In order to more precisely test the sensitivity of the lux transformants to lidocaine/prilocaine, cultures of the ATCC 25175 transformant were treated with varied doses of lidocaine/prilocaine (0 – 100 g/ml), combined with either near-optimal or suboptimal doses of minocycline hydrochloride (1 g/ml and 0.1 g/ml, respectively). In this experiment, we examined doses of lidocaine/prilocaine between 0-100 g/ml, in the absence or presence of 1 g/ml minocycline hydrochloride (Figure 4, Panels A and B, respectively). Consistent with the results of the UA159 transformant described above, we observe with the ATCC 25175 transformant, that lidocaine/prilocaine has weak antimicrobial activity when used alone at doses above 100 g/ml (unpublished observations), and that no reductions in absorbance occurred using lidocaine/prilocaine at doses < 100 g/ml (Figure 4, Panel A). As described above in the case of the UA159 transformant, when lidocaine/prilocaine (25-100 g/ml) is applied in conjunction with minocycline hydrochloride (1 g/ml) in the ATCC 25175 transformant, we observe that lidocaine/prilocaine in this intermediate dose range does not have a contraindicative effect on the antimicrobial activity of minocycline hydrochloride, when minocycline hydrochloride is used at the near-optimal dose of 1 g/ml (Figure 4, Panel B). Minocycline hydrochloride at that concentration will result in immediate and sustained reduction in absorbance. When minocycline hydrochloride is lowered to the suboptimal dose of 0.1 g/ml, we observe that the combined effect of minocycline hydrochloride plus lidocaine/prilocaine at 100 g/ml results in reductions in absorbance (Figure 4, Panel C). Other lower concentrations of lidocaine/prilocaine (< 100 g/ml, when used with the suboptimal dose of minocycline hydrochloride (0.1 g/ml) do not result in reductions in absorbance (Figure 4, Panel C). We conclude from these experiments that lidocaine/prilocaine at 100 g/ml, when used in conjunction with suboptimal doses of minocycline hydrochloride, adds measureable bacteriostatic activity (Figure 4, Panel C).
Minocycline Hydrochloride and Antiseptic Chlorhexidine Display Additive
Antimicrobial Activity in the UA159 Transformant
The UA159 transformant, when treated with chlorhexidine (0.01%) in conjunction with either high (1 g/ml) or low (0.1 g/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 (Figure 5, Panels A and B). When examining absorbance values at 5.5 hours, we find that the absorbance values of cultures treated with minocycline hydrochloride (1.0 g/ml) plus chlorhexidine (0.01%) or minocycline hydrochloride (1.0 g/ml) alone were statistically different from the absorbance values of untreated cultures (p<10-6 in both cases with Bonferroni correction; Figure 5, Panel A). When
comparing absorbance values at 5.5 hours for cultures treated with minocycline hydrochloride (1.0 g/ml) with or without chlorhexidine (0.01%), we find that these values are nearly statistically distinct (p=0.059; Figure 5, Panel A). Similarly when examining data with minocycline hydrochloride at 0.1 g/ml (Figure 5, Panel B), we observe statistical differences in absorbances between cultures treated with minocycline hydrochloride (0.1 g/ml) plus
chlorhexidine (0.01%) versus untreated cultures (p=0.00017 including Bonferroni correction), and cultures treated with minocycline hydrochloride (0.1 g/ml) with or without chlorhexidine (0.01%) (p=0.0018 including Bonferroni correction).
Figure 4. Panels A and B: Matched experiment examining the absorbance measurements of the lux ATCC 25175 transformant treated with varying doses of lidocaine/prilocaine (0 – 100 g/ml), in the absence (Panel A) or presence (Panel B) of minocycline hydrochloride (1 g/ml). Panel C: Absorbance measurements of the lux ATCC 25175 transformant treated with varying doses of lidocaine/prilocaine (0 – 100 g/ml), in the presence of suboptimal doses of minocycline hydrochloride (0.1 g/ml). Absorbances were measured at 600 nm.
Figure 5. Panels A and C: Matched experiment examining absorbance (Panel A) and bioluminescence (Panel C) of the lux UA159 transformant treated with minocycline hydrochloride (1.0 g/ml) or chlorhexidine alone or in combination. Panels B and D: Matched experiment examining absorbance (Panel A) and bioluminescence (Panel C) of the lux UA159 transformant treated with minocycline hydrochloride (0.1 g/ml) or chlorhexidine alone or in combination. Absorbances were measured at 600 nm. Symbols A and CHX are reflective of Arestin and chlorhexidine.
