Conlaining Lidocaine Or Benzocaine Mark E

Anesth Prog 35:9-13 1988

Antimicrobial Properties of Topical Anestletc Liquids Conlaining Lidocaine or Benzocaine Mark E. Morrow, DDS, MSD,* and Charles W. Berry, PhDt *Department of Pediatric Dentistry, tDepartment of Microbiology, Baylor College of Dentistry, Dallas, Texas

Six species of microorganisms commonly found ganisms have been isolated from the oral cavity,3 it is within the oral cavity were exposed for either one logical that an antiseptic agent should be wiped on the minute or two hours to 5% lidocaine liquid mucosa before the administration of an injectable local topical anesthetic and benzocaine liquid topical anesthetic in the dental setting. However, few dentists anesthetic. Mixtures of microorganisms and currently follow this regimen.4 Instead, the use of a anesthetics were diluted and plated onto a brain topical anesthetic just before administration of a local heart infusion medium. Reduction in cell viability anesthetic to block the pain of the needle puncture is very was 73-100% after exposure to the anesthetic common.5 agents when compared with the saline/buffer The possible antimicrobial properties of local anesthe- controls. A significant reduction (p < .005) in cell tic agents were originally described by Jonnesco.6 He growth by Streptococcus mutans, S. sanguis, S. indicated that there was no need to sterilize local anesthe- mitis, S. salivarius, Actinomyces viscosus, and tics used for spinal anesthesia because of their antiseptic Candida albicans was associated with a one- characteristics. The earliest work documenting the in vitro minute and two-hour exposure to lidocaine, antimicrobial activity of anesthetic agents was reported by benzocaine, 5% lidocaine, and the benzocaine Schlegal and Swan,7 who described the inhibition of vehicle control. Five percent lidocaine reduced growth of Micrococcus and Pseudomonas with 0.2% be- growth of the test orgainisms more than noxinate. Likewise, the effect of anesthetic agents on benzocaine in one-minute exposures to S. microorganisms found in clinical specimens was recog- mutans, A. viscosus and S. salivarius and with a nized.8-1 Each of these studies concluded that local two-hour exposure to S. salivarius. anesthetics reduced microbial growth. Sculley and Dun- Five percent lidocaine was bacteriocidal or ley12 were the first to report the antibacterial efficacy of fungicidal to all microorganisms for both time several anesthetic preparations used in dentistry. They periods whereas, benzocaine liquid topical examined the antimicrobial activity of two percent lido- anesthetic was predominately bacteriostatic or caine with and without epinephrine against 10 different fungistatic after one-minute exposures and species of nonoral microorganisms. Lidocaine with and bacteriocidal or fungicidal after two hours. without epinephrine demonstrated antimicrobial prop- The results indicated that two dental liquid erfies. topical anesthetics containing lidocaine or Because the application of topical anesthetic agents is benzocaine possessed considerable antimicrobial the only usual pre-injection preparation of the oral mu- activity to selected oral microorganisms. The cosa, the purpose of this study is to report the in vitro exclusive use of a topical liquid anesthetic may be antimicrobial properties of two commercially prepared an adequate means to render the oral mucosa topical anesthetic agents against microorganisms com- aseptic before injection of a local anesthetic. monly found in the oral cavity. The effect of the length of exposure of the test organisms to the topical anesthetic is also described. B efore the needle penetration of a local anesthetic injection, the tissue at the site of injection should METHODS be as aseptic as possible.",2 Because numerous microor- Two of the commercially available liquid topical anesthe- Received July 13, 1987; accepted for publication October 2, 1987. tics that frequently are used in the dental setting were Address correspondence to Dr. Charles W. Berry, 3302 Gaston obtained from the manufacturers. Five percent lidocaine Avenue, Dallas, TX 75246. (Xylocaine liquid, Astra Pharmaceutical Products Inc., C) 1988 by the American Dental Society of Anesthesiology ISSN 0003-3006/88/$3.50 9 10 Antimicrobial Properties of Topical Anesthetics Anesth Prog 35:9-13 1988

