Abdurrahman Aygül1, İsmail Öztürk2, Fatma Feriha Çilli3, Şafak Ermertcan4. 1. Çukurova University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Adana 01380, Turkey. Electronic address: aaygul@cu.edu.tr. 2. İzmir Katip Çelebi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, İzmir 35040, Turkey. 3. Ege University, Faculty of Medicine, Department of Medical Microbiology, İzmir 35040, Turkey. 4. Ege University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, İzmir 35040, Turkey.
Abstract
BACKGROUND: Via its virulence factors such as swarm differentiation, biofilm and hemolysin production, urease enzyme, Proteus mirabilis causes urinary tract infections (UTIs), especially in complicated cases. Anti-pathogenic compounds attenuate the virulence of bacteria without showing 'cidal' activity and carry the potential to be used in the prevention and treatment of infectious diseases. PURPOSE: Search for anti-pathogenic effects of quercetin, which is a widely known and biologically active phytochemical, on Proteus mirabilis was the purpose of this study. In this context, the potential inhibitory activity of quercetin on swarming motility and biofilm production of a wild-type strain, P. mirabilis HI4320, was investigated in both phenotypically and genotypically. METHODS: Quercetin's effect on swarming motility was examined on LB agar plates, containing quercetin at various concentrations, by measuring the swarming diameter. The effect on biofilm formation, on the other hand, was analyzed by staining the formed biofilm of the bacterium, exposed to quercetin at various concentrations, with crystal violet and reading spectrophotometrically. Differences in expression levels of selected genes involved in swarming regulation were determined by real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mechanism of inhibitory action on swarming. Further investigations were carried out repeating swarming assays with the clones that derived from the wild-type strain by a TA system kit for direct one-step cloning and overexpressing the relevant genes. RESULTS: Our study revealed that quercetin inhibited swarming motility while activating biofilm production of P. mirabilis in direct proportion to the dose. Although all selected genes are inhibited in the same manner in liquid medium, and no significant differences could be detected in solid medium as demonstrated by RT-qPCR, experiments repeated with the clones overexpressing flhC (a component of flagellar transcriptional activator), speB (an agmatinase enzyme) and ompF (an outer membrane porin) genes showed that the respective clones could restore swarming, compensating for the inhibitory effect of quercetin. CONCLUSION: Quercetin's inhibitory effect on P. mirabilis swarming was possibly due to interactions with components of swarming regulators, the genes expressing polyamine coding enzymes that trigger swarm differentiation, or active pump proteins.
BACKGROUND: Via its virulence factors such as swarm differentiation, biofilm and hemolysin production, urease enzyme, Proteus mirabilis causes urinary tract infections (UTIs), especially in complicated cases. Anti-pathogenic compounds attenuate the virulence of bacteria without showing 'cidal' activity and carry the potential to be used in the prevention and treatment of infectious diseases. PURPOSE: Search for anti-pathogenic effects of quercetin, which is a widely known and biologically active phytochemical, on Proteus mirabilis was the purpose of this study. In this context, the potential inhibitory activity of quercetin on swarming motility and biofilm production of a wild-type strain, P. mirabilis HI4320, was investigated in both phenotypically and genotypically. METHODS:Quercetin's effect on swarming motility was examined on LB agar plates, containing quercetin at various concentrations, by measuring the swarming diameter. The effect on biofilm formation, on the other hand, was analyzed by staining the formed biofilm of the bacterium, exposed to quercetin at various concentrations, with crystal violet and reading spectrophotometrically. Differences in expression levels of selected genes involved in swarming regulation were determined by real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mechanism of inhibitory action on swarming. Further investigations were carried out repeating swarming assays with the clones that derived from the wild-type strain by a TA system kit for direct one-step cloning and overexpressing the relevant genes. RESULTS: Our study revealed that quercetin inhibited swarming motility while activating biofilm production of P. mirabilis in direct proportion to the dose. Although all selected genes are inhibited in the same manner in liquid medium, and no significant differences could be detected in solid medium as demonstrated by RT-qPCR, experiments repeated with the clones overexpressing flhC (a component of flagellar transcriptional activator), speB (an agmatinase enzyme) and ompF (an outer membrane porin) genes showed that the respective clones could restore swarming, compensating for the inhibitory effect of quercetin. CONCLUSION:Quercetin's inhibitory effect on P. mirabilis swarming was possibly due to interactions with components of swarming regulators, the genes expressing polyamine coding enzymes that trigger swarm differentiation, or active pump proteins.