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© Borgis - Nowa Stomatologia 4/2004, s. 176-179
Joële Luc1, Elżbieta Dybiżbańska2, Christine Roques3
Bactericidal and fungicidal activity of selected mouthrinses - an in vitro study
1Fonderephar, Toulouse, Francja
2z Zakładu Stomatologii Zachowawczej IS AM w Warszawie
Kierownik Zakładu: prof. dr hab. Maria Wierzbicka
3Laboratoire de Bacteriologie, Virologie et Microbiologie Industrielle, Toulouse, Francja
Dental caries as well as periodontal diseases are disorders of bacterial origin. The microorganisms responsible for caries are bacteria of the Streptococcus mutans and Lactobacillus acidophilus species. Among the microorganisms considered pathogenic for the periodontium are Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannarella forsythensis, while Prevotella intermedia, Campylobacter rectus, Peptostreptococcus micros, Fusobacterium, Eubacterium and Treponema species, as well as yeast-like fungi, are classified as potentially pathogenic (1). The above mentioned bacteria are deposited on the tooth surface forming a biofilm, generally known as plaque. Plaque control plays a fundamental role in the prevention of dental caries and periodontitis. Despite of this, mechanical methods of plaque control are often insufficient, and the supplemental use of chemical agents is recommended. It is often the case that manual skills are deficient, in individuals with physical or mental handicap, as well as following oral surgical procedures (2, 3). The aim of chemical plaque control is to prevent the proliferation of microorganisms (bacteriostatic agents), or the eradication of the microorganisms already present in the biofilm, as well as the newcomers (bactericidal agents) (4). The most frequently used carrier of an antibacterial chemical agent is a mouthrinse. Other carriers used include toothpaste, sprays, irrigation liquids, chewing gum, lozenges and dental varnishes (5). Among the most frequently used substances in plaque control are chlorhexidine, belonging to the bis-biguanides, as well as cetylpiridinium chloride, a quaternary ammonium compound, phenolic compounds such as triclosan, oxidizing agents e.g. hydrogen peroxide, fluoride compounds e.g. a combination of fluoroamines and stannous fluoride, metal salts e.g. tin, zinc, and others e.g. hexetidine (5).
The effectiveness of the chemical agents used for plaque control has been assessed through in vitro and in vivo assays. For the in vitro studies bacteriological tests were used, which assess the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the tested agents (4).
The aim of this study was an in vitro assessment of the bactericidal and fungicidal action of eight mouthrinses used in home oral hygiene, with reference to selected species of bacteria and yeast most frequently observed in dental caries and periodontitis.
The tested mouthrinses are presented in Table 1. The bactericidal and fungicidal action of the rinses was evaluated with respect to standard strains of the following microorganisms: Streptococcus mutans CIP 103220T, Lactobacillus acidophilus CIP 7316T, Fusobacterium nucleatum CIP 101130T, Prevotella intermedia CIP 103607 and Actinobacillus actinomycetemcomitans CIP 52109, as well as Candida albicans IP 4872, originating from the microorganism collection of l´Institut Pasteur (Paris).
Table 1. Mouthrinses tested.