Similar reductions in bioluminescence activity were also observed, with additive effects when both antimicrobial agents were deployed together (Figure 5, Panels C and D). When using bioluminescence values obtained at 4 hours for cultures treated with minocycline hydrochloride at 1.0 g/ml (Figure 5, Panel C), and conducting multiple pair-wise comparisons, we find that all comparisons with the exception of the comparison between the untreated and chlorhexidine-treated cultures were statistically different with p values approaching zero (p<10-10 including Boneferroni correction). Similar statistically-significant
of minocycline hydrochloride (0.1 g/ml; Figure 5, Panel D). In these comparisons, statistically-significant differences were found between untreated cultures and cultures treated with minocycline hydrochloride alone (0.1 g/ml) or minocycline hydrochloride plus chlorhexidine (0.01%) (p=0.012 and p<10-8, respectively including Bonferroni correction),
between cultures treated with minocycline hydrochloride (0.1 g/ml) alone or with chlorhexidine (0.01%) (p=10-6 including Bonferroni correction) or between cultures treated
with chlorhexidine (0.01%) alone or with minocycline hydrochloride (0.1 g/ml) (p=0.00018 including Bonferroni correction).
D
ISCUSSIONThe treatment of periodontal disease commonly involves mechanical and surgical interventions, such as scaling and root planing (SRP). SRP is now often augmented with adjunct antimicrobial therapies to treat supragingival and subgingival plaque, including the use of chlorhexidine [18,21,22]. More targeted antimicrobial adjunct therapies have recently emerged as additional tools for the treatment of subgingival plaque and periodontitis. These include the slow-release minocycline hydrochloride, which has shown some success when used in conjunction with standard SRP procedures [18,22].
Adverse drug interactions are a growing concern for all aspects of patient care, including periodontology. Pharmacodynamic drug interactions, including competition by enzyme inhibition and substrate bioavailability, may potentially antagonize the effectiveness of specific drugs [64,65]. We have sought to identify potential antagonistic or complementary effects of two commonly used products, one the anesthetic lidocaine/prilocaine, the other the antimicrobial agent chlorhexidine, on the bacteriostatic activity of the antibiotic minocycline hydrochloride, used as an adjunct therapy after scaling and root planing. Even though the application of minocycline hydrochloride and lidocaine/prilocaine are temporally distinct, these two agents are applied subgingivally in the same periodontal sulcus within a short time of each other, and interaction is plausible. The two antimicrobial agents chlorhexidine and minocycline hydrochloride are designed for use during the extended period of time following SRP.
There are known antagonistic interactions between bactericidal and bacteriostatic compounds [64,65]. Bactericidal antibiotics are dependent on active cell replication, and bacteriostatic antibiotics, when used in conjunction with bactericidal agents, will inhibit replication and diminish the effectiveness of the bactericidal agent. For example, there are well-documented instances where simultaneous administration of penicillin with either tetracycline or erythromycin is less effective than using penicillin alone [64,65].
The potential interactions between minocycline hydrochloride and lidocaine/prilocaine, and between minocycline hydrochloride and chlorhexidine, were determined using the lux biosensor system, where lux gene recombinants from Photorhabdus luminescens were transformed into the oral bacteria Streptococcus mutans. This system can serve as sensitive real-time biosensors in the determination of antimicrobial activity and can rapidly monitor inhibition of bacterial metabolism. The application of the lux biosensor system in this study represents its first use in examining drug interactions in dentistry.
Streptococci were selected as host strains for transformation using lux gene recombinants, because they are among the most common colonizers in plaque biofilm, which would be reforming in the period following SRP when minocycline hydrochloride is designed to be effective, and in fact may be beneficial in shifting the reforming biofilm away from periodontal pathogens [56]. Some streptococci species are initiators of biofilms, and are required for the colonization and expansion of subsequent periodontal biofilm [66-69]. Mutans streptococci, including S. mutans, have also recently been identified in the subgingival plaque of patients with periodontitis [56]. Streptococci were also selected because of the availability of known transformation protocols for these strains and ease of growth in culture, and because minocycline hydrochloride is a broad-spectrum antibiotic that inhibits common protein synthesis components found in both gram-positive streptococci and gram- negative periodontal bacteria [18-21].
