Effect of nisin and p-coumaric acid on autoinducer-2 activity, biofilm formation, and sprE expression of Enterococcus faecalis.

Quorum sensing (QS) is an inter- and intracellular communication mechanism that regulates gene expression in response to population size. Autoinducer-2 (AI-2) signaling is a QS signaling molecule common to both Gram-negative and Gram-positive bacteria. Enterococcus faecalis is one of the leading causes of nosocomial infections worldwide. There has been an increasing interest in controlling infectious diseases through targeting the QS mechanism using natural compounds. This study aimed to investigate the effect of nisin and p-coumaric acid (pCA), on biofilm formation and AI-2 signaling in E. faecalis. Their effect on the expression of the QS-regulated virulence encoding gene sprE was also investigated. Nisin exhibited a MIC ranging from 0.25 to 0.5 mg/mL, while the MIC of pCA was 1 mg/mL. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used to determine AI-2 activity in E. faecalis strains. Nisin was not effective in inhibiting AI-2 activity, while pCA reduced AI-2 activity by ≥ 60%. Moreover, pCA and nisin combination showed higher inhibitory effect on biofilm formation of E. faecalis, compared to the treatment of pCA or nisin alone. qRT-PCR analysis showed that nisin alone and the combination of nisin and pCA, at their MIC values, led to a 32.78- and 40.22-fold decrease in sprE gene expression, respectively, while pCA alone did not have a significant effect. Considering the demand to explore new therapeutic avenues for infectious bacteria, this study was the first to report that pCA can act like a quorum sensing inhibitor (QSI) against AI-2 signaling in E. faecalis.

Regulation of biofilm formation by marT in Salmonella Typhimurium.

In this study, we aimed at identifying the regulatory role of marT gene, known as the regulator of misL, on 15 different biofilm-related genes in S. Typhimurium 14028 strain. We also tested the strains for their ability to form biofilm and determined the adherence characteristics of the wild type and the mutant strains of the organism on Caco-2 and HEp-2 cells. For comparative analyses of the candidate genes, individual gene mutations were created via antibiotic gene cassette insertion into each gene of interest. marT gene was cloned behind an arabinose inducible BAD promoter in order to control marT expression. This recombinant plasmid was transfer into each of the 15 mutant strains to investigate the level of expression of each single gene in the presence and absence of marT induction. Besides determination of variations in biofilm formation by each mutant strain, the attachment characteristics of them onto Caco-2 and HEp-2 cell lines were also reported. As a result of attachments experiments on polystyrene surfaces, it was determined that the biofilm production capacity of each mutant strain decreased in a statistically significant manner (p < 0.05). QRT-PCR trials indicated that the marT gene regulates the expression of 14 genes, namely fimA, fimD, fimF, fimH, stjB, stjC, csgA, csgD, ompC, sthB, sthE, rmbA, fliZ and yaiC, in a positive manner. QRT-PCR studies were also revealed that the MarT protein positively regulates its own promoter. When the adherence characteristics of the mutant strains and the wild-type were investigated by using Caco-2 and HEp-2 cells, it was determined that the single gene mutations did have no effect on bacterial adhesion. In view of our mutational analyses and biofilm formation studies, it was concluded that fliZ, ompC, rmbA, stjB and stjC genes are related with biofilm formation in Salmonella, besides other cellular functions of them. Taken together, our data suggested that the regulatory role of MarT protein is not only restricted to the regulation of misL gene expression, but it rather acts as a general regulator on the biofilm-related genes in Salmonella.