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. 2013 Nov 2:2013:378492.
doi: 10.1155/2013/378492. eCollection 2013.

Investigation of biofilm forming ability in Staphylococci causing bovine mastitis using phenotypic and genotypic assays

Samah F Darwish  1 , Hanaa A E Asfour
Affiliations

Investigation of biofilm forming ability in Staphylococci causing bovine mastitis using phenotypic and genotypic assays

Samah F Darwish et al. ScientificWorldJournal. .

Abstract

A total of 40 S. aureus and 68 coagulase negative Staphylococcus (CNS) isolates from bovine subclinical mastitis were investigated for their ability to form biofilm as one of the most important virulence factors.Using Congo Red Agar (CRA) method, 32.5%, 35%, and 32.5% of S. aureus strains were strong, intermediate, and negative biofilm producers, while in CNS the percentages were 29.5%, 42.6%, and 27.9%, respectively. By microtiter plate (MTP) method, 52.5%, 27.5%, and 20% of S. aureus isolates were strong, moderate, and weak biofilm producers, while in CNS the percentages were 44%, 30.9%, and 19.2%, respectively. Indian ink staining was used to detect the EPS layer of biofilm producers. All isolates were screened for presence of biofilm related genes, eno, icaA, icaD, and bap. In S. aureus isolates, the positive rates of eno, icaA, icaD, and bap genes were 75%, 15%, 62.5%, and 2.5% while in CNS were 92.6%, 5.9%, 47.1%, and 4.4%, respectively. The eno gene had the highest rate while the bap gene had the lowest rate. Presence of icaA and icaD genes was not always correlated with biofilm production. This study demonstrated high prevalence of Staphylococcus biofilm producers among bovine mastitis in Egypt. Therefore, attention must be paid toward implementation of new ways for effective treatment of such infections.

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Figures

Figure 1
Figure 1
Screening of biofilm (slime) producer Staphylococci using Congo red agar plate method ((a) dry black colonies; (b) smooth black colonies; (c) dry red colonies; (d) smooth red colonies).
Figure 2
Figure 2
2 Microtiter plate method showing none, strong, moderate, and weak biofilm producers differentiated by crystal violet stain in 96-well tissue culture plate.
Figure 3
Figure 3
Morphology of some of Staphylococcus isolates as observed after staining with Indian ink. The arrows pointed to capsular-like EPS (light microscopy ×100).
Figure 4
Figure 4
Agarose gel electrophoresis of PCR products stained with ethidium bromide. (a) eno gene (302 bp), M: 100 bp ladder DNA marker, 1–8: positive samples, N: negative control. (b) icaA gene (1315 bp), M: 1 kb plus DNA marker, 2, 3, 5, 7, 8: positive samples, 1, 4, 6, 9, 10: negative samples, N: negative control. (c) icaD gene (381 bp), M: 100 bp ladder DNA marker, 2–5, 7–10: positive samples, 1, 6: negative samples, N: negative control. (d) bap gene (971 bp), M: 100 bp ladder DNA marker, 1, 3: positive samples, 2, 4–9: negative samples, N: negative control.
Figure 5
Figure 5
Prevalence of biofilm related genes in S. aureus and CNS isolated from bovine mastitis milk.
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