Worchester, Massachusetts), an amide-type local anes- the C. albicans plates and tubes were incubated for 48 thetic, contains a mixture of 5% lidocaine, propylene hours aerobically, all at 37°C. Subsequently, the colonies glycol, glycerine, and flavoring agents. Benzocaine (Hur- on each plate were counted using a Quebec Colony ricane liquid, Beutlich Inc., Niles, Illinois), an ester-type Counter (American Optical Co., Buffalo, New York) and local anesthetic, contains 20% benzocaine with flavoring a hand-held counter. The broth tubes were observed for and coloring agents in a polyethylene glycol base. positive or negative growth. Six species of oral microorganisms (Streptococcus Bacteriostatic or bacteriocidal properties were deter- mutans, Streptococcus sanguis, Streptococcus mitis, mined by the ratio of microorganisms killed or altered by Streptococcus salivarius, Actinomyces viscosus, and Can- the test material when compared to a sterile control dida albicans) were chosen for study because of their medium. By definition, if a test material killed 99.9% of presence on the oral mucosal surfaces, or in the saliva or the microorganisms, the substance was described as gingival crevice exudate that coats the mucosal surfaces bacteriocidal; if less than 99.9%, the agent was bacterio- onto which topical anesthetics are applied before injec- static. 13 tion of local anesthetics. Subcultures of these microor- Two negative controls of sterile 0.85% saline and ganisms were acquired from the Baylor College of Den- sterile 0.1 M phosphate buffer was used in place of the tistry, Department of Microbiology, stock culture two anesthetic preparations. Also, Hurricaine vehicle collection and maintained on brain infusion agar (Difco without benzocaine and a 5%, by weight, lidocaine/ Inc., Chicago, Illinois). polyethylene glycol mixture served as vehicle controls. The cell populations for each assay were propagated Triplicate analyses were carried out with each exper- by inoculation of brain heart infusion broth. The strepto- iment. coccal organisms were incubated in an atmosphere en- The Peritz' F test14 was utilized to determine the level riched with carbon dioxide, A. viscosus was grown of statistical significance between the antimicrobial action anaerobically and C. albicans was propagated aerobically of the anesthetic agents and their control solutions. at 37°C for 24 hours. Cell pellets were obtained by refrigerated centrifugation and were rinsed with either sterile 0.85% saline (Streptococcus) or 0.1 M buffered RESULTS phosphate solution (Actinomyces and Candida). A Coul- ter Counter (Coulter Electronics, Hialeah, Florida) was Six species of oral microorganisms were exposed for one used to confirm cell inoculum concentrations, that were minute and two hours to both 5% lidocaine liquid topical found to be between 2.3 and 7.4 x 107 cells/mL. anesthetic or benzocaine liquid topical anesthetic. The A procedure described by Sculley and Dunley was mixture of the microorganisms and anesthetics were modified and used for this experiment.12 Specifically, a diluted and subsequently plated to brain heart infusion 0.01 mL portion of each of the six cultures, harvested in agar and broth medium to observe for organism via- logarithmic growth phase, was placed into 1.99 mL of bility. each undiluted anesthetic (as received from the manufac- The growth of all six species of microorganisms was turer) and was kept at room temperature for either one significantly reduced (p < .005) by exposure to either minute or two hours. After the appropriate reaction time, 5% lidocaine liquid topical anesthetic or benzocaine a 0.01 mL aliquot of the reactants was removed with a liquid topical anesthetic for one minute or two hours fixed-volume pipette and transferred into 9.99 mL of when compared with sterile saline/buffer controls (Table 0.85% sterile saline for streptococcal species and 0.1 M 1). There was no significant difference (p < .05) in the buffered phosphate solution for A. viscosus and C. killing ability of either topical anesthetic on S. sanguis, S. albicans. The tubes containing cells, anesthetic and dilu- mitis, or C. albicans for both periods. There was also little ent were tightly capped and thoroughly agitated on a difference (p < .05) between lidocaine or benzocaine for vortex mixer (Scientific American Co., McGaw Park, two hour exposures to A. viscosus, S. mutans, and S. Illinois) for 15 seconds to promote cell dispersion. To salivarius. However, the antibacterial effect of 5% lido- determine the bacteriostatic properties of each anesthetic, caine liquid topical anesthetic was significantly greater (p brain heart infusion pour plates were made with 0.1 mL < .005) than benzocaine liquid topical anesthetic in one of each diluted reaction solution. Another 0.1 mL aliquot minute exposures to A. viscosus, S. mutans, and S. of reaction mixture was placed into 8.0 mL of brain heart salivarius. The 5% lidocaine/propylene glycol and ben- infusion broth. The plates and broth tubes containing zocaine vehicle also significantly reduced (p < .005) the streptococcal organism were incubated for 24 hours in a viability of all six microorganisms for both exposure carbon dioxide enriched environment. A. viscosus plates periods when compared with their saline/buffer controls. and tubes were incubated for 72 hours anaerobically and The term bacteriocidal or fungicidal was given to those Anesth Prog 35:9-13 1988 Morrow et al. 11

