Wang and Ren AMB Expr (2017) 7:204 DOI 10.1186/s13568-017-0505-z ORIGINAL ARTICLE Open Access Controlling Streptococcus mutans and Staphylococcus aureus bioflms with direct current and chlorhexidine Hao Wang1,2 and Dacheng Ren1,2,3,4* Abstract Microbial bioflms formed on biomaterials are major causes of chronic infections. Among them, Gram-positive bac- teria Streptococcus mutans and Staphylococcus aureus are important pathogens causing infections associated with dental caries (tooth-decay) and other medical implants. Unfortunately, current antimicrobial approaches are inefec- tive in disrupting established bioflms and new methods are needed to improve the efcacy. In this study, we report that the bioflm cells of S. mutans and S. aureus can be efectively killed by low-level direct current (DC) and through synergy in concurrent treatment with DC and chlorhexidine (CHX) at low concentrations. For example, after treatment with 28 µA/cm2 DC and 50 µg/mL CHX for 1 h, the viability of bioflm cells was reduced by approximately 4 and 5 logs for S. mutans and S. aureus, respectively. These results are useful for developing more efective approaches to control pathogenic bioflms. Keywords: Bioflm, Electrochemical control, Chlorhexidine, Synergistic efects Introduction mitis, and S. salivarius, due to its acid tolerance and thus Bioflms are formed by microbial cells embedded in a the capability to live in low pH environment of oral cavi- matrix comprised of extracellular polymeric substance ties (Bender et al. 1986; Harper and Loesche 1984; Kreth (EPS) containing polysaccharide, proteins, and DNA. et al. 2005). S. mutans expresses multiple exoenzymes Te presence of this extracellular matrix provides pro- (glucosyltransferases) that make it the primary EPS pro- tection to microbial pathogens from antimicrobials to ducer in oral cavity (Falsetta et al. 2014), while it is also host immune cells/factors (Liu et al. 2016; Hall and Mah highly acidogenic and aciduric. S. mutans can rapidly 2017). Bioflms can form on both biotic and abiotic sur- colonize tooth surface and establish cariogenic bioflms faces and are common causes of chronic infections with extracellular polysaccharides (EPS). Tis acidifes including dental plaques (Smith et al. 2011; Song et al. the local microenvironment and promotes the growth of 2015). Te protection of EPS plus the dormancy of bio- an acidogenic microbiota, facilitating the development of flm cells render these multicellular structures extremely dental caries (Falsetta et al. 2012, 2014). difcult to eradicate (Kouidhi et al. 2015; Smith et al. Staphylococcus aureus is also an abundant Gram- 2011; Song et al. 2015). positive bacterium, which usually harbors in the nasal Streptococcus mutans is a Gram-positive bacterium passages and ears of patients (Smith et al. 2011). Previ- commonly found in human dental bioflms. It is a domi- ous studies have shown that S. aureus is not only a sig- nant species with higher biomass in dental bioflms than nifcant cause of many localized and systemic infections other Streptococcus species, including S. sanguinis, S. such as osteomyelitis (Lew and Waldvogel 2004), chronic wound infection (Hansson et al. 1995), and chronic rhi- nosinusitis (Stephenson et al. 2010), but also has a strong *Correspondence: [email protected] 1 Department of Biomedical and Chemical Engineering, Syracuse connection to dental implant infections (Salvi et al. 2008; University, Syracuse, NY 13244, USA Harris et al. 2004). Te established bioflms of S. aureus, Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wang and Ren AMB Expr (2017) 7:204 Page 2 of 9 especially the methicillin-resistant S. aureus (MRSA), was used to inoculate a petri dish containing 25 mL of are highly tolerant to common antimicrobial treatments BHI medium and acrylic coupons. Te culture was incu- (Jones et al. 2001; O’Donnell et al. 2015; Lewis et al. bated at 37 °C for 48 h without shaking. Ten the cou- 2015). pons with bioflms were removed from petri dish and Few approaches are currently available for control- washed three times with 0.85% NaCl solution to remove ling cariogenic bioflms (Liu et al. 2016). Chlorhexidine all planktonic cells and only retained the frmly attached (CHX) is considered the “gold standard” for oral antimi- cells for DC and CHX treatments. Te S. aureus bioflm crobial therapy (Jones 1997). However, use of high dose samples were prepared in the same way except that the CHX has adverse side efects such as tooth staining and medium was LB plus 10 µg/mL chloramphenicol and calculus formation. Also, CHX is not recommended for the incubation time was reduced to 24 h due to a higher long term daily therapeutic use (Flotra et al. 1971). In growth rate of S. aureus. 