title: Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA).
abstract: Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times.
authors.affiliation: Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. salmanatshan@yahoo.com
authors.forename: Salman Sahab
authors.initials: SS
authors.lastname: Atshan
authors.forename: Mariana Nor
authors.initials: MN
authors.lastname: Shamsudin
authors.forename: Arunkumar
authors.initials: A
authors.lastname: Karunanidhi
authors.forename: Alex
authors.initials: A
authors.lastname: van Belkum
authors.forename: Leslie Than Thian
authors.initials: LT
authors.lastname: Lung
authors.forename: Zamberi
authors.initials: Z
authors.lastname: Sekawi
authors.forename: Jayakayatri Jeevajothi
authors.initials: JJ
authors.lastname: Nathan
authors.forename: King Hwa
authors.initials: KH
authors.lastname: Ling
authors.forename: Johnson Shueh Chong
authors.initials: JS
authors.lastname: Seng
authors.forename: Alreshidi Mateg
authors.initials: AM
authors.lastname: Ali
authors.forename: Salwa A
authors.initials: SA
authors.lastname: Abduljaleel
authors.forename: Rukman Awang
authors.initials: RA
authors.lastname: Hamat
mesh_terms: D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013203:Staphylococcal Infections / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger
keywords: Adhesion; Biofilm; Biofilm related gene expression; SEM; Staphylococcusaureus; qPCR