REAL‐TIME POLYMERASE CHAIN REACTION QUANTIFICATION OF SALMONELLA TYPHIMURIUM HILA EXPRESSION DURING AGITATION AND STATIC INCUBATION

Abstract

ABSTRACTSalmonella virulence is influenced by a combination of environmental stimuli including different levels of pH, oxygen availability and osmolarity and involves a coordinated genetic response. The expression of hilA in Salmonella Typhimurium, considered a key regulator of the bacterial virulence, was determined as relative to the expression of the endogenous control gene rsmC using the comparative Ct (ΔΔCt) real‐time polymerase chain reaction (RT‐PCR) method. Enhanced osmolarity (total of 2% NaCl) significantly increased hilA expression in cells grown at pH 8 but not at pH 6. The determination of hilA expression in bacterial cells under oxygen limitation, for all experiments, resulted in approximately twofold higher levels compared to agitated cells. The effect was more pronounced when static incubation was combined with complete removal of NaCl. Overall results support that while oxygen limitation can trigger S. Typhimurium virulence, the effect may be enhanced by other extremes for S. Typhimurium growth. By using RT‐PCR, S. Typhimurium virulence gene expression can be quantified directly and more rapidly under a wide variety of environmental and laboratory conditions.PRACTICAL APPLICATIONS Salmonella Typhimurium pathogenicity is influenced by various factors. Historically, hilA expression which is a key regulator of bacterial virulence has been determined by colorimetric β‐galactosidase assay. The method, however, requires a preconstruction of an appropriate gene fusion and is limited by its sensitivity. This study demonstrated the utility of quantitative RT‐PCR method in determining virulence gene expression under a variety of laboratory conditions. The results confirmed oxygen limitation as a potential stimulant of S. Typhimurium hilA expression, which can also be triggered by other extremes for the bacteria growth.