The chemical components of growth media and their supplements such as NaCl, ethanol, glucose and sub-inhibitory concentrations of antibiotics, strongly affect biofilm development (Rohde et al., 2001). In particular, the presence of glucose in the growth medium has been reported to enhance biofilm formation (Stepanović et al., 2007). Various methods have been introduced to measure the biofilm-forming capability of S. aureus isolates, among which, MtP is one of the most common methods in bacteriology laboratory. In this technique, TSB medium containing various glucose concentrations is often used to study the adherence ability of S. aureus to 96-well polystyrene tissue culture plates (Dhanawade et al., 2010).
In the present study, the ability of biofilm formation by S. aureus strains was investigated in four bacterial culture media (NB, TSB, MHB and BHIB) containing various glucose supplements (0, 0.25, 0.5, 1 and 2%) using MtP assay. The results showed that among all of the tested broth media, TSB was the most suitable medium to examine the ability of biofilm formation. However, some studies reported that bacterial biofilm formation in BHIB is sometimes occurred better than TSB (Kennedy and O'Gara, 2004; Mathur et al., 2006; Stepanović et al., 2007). Nevertheless, it has also been shown that some staphylococcal strains produce greater biofilm in TSB compared to BHIB (Knobloch et al., 2002). Moreover, researchers commonly use TSB with various glucose supplements for evaluation of biofilm formation by S. aureus strains (Kennedy and O'Gara, 2004). It has been reported that addition of glucose to culture media lead to the increase of the ability of staphylococci biofilm formation (Knobloch et al., 2002; Mathur et al., 2006). In agreement with the previous findings, the present study showed significant increase in S. aureus biofilm production when the bacterium exposed to the increasing concentrations of glucose. The best concentration of glucose for biofilm formation of S. aureus on polystyrene surfaces in all of the tested culture media was 1%.
In addition, Congo red tube test and real time-PCR were used to detect extracellular exopolysaccharide and demonstrate icaA expression, respectively. RT-PCR results showed that icaA was expressed in all of the media with different glucose concentrations but the most expression occurred when one percent glucose was added to the medium (P < 0.05). Within the icaADBC operon, the icaA gene encodes the enzyme N-acetylglucosaminyl transferase, capable to catalyse the synthesis of poly-N-acetylglucosamine polymer which is called PIA and is important for cell to cell adhesion and accumulation (Gerke et al., 1998). The icaADBC and products of the ica locus [icaR (regulatory) and icaADBC (biosynthetic) genes] have been demonstrated to be necessary for biofilm formation (Croes et al., 2009). Consequently, in the present study, the expression of icaA locus was evaluated in different culture media supplemented with 0, 0.25, 0.5, 1 and 2% glucose. The results revealed that among all culture media (TSB, MHB, NB and BHIB) the most icaA expression level was detected in 1% glucose addition. In addition, the color of Congo red tube culture was not changed was not changed by glucose addition, indicating that this medium is not suitable for measurement of precise amount of PIA or measurement of icaADBC operon expression.
The results of the present study revealed that in fact, glucose increases the ability of biofilm formation by promoting icaA transcription and subsequently, PIA production. Hence, additional ica-independent factors may be involved in increasing of phenotypic biofilm formation by staphylococci. For instance, it was reported that Agr system is down-regulated by addition of glucose (Regassa et al., 1992), and S. aureus agr mutants also show enhanced biofilm formation (Vuong et al., 2000). Therefore, glucose-induced promotion of biofilm formation may be related to a suppression mechanism of agr. Using phenotypic analysis of wild type and mutant strains of S. aureus, Lim et al also described that rbf gene is another gene that mediates the biofilm formation of S. aureus at the multicellular aggregation stage in an ica independent manner (Lim et al., 2004), suggesting that the genetic basis of biofilm formation in staphylococci is multifactorial, and much remains to be explored.
In conclusions, the present study suggested that among various examined media, TSB supplemented with 1% glucose was the most appropriate medium for evaluation of biofilm formation of S. aureus strains. However, further studies are necessary to elucidate the precise mechanisms of increasing S. aureus biofilm formation following glucose induction to culture media.