Investigation of Biofilm Forming Ability in Staphylococci Causing Bovine Mastitis Using Phenotypic and Genotypic Assays

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.

Detection of Biofilm Production Capability and icaA/D Genes Among Staphylococci Isolates from Shiraz, Iran

“…The results of this study and previous studies indicate a high prevalence of the icaA/D genes among staphylococci isolates, and that their presence is not necessarily associated with in-vitro formation of biofilm (36). In the current study, the proportion of S. epidermidis isolates that were positive for icaA/D genes in PCR method was higher than the proportion of those that were positive in CRA method.…”

Identification and production of biofilm by Staphylococcus aureus isolated from buffalo milk and milking environment

“…No sample was positive for icaA, but seven (21.8%) samples were only positive for icaD, suggesting that biofilm can be formed even when only one ica loci gene is present. These results are like those obtained by Darwish and Asfour (2013) with bovine milk samples. These authors observed prevalence rates of icaA and icaD genes of 15.0 and 62.5%, respectively.…”

“…Although, other authors reported lower sensitivity and specificity of CRA biofilm detection (Taj et al, 2012;Darwish and Asfour, 2013), and also this test may not be reliable for detection of biofilm (Taj et al, 2012), in this study, this test demonstrated a good correlation with gold standard microtiter assay. Biofilm producer samples were found to be simultaneously positive for the presence of icaA and icaD genes in nine (28.1%) of the 32 samples.…”

“…The lower detection of icaA, and therefore the differences in the prevalence rates can be attributed to the variation in DNA sequences. This may lead to failed amplification of the genes in some isolates and consequently false negative results (Tormo et al, 2005;Ferrer et al, 2012;Darwish and Asfour, 2013). Since ica locus is responsible for the production of intracellular adhesion polysaccharide (PIA) (Krewer et al, 2015) and thus forming the multiple layer of cell in a slime matrix characteristic of the staphylococcal biofilm (Darwish and Asfour, 2013), the presence of icaA or icaD negative strain, with biofilm production in Congo Red Agar and microplate assay isolates, can be accounted for by an ica gene independent control of slime production/adhesion mechanism (Liberto et al, 2009).…”

Antibiogram of CoPS and CoNS Isolated from Bovine Mastitis in Banaskantha District of Gujarat, India

“…CoNS are normal inhabitant of udder skin, teat canal and milkers' hand; thus, they are regarded as opportunistic, nosocomial and emerging pathogen Taponen and Pyorala, 2009;El-Jakee et al, 2013). Effectiveness of antibiotic therapy to treat bovine mastitis has been demonstrated to be compromised due to induction of antibiotic resistance and formation of biofilms which can be produced by both CoPS and CoNS Darwish andAsfour, 2013: Tiwari et al, 2013;Al-Rubaye et al, 2016). Among many reasons behind failure of successful treatment of bovine mastitis, one is the failure of appraisal of the antimicrobial resistant pattern of pathogens involved (Du Preez, 2000).…”