary gland. So, the present study was carried out for screening of such genes in organisms isolated from bovine mastitis cases.
Materials and Methods
Isolation of S. aureus DNA
Bacteria from 35 affected milk samples were isolated on selective media. Among isolated bacteria 15 were identified as S. aureus by morphologic characteristics and biochemical analysis. Pure colonies of isolated organisms were allowed to grow in BHI broth for 18 hours. Bacterial lysis was achieved by direct heat application immediately followed by snap chilling in crushed ice. Centrifugation of broth was done to isolate bacterial genome for 10 minutes at 3000 rpm and 4oC temperature. From the supernatant 5 µl of liquid was taken in the PCR tubes as DNA templates used for PCR assays.
Amplification of Adhesion Genes
PCR amplification of adhesion genes fnbB (fibronectin binding protein B) and fib (fibrinogen binding protein) was accomplished by using following primers.
Table 1: Nucleotide primers of fib and fnbB genes of S. aureus
|Fibronectin Binding Protein B
|Forward||GTA ACA GCT AAT GGT CGA ATT GAT ACT||Tristan et al., 2003|
|Reverse||CAA GTT CGA TAG GAG TAC TAT GTT C|
|Fibrinogen Binding Protein
|Forward||CTA CAA CTA CAA TTG CCG TCA ACA G|
|Reverse||GCT CTT GTA AGA CCA TTT TCT TCA C|
The thermal cycling conditions for fnbB gene included an initial denaturation step (5 min at 940C) followed by 30 cycles of amplification (1 min denaturation at 940C, 1 min annealing at 580C and 1 min extension at 720C). The reaction was terminated with a 10 min incubation step at 720C. Cycling conditions for fib gene were same as above except annealing step was performed at 470C.
Result and Discussion
Screening of isolated for presence of adhesion genes by PCR technique gave good result. Fibgene was detected in 5 (33.33%, n=15) bacterial samples, whereas fnbB gene was detected in 1 (6.67%, n=15) bacterial sample isolated from mastitic milk. The PCR amplification of fib gene revealed 404 bp product (Fig. 1) using previously published oligonucleotide primer sequence (Table 1), whereas amplification of fnbB gene reveal 524 bp product (Fig. 2).
|Fig. 1: PCR amplification of fib gene recovered 404 bp products in 1, 2, 3 and 4 lane.||Fig. 2: PCR amplification of fnbB gene recovered 524 bp products in 1 lane.|
Staphylococcal surface adhesins such as fibronectin, fibrinogen binding proteins etc. have contribution in the persistence of intramammary bacterial infection by adhering to subepithelial tissue components after bacterial invasion and tissue damage (de Bentzmann et al., 2004). The organism has been shown to interact with fibrinogen through fibrinogen binding protein during intramammary infection is helping in colonization (Khoramrooz, et al., 2016). The fibrinogen binding protein probably have important role in pathogenicity by allowing bacterial to avoid host defenses and by acting as adhesins. Fibronectin binding proteins are essential for adhesion and invasion of the bovine mammary epithelial cells. Dziewanowska et al., (1999) reported the mutant devoid of fibronectin binding protein loses the ability to adhere and invade the bovine mammary epithelial cell line MAC-T by 95% whereas, Brouillette et al., 2003, described that the presence of fibronectin binding protein on S. aureus increases the capacity of the bacteria to colonize in the mammary gland in mouse model mastitis. S. aureus generally causes chronic and subclinical mastitis in lactating dairy animals. These adhesive molecules help in adhering to the mammary epithelial cells and it is generally believed that it is the decisive step in infection process and preventing the bacteria from flowing out of the gland during milking. Hensen et al., 2000, noted that these adhesin molecules contribute to the chronic nature of the disease and the author further described it could be due to the low efficacy of the antibiotic therapy. Kuzma et al., 2006, reported that 92.06% pathogenic S. aureus isolates from bovine mastitis bears fib gene whereas, 48.82% bears fnbB gene. In the present study the frequency of fib gene was much higher in pathogenic isolates than the fnbB gene.
Snap chilling method for acquiring PCR templates is one of the simple method for PCR amplification of S. aureus genome. Screening of S. aureus for presence of adhesion genes via PCR amplification is superior method.