1994, Costerton et al. (1994) reported bacterial killing by synergistic efects between low-level electric currents and Electrochemical treatment antibiotics, a phenomenon named “bioelectric efects”. Te experimental system for DC treatment is the same Since 1990s, direct currents (DCs) ranging from μA to as we described previously (Niepa et al. 2012, 2016b). mA have been reported for their bactericidal efects Briefy, an electrochemical cell was constructed with after a relatively long period (from several hours to days) two electrodes on the opposite sides of a plastic cuvette of treatment time (Costerton et al. 1994; del Pozo et al. (Termo Fisher Scientifc, Pittsburg, PA, U.S.). DC was 2009; Schmidt-Malan et al. 2015; Spadaro et al. 1974) generated using a potentiostat (Potentiostat WaveNow, either by DC alone or with antibiotics together (Wattana- Pine Research Instrumentation, Raleigh, NC, U.S.) in the karoon et al. 2000; Niepa et al. 2012, 2016a). Recent stud- three electrode system with a silver wire (0.015” diame- ies reported that mA level DC could enhance the killing ter, A-M Systems, Sequim, WA, U.S.) placed in bleach for efect of 0.2% (200 µg/mL) chlorhexidine on bioflms of 30 min to create an Ag/AgCl reference electrode. Te DC Gram-negative Porphyromonas gingivalis, although there level and voltage across the electric feld were monitored was no bactericidal efect by DC alone (Lasserre et al. and recorded using the AfterMath software (Potentiostat 2015). To explore the potential of lower levels of DC and WaveNow, Pine Research Instrumentation, Raleigh, NC, CHX in killing dental bioflms of Gram-positive bacteria, U.S.) in the galvanostatic mode during the treatment. we conducted this study using S. mutans and S. aureus as model species. We demonstrate that stainless steel DC treatment of bioflms electrode derived DC and CHX have strong synergy in Each DC treatment was carried out in 3 mL 0.85% killing S. mutans and S. aureus bioflms; and the levels NaCl solution. First, a sterile SS304 electrode of DC and CHX appear to be lower than other reported (3.5 cm × 0.95 cm × 0.05 cm) was inserted into a cuvette, systems. followed by an acrylic coupon with S. mutans or S. aureus bioflm attached. Another sterile SS304 electrode Materials and methods was then inserted on the opposite side. Te bioflm was Bacteria strains and growth media treated galvanostatically with direct current (DC) for 1 h Staphylococcus mutans Clarke strain (ATCC 25175) was in the absence or presence of CHX (MP Biomedicals, cultured in brain heart infusion (BHI) broth (BD Bio- Solon, OH, U.S.). Samples treated with DC or CHX alone sciences, San Jose, CA, U.S.) (Murchison et al. 1982). and untreated samples were used as controls. After treat- Te S. aureus ALC2085 (strain RN6390 containing ment, each acrylic coupon was transferred to a 10 mL pALC2084) was obtained from Dr. Karin Sauer at Bing- tube containing 5 mL 0.85% NaCl solution. Te bioflm hamton University (Sauer et al. 2009) and cultured in cells were removed from the surface by gentle sonica- Lysogeny broth (LB) (Sambrook and Russell 2001) con- tion for 1 min. Te number of viable cells detached from taining 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L acrylic coupons was quantifed by counting colony form- NaCl, supplemented with 10 µg/mL chloramphenicol ing units (CFUs) in the solution. (Sigma-Aldrich, St. Louis, MO, U.S.). Both strains were To further evaluate the efects in an environment simi- routinely cultured overnight at 37 °C with shaking at lar to that of oral cavity, the test medium was replaced 200 rpm. with artifcial saliva medium or a mixture of 0.85% NaCl and artifcial saliva medium (2:1). Te recipe of artifcial Bioflm formation saliva from Pratten et al. (1998) was followed. It contains Bioflms were formed on acrylic coupons 2 g/L yeast extract, 5 g/L peptone, 2.5 g/L type III hog (3.5 cm × 0.5 cm × 0.1 cm; McMaster-Carr, Aurora, OH, gastric mucin, 0.2 g/L NaCl, 0.2 g/L KCl, and 0.3 g/L U.S.). Briefy, 25 µL of an overnight culture of S. mutans CaCl2, supplemental with 1.25 mL of sterile 40% urea. Wang and Ren AMB Expr (2017) 7:204 Page 3 of 9 Te CHX was tested at 50 µg/mL to 500 µg/mL. Te followed by Tukey test. Statistical signifcance was set as treatment process was the same as described above for p < 0.05. All analyses were performed using SAS 9.4 soft- 0.85% NaCl solution.