Prevalence of adhesion and regulation of biofilm-related genes in different clones of Staphylococcus aureus

Abstract

Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto the polystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR.

Figure 1

Colony morphologies of clinical S. aureus isolates on the modified Congo red agar medium.

Figure 2

Comparing the differences in the molecular determination of the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and biofilm genes between MSSA and MRSA isolates. bbp: sialoprotein binding protein, fnbB: fibronectin binding protein B, and cna: collagen binding protein. The intercellular adhesion biofilm genes: icaADBC, fibronectin binding protein A (fnbA), laminin binding protein (eno), elastin binding protein (ebpS), and clumping factors A and B (clfA and B), their distribution did not differ between clones associated with MSSA and those with MRSA. Spa refers to Staphylococcal surface protein A. MLST* refers to multilocus sequence typing. SCCmec*refers to staphylococcal cassette chromosome. MSSA* refers to methicillin-senstive Staphylococcus aureus. MRSA* refers to methicillin-resistant Staphylococcus aureus.

Figure 3

(a, b) Multiplex PCR amplification of MSCRAMMs and biofilm genes of MRSA and MSSA clinical isolates. (a) is related to the amplified bands of 10 genes of icaA, icaD, icaB, icaC, bbp, fib, eno, clfA, clfB, ebbps, and finbA at 1100, 900, 574, 404, 301, 288, 203, 180, and 128 bp, respectively, in one tube reaction, all these genes were determined in 5 different clones of MRSA and MSSA: MRSA-spa-t091(L1), MSSA-spa-t159(L2), MSSA-spa-t14413(L3), MSSA-spa t3204(L4), MSSA-spa-t4085(L5). (b) is related to all the 10 amplified genes mentioned in (a) except bbp, which was replaced with finbB at 523 bp and was found in 16 different clones of MRSA: MRSA-spa-t932(L1), t037(L2, L3, L4), t421(L5, L6, L7), t41509(L8, L9, L10), t2575(L11), t138(L12, L13), and t4213(L14, L15, L16). 16sRNA at product size 455 bp was used as internal positive control. M is DNA ladder marker.

Figure 4

Products of RT-PCR performed on RNA isolated from MSSA and MRSA clones. The presence of RT-PCR products indicates expression of 13 adhesion and biofilm genes. All the genes in this figure were prepared by cutting and pasting from original amplified products in the gel for each gene determined in this study. M100 is DNA base pair size marker.