Bioluminescent lux gene biosensors in oral streptococci: Determination of complementary antimicrobial activity of minocycline hydrochloride with the anesthetic lidocaine/prilocaine or the antiseptic chlorhexidine

Background: Plaque-induced periodontitis is gingival inflammation at sites undergoing loss of connective tissue, apical migration of junctional epithelium and loss of alveolar bone. Non-surgical treatment of plaque-induced periodontitis typically involves removal of biofilm conducted through mechanical scaling and root planing (SRP) procedures. The antibiotic minocycline hydrochloride, delivered as a sustainedrelease product¹ used for professional subgingival administration into periodontal pockets, has been shown to be beneficial as an adjunct to conventional SRP. Use of chlorhexidine rinse is also a typical adjunct therapy to SRP procedures for chemical control of supragingival plaque. Lidocaine (2.5%) and prilocaine (2.5%)² provides localized anesthesia for SRP. The objective of this study is to develop and use bioluminescent recombinants of oral streptococci in determining the potential antibacterial activity of minocycline hydrochloride used either alone or in combination with the anesthetic lidocaine/prilocaine, or with the antiseptic chlorhexidine. Methods: Recombinant plasmids containing the bioluminescence-generating lux gene from Photorhabdus luminescens were transformed into the oral bacterium Streptococcus mutans, strains UA159 and ATCC 25175. Transformants were verified as S. mutans derivatives by selection and growth on mitis salivarius agar supplemented with bacitracin, in addition to an antibody test directed specifically against S. mutans cell wall proteins and polymerase chain reaction experiments targeting sequences in the S. mutans glucosyltransferase (gtf) gene. S. mutans transformants were then subjected to growth analysis for comparison of absorbance and bioluminescence activity. Minocycline hydrochloride and lidocaine/prilocaine, or minocycline hydrochloride and chlorhexidine were used in combination to determine the potential interactive effects of these agents on the antibacterial activity of minocycline hydrochloride. Results: Using two distinct S. mutans transformants representing both strains UA159 and ATCC 25175, we observed rapid and pronounced bacteriostatic activity when using high doses of minocycline hydrochloride (>1 µg/ml), which were statistically distinct from untreated cultures (p=0.000058) when measured at the peak of metabolic activity. Reduced bacteriostatic activity was seen using lower doses. When lidocaine/prilocaine at doses >100 µg/ml is used in conjunction with minocycline hydrochloride, we observed an additive antibacterial effect. The S. mutans transformant strain UA159, when treated with chlorhexidine (0.01%) in conjunction with either high (1 µg/ml) or low (0.1 µg/ml) doses of minocycline hydrochloride, displayed reduced levels of cell mass accumulation, as measured by absorbance, that were additive when both antimicrobial agents were deployed. Bioluminescence determinations, which are a direct measure of metabolic activity and an indirect measure of cell number when cells are in logarithmic stage of growth, displayed similar reductions when cultures were treated with minocycline hydrochloride and chlorhexidine used singularly or in combination. Conclusions: The S. mutans lux transformants serve as sensitive biosensors in the determination of antimicrobial activity, and can rapidly monitor inhibition of bacterial metabolism. We conclude that the anesthetic lidocaine/prilocaine does not interfere with the potent bacteriostatic activity of minocycline hydrochloride, and actually has an additive antibacterial effect. The potent bacteriostatic activity of minocycline hydrochloride can also be complemented with the addition of chlorhexidine. The application of the lux biosensor system in the assessment of minocycline hydrochloride and lidocaine/prilocaine, or minocycline hydrochloride and chlorhexidine, represents its first use in examining antimicrobial drug interactions in periodontology.