Expression of the Biofilm-Associated Genes in Methicillin-Resistant Staphylococcus aureus in Biofilm and Planktonic Conditions

Abstract

The role of genes that are essential for development of Staphylococcus aureus biofilm during infection is not fully known. mRNA from two methicillin-resistant S. aureus strains that formed weak and strong biofilm on polystyrene plates were isolated at five time points from cells grown in biofilm and planktonic culture. Quantitative real-time PCR analysis showed that the expression levels of investigated genes under biofilm conditions were significantly higher than under planktonic conditions. The expression levels of the gene encoding elastin binding protein (ebps) and laminin binding protein (eno) were significantly increased in biofilm at 3 h, both in strongly and weakly adhering strain. The peak expression of fib gene encoding fibrinogen binding protein was found at 6 and 8 h in the case of strongly and weakly adhering strain, respectively. The expression of icaA and icaD genes in both strains was significantly higher under biofilm conditions when comparing to planktonic cells during 12 h. The expression level of the genes encoding binding proteins and the glucosamine polymer polysaccharide intercellular adhesin (PIA) slowly decreased after 24 h. Finally, we found that the expression levels of genes encoding binding factors in weakly adhering strain were significantly lower than in strongly adhering strain.

Keywords: MRSA; biofilm; expression of genes; quantitative real-time PCR.

Figure 1

Electrophoresis in 1.5% agarose gel PCR products obtained by using specific primers for icaA, icaD, eno, fib and ebps genes. Lines M: molecular weight marker (500, 400, 300, 200, 100 bp; GenoPlast Biochemicals, Poland); Lines 1–3: products (188 bp) specific for icaA gene; Lines 4–6: products (198 bp) specific for icaD gene; Lines 7–9: products (302 bp) specific for eno gene; Lines 10–12: products (404 bp) specific for fib gene; and, Lines 13–15: products (186 bp) specific for ebps gene.

Figure 2

Biofilm formation on polystyrene 96-well plates by methicillin-resistant Staphylococcus aureus (MRSA) strains. The weak, moderate and strong biofilm on polystyrene microtiter plate surface stained with 1% crystal violet after 48 h of incubation MRSA strains (A). The quantitative analysis of biofilm production by measuring the optical density of stained biofilms at 492 nm (B). Whiskers indicate standard deviations.

Figure 3

Transcriptional profiling of eno (A), ebps (B), and fib (C) genes in the examined MRSA strains in planktonic growth (PL) and biofilm conditions (BIO). Data were normalized to rpoB (reference) gene. Results are presented as n-fold changes ± standard deviation (SD) in relation to the calibrator (planktonic cells at 6 h of growth period of MRSA strain no. 156, which was the moderate biofilm producer). Different letters (a, b, c, d, e, f, de, ef, cd, bc) denote significant differences in target gene expression among investigated MRSA samples (Tukey’s test; p < 0.05). W—weak producer of biofilm; S—strong producer of biofilm; 3, 6, 8, 12, 24—time of bacterial growth (h).

Figure 4

Abundance of eno (A), epbs (B), and fib (C) transcripts under biofilm conditions (BIO) versus planktonic cells (PL). Data were normalized to rpoB (reference) gene. Results are presented as n-fold changes ± standard deviation (SD) in relation to the calibrator (planktonic cells at 6 h of growth period of MRSA strain no. 156, which was the moderate biofilm producer). Different letters (a, b, c, d, e, cd) denote significant differences in target gene expression among investigated MRSA samples (Tukey’s test; p < 0.05). W—weak producer of biofilm; S—strong producer of biofilm; 3, 6, 8, 12, 24—time of bacterial growth (h).

Figure 5

Transcriptional profiling of icaA (A) and icaD (B) genes in the examined MRSA strains in planktonic growth (PL) and biofilm conditions (BIO). Data were normalized to rpoB (reference) gene. Results are presented as n-fold changes ± standard deviation (SD) in relation to the calibrator (planktonic cells at 6 h of growth period of MRSA strain no. 156, which was the moderate biofilm producer). Different letters (a, b, c, d, e, f, bc) denote significant differences in target gene expression among investigated MRSA samples (Tukey’s test; p < 0.05). W—weak producer of biofilm; S—strong producer of biofilm; 3, 6, 8, 12, 24—time of bacterial growth (h).

Figure 6

Abundance of icaA (A) and icaD (B) transcripts under biofilm conditions (BIO) versus planktonic cells (PL). Data were normalized to rpoB (reference) gene. Results are presented as n-fold changes ± standard deviation (SD) in relation to the calibrator (planktonic cells at 6 h of growth period of MRSA strain no. 156, which was the moderate biofilm producer). Different letters (a, b, c, d, e, f) denote significant differences in target gene expression among investigated MRSA samples (Tukey’s test; p < 0.05). W—weak producer of biofilm; S—strong producer of biofilm; 3, 6, 8, 12, 24 – time of bacterial growth (h).