Table 1. Effect of lidocaine and benzocaine topical anesthetic preparations on microbial growth. Number of tubes _ with growth X CFU/plate* % Reductiont

Organism/Test Condition 1 min 2 hr 1 min 2 hr 1 min 2 hr Streptococcus mutans Lidocaine preparation Ot 0 <1 0 100 100 Benzocaine preparation 3% 0 324 0 73 100 5% Lidocaine 0 0 0 0 100 100 Benzocaine vehicle 0 0 0 0 100 100 Saline 3 3 >1200 >1200 0 0 Streptococcus sanguis Lidocaine preparation 0 0 0 0 100 100 Benzocaine preparation 0 0 0 0 100 100 5% Lidocaine 0 0 0 0 100 100 Benzocaine vehicle 0 0 0 0 100 100 Saline 3 3 37 33 0 0 Streptococcus mitis Lidocaine preparation 0 0 0 0 100 100 Benzocaine preparation 1 0 0 <1 100 99.1 5% Lidocaine 0 0 0 0 100 100 Benzocaine vehicle 0 0 0 0 100 100 Saline 3 3 34 38 0 0 Streptococcus salivarius Lidocaine preparation 0 0 <1 0 99.9 100 Benzocaine preparation 3 3 96 4 90.4 99.6 5% Lidocaine 0 0 0 0 100 100 Benzocaine vehicle 3 0 46 0 95.5 100 Saline 3 3 1012 1208 0 0 Actinomyces viscosus Lidocaine preparation 1 0 <1 0 99.9 100 Benzocaine preparation 1 0 271 18 72.9 98.2 5% Lidocaine 0 1 0 0 100 100 Benzocaine vehicle 0 0 47 4 95.3 99.6 Phosphate buffer 3 3 >1000 >1000 0 0 Candida albicans Lidocaine preparation 0 0 0 0 100 100 Benzocaine preparation 1 0 2 <1 99.3 99.9 5% Lidocaine 0 0 0 0 100 100 Benzocaine vehicle 3 0 35 2 88 98.8 Phosphate buffer 3 3 294 177 0 0

