Optimising biofilm growth of S. pyogenes fro RNA extraction to study the expression of the Bfr-like regulator VicR
Al Rujaibi, Sharifa
Cardiff Metropolitan University
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Streptococcus. pyogenes (group A streptococcus) is the most common aetiological agent of a variety of diseases in humans ranging from mild infection like a sore throat to life threatening diseases such as necrotizing fasciitis , scarlet fever, and glomerulonephritis (1). Like many other bacteria S. pyogenes relies on Two-Component Signal transduction Systems (TCSTS) to regulate the expression of virulence factors in response to environmental stimuli (1). TCSTS are essential for bacteria for survival and forming biofilms (2). In recent years the increasing development of bacterial antibiotic resistance and treatment failure has been become the major health problem (2). This is most likely to be due to the ability of many bacteria to acquire new resistance and to grow as a biofilm, which are inherently resistant to antibiotics (3). The main aim of this study was to optimise conditions to extract RNA from biofilms of S. pyogenes to study the expression of Bfr-like regulator vicR, which is thought to be the master regulator of biofilm associated genes in S. pyogenes. Different inoculum sizes of S.pyogenes isolate (MGAS 6180) was used to optimize biofilm growth. Biofilm biomass was measured and the actual cfu/ml calculated using the method of Miles and Misra. The optimum inoculum size was determined and used to grow large scale biofilms. The cfu/ml was calculated for the large scale biofilms and found to be equal to 5 x 108 cfu/ml; this is sufficient for RNA extraction (1x106 cfu/ml required). In this study the best conditions to grow cells planktonically and as a biofilm to extract RNA were successfully optimized and the vicR gene was amplified by PCR. These optimization experiments can be used in the future to study the expression of vicR in the biofilm state compared to planktonic cells.
BSc (Hons) Biomedical Science
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