Transformants that carry significant plasmid loads might be expected to have slowed growth kinetics because of the increased metabolic burden to replicate additional nucleic acids. Interestingly, this does not appear to be the case with the lux mutans transformants, where the saturating cell mass accumulation based on absorbance, and the duration of time necessary to obtain saturation, does not appear to be different from what occurs in nontransformed S. mutans (compare Figure 1, Panel C with Figure 2, Panel A).
The lux A-E operon reconstitutes an aldehyde-recycling pathway [60] and using bioluminescence can detect changes in metabolic activity before changes in cell mass occur. In this regard, we have determined that bioluminescence activity decreases as the culture enters stationary phase, reflective of the diminished metabolic activity of the culture at that stage, and occurs well in advance of the observed plateau in cell mass accumulation, which is an indirect measure of cell number (Figure 2, Panels C and D). Bioluminescence and the assessed metabolic activity of the culture approaches zero during prolonged periods of stationary phase (Figure 2, Panels C and D). Other similar systems using ATP-driven bioluminescence, also conducted in our laboratory [70], measures ATP content in bacterial cells using a luciferase-luciferin substrate, and like the lux biosensor system also demonstrates a dramatic decrease in metabolic activity during stationary phase of growth.
We have verified the two transformants used in this study as S. mutans derivatives, using the criteria of growth on MSA with bacitracin inhibitors, PCR using specific S. mutans primers and immunoreactivity using antibodies directed at unique S. mutans cell wall components. In spite of differences in utilization of metabolic substrates and acidification profiles (Table 1), both S. mutans transformants used in this study, lux UA159 and lux ATCC 25175, have near equivalent sensitivities to minocycline hydrochloride, where 1 g/ml minocycline hydrochloride was noted to be the minimum effective dose for immediate and sustained reduction of accumulated cell mass as measured by absorbance (Figure 2). In addition, the influence of the lux recombinant plasmid in transformed cells on minocycline sensitivity is negligible, since both nontransformed and transformed mutans cells exhibit the same minimum effective dose of 1 g/ml minocycline hydrochloride for sustained reduction of accumulated cell mass (compare Panel A versus Panel B of Figure 2). This reduction of accumulated cell mass is reproducible in an independently conducted experiment (Figure 2, Panel C) using an intermediate range of minocycline hydrochloride (0-10 g/ml), where reduction in bioluminescence is also observed at the same dose of minocycline hydrochloride
(Figure 2, Panel D; 1 g/ml and 10 g/ml minocycline hydrochloride curves are merged as overlapping line graphs).
In additional experiments, we demonstrate that lidocaine/prilocaine has weak antimicrobial activity at the higher doses examined (200 g/ml) in mutans transformants (Figure 3, Panel A). Minocycline hydrochloride at the optimal concentration of 1.0 g/ml added to all doses of lidocaine/prilocaine resulted in the sustained reduction of the mutans transformant both in cell mass accumulation and bioluminescence (Figure 3, Panels B and C, respectively). Thus, in these instances, the addition of lidocaine/prilocaine does not have a contraindicative effect on the antimicrobial activity of minocycline hydrochloride. In some experiments, we used reduced amounts of minocycline hydrochloride (0.1 g/ml), where the bacteriostatic activity of minocycline hydrochloride was purposefully weak, in order to confirm that the addition of lidocaine/prilocaine (at 200 g/ml) had augmented antimicrobial activity. Interestingly, the dose of lidocaine/prilocaine used in the lux biosensor system (200 g/ml) is much lower than the dose applied in the periodontal pocket (each cartridge contains 1.7 g of gel which contains 42.5 mg of lidocaine and 42.5 mg of prilocaine), where lidocaine/prilocaine is mixed with crevicular fluid and saliva, and one may presume that the effective dose of lidocaine/prilocaine in the periodontal pocket would be less than the applied dose.