* Mean of colony forming units per plate after incubation at 37°C; t Compared to saline/buffer control X CFU/plate; t No visible growth after incubation at 37°C for 24 hours; % Visible growth after incubation at 37°C for 24 hours. anesthetics that inhibited 99.9% or more of the microorganisms for both time periods. Benzocaine liquid topical ganisms compared to the sterile saline or phosphate anesthetic was bacteriocidal or fungicidal in one minute buffer control plates. Anesthetics killing less than 99.9% to S. sanguis and S. mitis in two hours for S. mutans, S. were labelled bacteriostatic or fungistatic. The percent sanguis, and C. albicans. However, benzocaine liquid reduction in viable cells remaining after exposure to the topical anesthetic was only bacteriostatic or fungistatic in anesthetic agents, when compared with the sterile control one minute to S. salivarius, S. mutans, C. albicans, and solutions, was 73-100 percent. Five percent lidocaine A. viscosus and in two hours to S. mitis, S. salivarius, and liquid topical anesthetic demonstrated bacteriocidal or A. viscosus. The 5% lidocaine/polyethylene glycol solu- fungicidal properties against all six strains of microor- tion demonstrated bacteriocidal or fungicidal properties 12 Antimicrobial Properties of Topical Anesthetics Anesth Prog 35:9-13 1988 against all six strains of microorganisms for both time tested in this experiment. A 5%, by weight, solution of periods. The benzocaine vehicle without benzocaine was lidocaine and polyethylene glycol was also bacteriostatic bacteriocidal within one minute to S. mutans, S. sanguis, or bacteriocidal to the six microorganisms tested. Like- and S. mitis, and in two hours for S. mutans, S. sanguis, wise, it has been previously reported that 2% lidocaine in S. mitis, and S. salivarius. However, the benzocaine a sterile water medium possessed antimicrobial proerties vehicle was only bacteriostatic or fungistatic within one against nonoral microorganisms.12 A 1% solution of minute or two hours to A. viscosus and C. albicans. lidocaine was reported to reduce growth one log unit in 6 of 26 strains of bacteria.23 Also, at 0.5 and 1% concentra- tion of lidocaine alone, growth of Neisseria was inhib- DISCUSSION ited.24 This study simulates the conditions in a clinical dental Other investigators have described the antimicrobial practice by testing topical anesthetic preparations against properties of local anesthetic agents containing selected oral microorganisms for a short time period. A lidocaine.8-12 It has been suggested that the use of these one-minute exposure to either lidocaine or benzocaine agents may significantly alter microbial population on liquid topical anesthetic was sufficient to lower the micro- surfaces that they contact, especially the bronchial tree bial populations by 73-100% when compared with during bronchoscopy procedures with topical anesthe- controls without anesthetic. The antimicrobial properties sia.9 The present investigation, however, was the first to of liquid topical anesthetic preparations may be of con- examine the antimicrobial effect of two commercially siderable importance in the practice of dentistry because prepared dental topical anesthetics, Xylocaine, (lido- most dentists use topical anesthetics just before the caine) and Hurricane (benzocaine), against selected mi- injection of local anesthetics. Therefore, the antimicrobial croorganisms that are indigenous to the oral cavity. These activity of topical anesthetics before injection may explain products were found to possess antimicrobial properties the very low incidence of postinjection infections seen against each of the six microorganism strains tested. clinically. Similar findings for lidocaine were observed by others This investigation demonstrated that six species of using nonoral bacterial test strains,9-12 or anesthetic microorganisms commonly found in the oral cavity were sprays against oral microbes.15 killed in vitro by a brief exposure to two commonly used Several hypotheses have been proposed to explain the topical anesthetic liquids. effect that anesthetic agents have on microorganism survival. Schmidt and Rosenkranz"6 described potential mechanisms of action for lidocaine and procaine when ACKNOWLEDGMENTS tested with over 1,200 different strains of microorganisms. Lidocaine inhibited protein synthesis to a The authors are grateful to Astra Pharmaceutical Prod- greater extent than the formation of DNA and RNA. ucts, Inc. and Beutlich, Inc. Pharmaceuticals for supply- Procaine affected both DNA and protein production and ing the Xylocaine and Hurricaine liquid topical anesthe- high doses of the drug were required to inhibit RNA tics used in this study. synthesis. It was concluded that the basis of action of these This study was funded in part by Baylor College of agents may involve the microbial cell wall or membrane. Dentistry Research Funds. In another study, the anesthetic directly altered perme- ability of the cytoplasmic membrane that caused a rapid REFERENCES efflux of potassium across the membrane.17 It was suggested that local anesthetics were generally more effec- 1. Bennett CR, Monheim's Local Anesthesia and Pain tive against gram-positive than gram-negative cells. 18 This Control in Dental Practice, St. Louis, MO: C. V. 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