MouthrinseActive ingredientConcentration used
Corsodyl0.20% chlorhexidine digluconatepure
Eludril0.10% chlorhexidine digluconatediluted: 1:2, 1:3
Hextril/Oralden0.10% hexetidinepure
Meridolfluoroamines + 0.025% stannous fluoridepure
Tantum verde0.15% benzydamine hydrochloridediluted: 1:2
Alodont0.005% cetylpiridinium chloridepure
Lacalut0.20% chlorhexidine digluconate, aluminium lactate, betain pure
Plak out0,12% chlorhexidine digluconatepure
Bacterial suspensions were prepared directly prior to use in sterile, distilled water at a concentration of 108-109 bacteria/ml, whereas for C. albicans – at a concentration of 107/ml. Bactericidal and fungicidal action was determined using the „dilution-neutralization” method (European Standard). The test was conducted by adding 1 ml of the microorganism suspension to 9 ml of the tested solution. In order to stimulate conditions in vivo in which there is interaction with organic components 0.3% bovine albumin was added to the test tube. Contact time was 1 minute at a temperature of 32°C. Contact was interrupted by transferring 1 ml from the test tube into 9 ml of a neutralizing substrate (10% polysorbate, 2% saponin, 2% lecithin, 0,5% sodium thiosulfate, soy-tryptone broth). Following 10 minutes of neutralization, the remaining live microorganisms were counted by incubating 1 ml of the suspension in Schaedler´s agar substrate (in the case of S.mutans), agar MRS substrate (for L. acidophilus), or agar plus malt extract (for C. albicans). In the case of F. nucleatum, P. intermedia and A. actinomycetemcomitans, counts were performed by plating 100 ml on Columbia sheep blood agar. The cultures were incubated at a temperature of 37°C for 48-72 hours under anaerobic or aerobic conditions for the bacteria and at a temperature of 30°C for C.albicans. After that, the colonies were counted. All tests were done twice.
Table 2 presents the number of colonies of the individual microorganism species (CFU/ml) following a 1-minute contact with the tested mouthrinses. Both of the chlorhexidine-containing rinses – Corsodyl and Eludril, at both of the applied concentrations: 1:2 and 1:3, demonstrated similar bactericidal and fungicidal action with respect to all of the microorganisms used. Hextril and Plak out demonstrated bactericidal and fungicidal action similar to Corsodyl and Eludril, with respect to S. mutans, L. acidophilus, F. nucleatum, A. actinomycetemcomitans and C. albicans; however, they did not show bactericidal action with regard to P. intermedia. Meridol acted as a bactericidal agent only with regard to S. mutans and F. nucleatum, while Tantum verde – only with regard to F. nucleatum. Alodont and Lacalut did not demonstrate bactericidal activity with respect to any of the tested strains.
Table 2. Number of colonies of specific microorganisms (CFU/ml) after 1 minute of contact with the tested mouthrinses.
CorsodylEludril 1:2Eludril 1:3Hextril/OraldenMeridolTantum verdeAlodontLacalutPlak out
S. mutans0-1 0-0 *1-3 0-2 *2-4 0-1 *1-7 0-0 *0-0 1-1 *> 300 > 300> 300 > 300> 300 > 3000-1 0-0 *
L. acidophilus0-0 4-6 *0-2 0-0 *0-0 0-0 *0-0 0-0 *246-264 150-163> 300 > 300> 300 > 300> 300 > 3000-0 0-0 *
F. nucleatum0-0 0-0 *0-0 0-0 *0-0 0-0 *0-0 0-0 *0-0 0-0 *3-4 0-0 *7-10 * > 300> 300 > 3000-0 0-0 *
P. intermedia0-0 0-1 *0-0 0-0 *0-0 0-0 *37-80 40-60> 300 > 300> 300 > 300> 300 > 300> 300 > 300> 300 > 300
A. actinomycetemcomitans0-0 0-0 *0-0 0-0 *0-0 0-0 *0-0 0-0 *> 300 > 300> 300 > 300> 300 > 300> 300 > 3000-0 0-0 *
C. albicans0-0 0-1 *0-0 0-0 *0-0 0-0 *0-0 0-0 *> 300 > 300> 300 > 300> 300 > 300> 300 > 3000-2 1-1
* Bactericidal / fungicidal activity
The results of the study presented have indicated a wide range of bactericidal and fungicidal action by mouthrinses containing chlorhexidine. Similarly, earlier studies have demonstrated the effectiveness of chlorhexidine in the inhibition of the growth of both the Gram-positive and Gram-negative microorganisms associated with dental caries and periodontitis, including S. mutans, A. actinomycetemcomitans, P. intermedia, F. nucleatum and P. gingivalis (6, 7), as well as C. albicans (8). Earlier studies have indicated a much wider and more homogenious spectrum of in vitro anti-bacterial activity for chlorhexidine than for hexetidine or cetylpiridinium chloride (9), as well as strong fungicidal action (8). The minimum inhibitory concentration (MIC) of chlorhexidine with respect to the microorganisms of the oral cavity has been established at 1-20 mg/ml (5). However, it is known that the microorganisms present within the biofilm behave differently from microorganisms contained in the suspension used for the laboratory test; for this reason the impact of chlorhexidine on the viability of microbes residing in plaque has also been evaluated. It has been concluded that following the rinsing of the oral cavity with a solution of chlorhexidine, the number of live microorganisms in plaque decreased to a greater extent than following a rinse with a solution of fluoroamines / stannous fluoride (10), and the ratio of aerobic to anaerobic microorganisms in plaque increased (11). Following six months of rinsing with chlorhexidine a positive influence was found on the subgingival microflora, namely, a reduction in the proportion of Gram-negative microorganisms (5).
