Lactobacillus acidophilus-derived biosurfactant effect on gtfB and gtfC expression level in Streptococcus mutans biofilm cells

Brazilian Journal of Microbiology (2011) 42: 330-339 ISSN 1517-8382 LACTOBACILLUS ACIDOPHILUS-DERIVED BIOSURFACTANT EFFECT ON GTFB AND GTFC EXPRESSION LEVEL IN STREPTOCOCCUS MUTANS BIOFILM CELLS *Arezoo Tahmourespour¹, Rasoul Salehi², Rooha Kasra Kermanshahi³ ¹Assistant Professor of Microbiology, Islamic Azad University-Khorasgan branch, Isfahan, Iran; ²Associated Professor of Genetics, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran; ³Professor of Microbiology, Alzahra University, Tehran, Iran. Submitted: January 28, 2010; Returned to authors for corrections: April 23, 2010; Approved: June 21, 2010. ABSTRACT Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus–derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque). The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus–derived biosurfactant fraction. Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus- derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings, we can suggest that the prepared biosurfactant may interfere with adhesion processes of S. mutans to teeth surfaces, provided additional evaluation produce satisfactory results. Key words: Biofilm formation, biosurfactant, gtfB/C, Lactobacillus acidophilus, real-time RT PCR, Streptococcus mutans INTRODUCTION and progression of dental caries (3, 6, 31). The S. mutans adhesion mechanism is related to synthesis of both extracellular Streptococcus mutans (S. mutans), harbouring the dental enzymes, glucosyl transferase (GTF) and fructosyl transferase biofilm, is one of the etiological factors of dental caries (16). Its (FTF) (7, 17, 26). These enzymes are responsible for the ability to adhere to the teeth surface is vital for the initiation synthesis of extra cellular polysaccharides such as glucans and *Corresponding Author. Mailing address: Assistant Professor of Microbiology, Khorasgan Branch, Islamic Azad University, Isfahan, Iran.; Add: no. 21, Sh. Salimian st., Daneshgah Bv., Azadi sq., Isfahan, Iran.; Fax: +98 311 5354061.; E.mail: / 330 Tahmourespour, A. et al. L. acidophilus-derived biosurfactant effect in S. mutans fructans. These polymers mediate adherence of S. mutans to the biochemical tests and rapid identification kit of Streptococci tooth surfaces and surfaces of bacteria. They also play an (Rap ID STR kit) and by PCR. L. acidophilus DSM 20079 as a important role in the formation of plaque, the precursor of probiotic source was cultured in MRS broth or Agar. dental caries (13, 27). -(1-3) and -(1-6)-linked glucan polymers are encoded by Biosurfactant production the genes gtfB, gtfC and gtfD. Recently, in vitro studies 15 mL of L. acidophilus cultured overnight was inoculated indicated that gtfB and gtfC are essential for the sucrose- to 600-ml of MRS broth and incubated for 24 hours. The cells dependent attachment of S. mutans cells to hard surfaces (2) but were harvested by centrifugation at 10,000 gtfD is dispensable (8, 43). Therefore, these genes have become °C, a potential target for protection against dental caries (7). resuspended in 100 ml of PBS. The lactobacilli were incubated Lactobacilli, as probiotic agents, are believed to interfere with pathogens by different mechanisms (22, 30). One of its washed twice in demineralized g for 5 min at 10 water, and were at room temperature for 2 hours with gentle stirring for biosurfactant production. Subsequently, the bacteria were removed by centrifugation, mechanisms is biosurfactant production. Biosurfactants, a structurally diverse group of surface and the remaining supernatant liquid was filtered through a active molecules synthesized by microorganisms, have attracted 0.22 mm-pore-size filter (Millipore). A 10-ml portion of the attentions in recent years. Because the reason, they had several supernatant was used immediately in the adhesion assay, and advantages on synthetic surfactants, such as low toxicity, the remainder was dialyzed against demineralized water at 4 °C inherent good biodegradability and ecological acceptability. in a Spectrapor membrane tube (molecular weight cutoff, 6'000 Biosurfactants include unique amphipathic properties derived to 8'000; Spectrum Medical Industries, Inc.), and was freeze- from their complex structures, which include a hydrophilic dried as in method of Velraed et al (38). moiety and a hydrophobic portion (37). The use of biosurfactants from probiotic bacteria as antimicrobial and/or Drop-collapse method anti-adhesive agents has been studied before and their ability to In order to test whether produced biosurfactant was able to inhibit adhesion of various microorganisms isolated from decrease the surface tension between water and hydrophobic explanted voice prostheses has been demonstrated (20). surfaces, the ability to collapse a droplet of water was tested as The present research focused on the influence of follows: 25 L of extracted biosurfactant was pipetted as a acidophilus)–derived droplet onto parafilm; the flattening of the droplet and the biosurfactant on the gtfB and gtfC genes expression level in S. spreading of the droplet on the parafilm surface was followed mutans biofilm cells by real time RT PCR for the first time. over seconds or minutes. Lactobacillus acidophilus (L. Subsequently, methylene blue (which had no influence on MATERIALS AND METHODS the shape of the droplets) was added to the water stain and supernatants for photographic purposes. The droplet was Bacterial strains and culture conditions The S. mutans strains used in this study were S. mutans allowed to dry and the diameter of the dried droplet was recorded (14, 34). ATCC35668 and dental plaque isolated S. mutans 22, with high ability of biofilm forming. Fourier transform infrared spectroscopy S. mutans strains were cultured on blood agar and mitis Freeze-dried biosurfactants (2 mg) were ground with 100 mg salivarius agar media and were incubated (...truncated)