As combination of phenotypic and genotypic methods would be recommended for identifying biofilm producing strains, the detection of 4 biofilm related genes and their incidence in Staphylococcus isolates were investigated using simplex PCR reactions (Figures (Figures44 and and5).5). Because of the absence of a reference positive control for each of the studied genes, apply for it the DNA of all isolates was first examined by Staphylococcus genus specific primers to monitor the quality of the DNA for amplification and to exclude any false negative results. All the 108 Staphylococcus isolates successfully amplified the specific 228bp of the 16S rRNA gene of genus Staphylococcus. This amplification confirmed all the isolates to be Staphylococci and ensuring the suitability of the DNA for amplification.
The intercellular adhesion (ica) locus, consisting of the genes icaADBC, has been reported to have a potential role as a virulence factor in the pathogenesis of mastitis in ruminants [4]. Among the ica genes, icaA and icaD have been reported to play a significant role in biofilm formation in S. aureus and S. epidermidis [32]. The prevalence rates of icaA and icaD genes were 15% and 62.5% in S. aureus isolates and 5.9% and 47.1% in CNS isolates, respectively (Figure 5). Although the majority of researches reported the icaA and icaD to be nearly similar in incidence [2, 4, 30], our results agreed withCiftciet al. [33] who found that 16 and 38 out of 59 strains were positive for icaA and icaD genes, respectively.
This difference in the prevalence rates can be attributed to variation in DNA sequences which may lead to failed amplification of the gene in some isolates leading to false negative results [34]. This was previously reported by Simojoki et al. [35] who excluded icaA and icaD results from their work because their primers, although were able to detect the genes in S. aureus isolates, were not able to detect the genes in known icaA and icaD positive S. epidermidis. Detection of icaA and icaD was not well correlated with biofilm production on MTP methods (Table 5). Presence of icaA or icaD negative biofilm positive isolates can be accounted for by an ica gene independent control of slime production/adhesion mechanism [36].
On the contrary, inability of Staphylococcus isolates that Carfilzomib were positive for icaA and/or icaD genes to produce biofilm in vitro can be due to point mutation in the locus and/or any other yet unidentified factors that negatively regulate polysaccharide intercellular adhesion synthesis or influence biofilm formation [1]. Also, some experimental evidence supports the development of new clones referred as biofilm negative with both icaA and icaD genes positive [37]. Additionally, lacking of icaA mRNA or icaD mRNA or both was reported to explain absence of phenotypic expression of biofilm [36]. Despite discrepancy between results of ica locus and biofilm phenotypic expression, Vasudevan et al.