Apart from the bacteriological studies, chlorhexidine has been the subject of many clinical trials. In a situation of several days of negligence in tooth brushing, a greater effectiveness of chlorhexidine has been noted with respect to the prevention of plaque formation, compared with a placebo group (12), with fluoroamines/stannous fluoride (13) as well as with cetylpiridinium chloride (14, 15). Together with an increase in the applied concentration of chlorhexidine from 0.01% to 0.05% Jenkins et al. have observed an increase in the inhibitory action with respect to the plaque index as well as to the area of the tooth covered with plaque, while at concentrations ranging from 0.05% to 0.20%, the increase in chlorhexidine concentration resulted in only a slight increase in the effectiveness of the mouthrinse (12). Also the studies by Keijser et al. as well as by Smith et al. have demonstrated a similar effectiveness of rinses containing chlorhexidine at concentrations of 0.12% and 0.20%, at a simultaneous maintenance of a similar total dose of chlorhexidine (16, 17). Herrera et al. concluded that rinses containing 0.12% chlorhexidine with alcohol and chlorhexidine with 0.05% cetylpiridinium chloride possess a stronger bactericidal action with respect to S. mutans than 0.12% chlorhexidine without alcohol (18).
Neglecting toothbrushing for a period of 1-3 weeks has been used to evaluate the influence of chlorhexidine on the development of gingivitis. It has been concluded that rinsing the oral cavity during this time using a solution 0.10%-0.20% chlorhexidine resulted in a reduction in plaque formation as well as in the development of gingivitis, as compared with a placebo group (11, 19, 20), as well as with a group using fluoroamines/stannous fluoride (10, 21). Extended use of chlorhexidine has led neither to a development of opportunistic microorganisms nor to permanent changes in the composition of the oral microflora. The observed temporary and slight increase in the resistance of the microorganisms to the rinses, manifested by an increase in the MIC value, returned to the original status 5 months after the use of the agent was terminated (5). A factor limiting the use of chlorhexidine may be the observed side-effects – discoloration of the teeth and restorations, as well as disorders of the sense of taste (3, 11, 22, 23), although these symptoms are less intense when lower concentrations of the agent are applied (3).
It is currently known that the microorganisms pathogenic to the periodontium are present not only in plaque, but also on the dorsal surface of the tongue, on the buccal mucosa and in other ecological niches, therefore the idea of disinfecting the entire oral cavity has been introduced in the treatment of periodontitis (19). It is aimed at preventing reinfection of the periodontal pockets following scaling, through the limitation of pathogenic microorganisms from the entire oral cavity and throat (1). To this end, it is generally recommended that the oral cavity be rinsed twice a day with 10-15 ml 0.12%-0.20% solution of chlorhexidine for a period of 1-2 weeks (19, 20). However the composition of the rinse may by important, as the present study showed that a 0.20% chlorhexidine rinse also containing aluminium lactate and betain lacked bactericidal and fungicidal activity. With regard to any of the species investigated and another containing 0.12% chlorhexidine did not show bactericidal action with regard to P. intermedia.
1. From all of the mouthrinses tested, bactericidal and fungicidal action with respect to all the species of microorganisms was observed in the case of chlorhexidine.
2. No difference in bactericidal or fungicidal action was noted between the rinses containing 0.20% and a 0.10% chlorhexidine, even after dilution of the latter.
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