The limited antimicrobial activity of lidocaine/prilocaine at the 100 g/ml dose in the ATCC 25175 transformant (Figure 4, Panel C) has been found to be reproducible in additional experiments (unpublished observations). The ATCC 25175 transformant appears to retain similar sensitivity profiles for lidocaine/prilocaine when administered with the suboptimal dose of minocycline hydrochloride (0.1 g/ml), but may be less sensitive than the UA159 transformant at the 100 g/ml dose (unpublished observations). Thus again, lidocaine/prilocaine does not appear to be contraindicative to the bacteriostatic activity of minocycline hydrochloride, and may in fact augment the antimicrobial effect of suboptimal doses of minocycline hydrochloride in the lux biosensor system (Figure 4, Panel C).
The mechanism of the antimicrobial activity of lidocaine/prilocaine is unknown. The anesthetic composite of lidocaine and prilocaine has been found to retain antimicrobial properties in skin creams used to treat human skin flora [71,72]. Both lidocaine and prilocaine are sodium channel blockers [73], and voltage-gated sodium ion channels have been found in bacteria [74]. One potential mechanism of action may constitute blockade of ion flux and resultant dysregulation of osmolarity, ultimately resulting in slow disruption of the cell. Interestingly, chlorhexidine, another antimicrobial agent used in the treatment of supragingival and subgingival plaque, is a chemical antiseptic with both bactericidal and bacteriostatic properties that is believed to act through membrane disruption [75]. Like the combination of minocycline hydrochloride and lidocaine/prilocaine, the combination of the bacteriostatic antibiotic minocycline hydrochloride and the antiseptic chlorhexidine display additive antimicrobial effects in the lux biosensor system (Figure 5), and that the majority of pair-wise comparisons are statistically distinct. Statistically-significant differences were found 1) between untreated cultures and cultures treated with minocycline hydrochloride alone (0.1 g/ml) or with minocycline hydrochloride plus chlorhexidine (0.01%) (p=0.012 and p<10-8, respectively including Bonferroni correction), 2) between cultures treated with minocycline hydrochloride (0.1 g/ml) alone or with chlorhexidine (0.01%) (p=10-6 including Bonferroni correction) or 3) between cultures treated with chlorhexidine (0.01%) alone or
with minocycline hydrochloride (0.1 g/ml) (p=0.00018 including Bonferroni correction). Our results are consistent with other studies that indicate chlorhexidine may enhance the penetration and intracellular activity of antibiotics in bacterial cells [76,77]. This may have important implications for the periodontist when considering use of minocycline hydrochloride antibiotic as an adjunct therapy for SRP, followed by use of chlorhexidine for generalized control of supragingival plaque. Using the lux biosensor system, there appears to be no contraindicative effect when using minocycline hydrochloride and chlorhexidine in combination.
C
ONCLUSIONWe have found that the lux biosensor system is an excellent monitor of the metabolic activity of the culture, and is especially useful when assessing single antimicrobial agents, or with combinations of multiple agents. We conclude that the anesthetic lidocaine/prilocaine, or the antiseptic chlorhexidine, does not interfere with the potent bacteriostatic activity of minocycline hydrochloride, and in fact has an additive antibacterial effect.
A
CKNOWLEDGMENTSThe lux recombinant was a kind gift of Dr. Vyv Salisbury (Faculty of Applied Sciences, University of the West of England, Bristol, United Kingdom). We also thank Anna Nguyen (Integrative Biosciences, OHSU) for assistance in final development of the figures, and Dr. Michael Leo (Affiliate Assistant Professor, Integrative Biosciences, OHSU) for advice on statistical determinations. We also thank our OHSU colleagues, Drs. Don Adams and Marvin Levin, for their critical review of this work, and for Dr. Don Adams as the reviewer of this manuscript. Portions of this work were conducted by VTT and SN in fulfillment of the research component of the OHSU Periodontology graduate certification program. AR was supported by a special research fellowship award from Dean Clinton and the OHSU School of Dentistry. WSM was supported as a 2008 OCTRI Student Research Fellow from the Oregon Clinical and Translational Research Institute, OHSU. RS was the recipient of the 2008 OCMID Student Fellowship for Caries Microbiology Research. This research was supported by Dentsply Pharmaceutical, with funds for supplies provided to TM, and use of the luminometer was kindly provided by Oral Biotech. WC, TM and CM are supported by the OHSU School of Dentistry.
R
EVIEWEDB
YDr. Don Adams, Professor, Department of Periodontology, Oregon Health & Science University School of Dentistry, 611 SW Campus Drive, Portland, OR 97239.
R
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