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Determination of the minimum inhibitory concentration of four medicaments used as intracanal medication

Aust Endod J 2007; 33: 107–111
Determination of the minimum inhibitory concentration of four
medicaments used as intracanal medication
Raul C. Pallotta, DDS, MDSc, PhD1; Mariangela S. Ribeiro, MMSc2; and Manoel E. de Lima Machado, DDS,MDSc, PhD3 1 Department of endodontics, University Cruzeiro do Sul, Sao Paulo, Brazil2 Department of Microbiology, PUCCAMP, Campinas, Brazil3 Department of Endodontics, University Camilo Castelo Branco, Sao Paulo, Brazil antibiotics, calcium hydroxide, iodine,iodoform, minimum inhibitory concentration.
The aim of this study was to determine the minimum inhibitory concentration(MIC) of iodoform, calcium hydroxide, IKI (iodine potassium iodine) and CFC (ciprofloxacin, Flagyl (metronidazole) and calcium hydroxide) required to kill Dr Raul C. Pallotta, R. Moreira de Godoi, S. aureus, Pseudomonas aeruginosa, Enterococcus faecalis and B. fragilis. In the 664, 2° andar – cj.07 – CEP 04266 – 060, experiment, medicaments were added to bacterial species into test tubes, in 10 Ipiranga, São Paulo, SP, Brasil. Email: different concentrations. The MIC was the lowest concentration of the drug atwhich bacterial growth could not be observed. In this investigation, CFC was the most effective medicament against all bacteria. All drugs were able toeliminate E. faecalis and B. fragilis, while IKI was not effective against S. aureus.
IKI and calcium hydroxide were not able to eliminate P. aeruginosa as well.
must be able to kill bacteria, either by acting directly onthem or by creating unsuitable conditions in which to The presence of microorganisms and their by-products in survive (19–21).
the root canal system provokes a host response, which The most widely used is calcium hydroxide (CH) can be demonstrated clinically and radiographically as (15,16,20–23). Its main mechanism of action is to raise periapical alterations (1–4). The main goal of endodontic the Ph sufficiently that few microorganisms are able to therapy is to decrease contamination allowing periapical survive (20). However, there are some strains that are resistant to the use of this drug (22,23). To improve its antimicrobial activity, CH may be used in association with mechanically during root canal preparation. However, ciprofloxacin and CFC (ciprofloxacin, Flagyl (metronida- they are often found in areas that are not instrumentable zole) and calcium hydroxide) (14,17). Ciprofloxacin is a (5–9). These bacteria, both inside the canal and in the bactericidal drug, which acts by blocking bacterial DNA adjacent periapical tissues, can organize themselves in replication. Additionally, metronidazole has a specific such a way to form a biofilm (4,5,7,10). Periapical biofilm selective toxicity for anaerobic bacteria as well as parasites is usually found in teeth with pulp necrosis and radio- graphically visible periapical lesions (3). In these cases, Another drug used to improve the antibacterial activity conventional endodontic therapy tends to fail in a higher is iodoform, which has been used successfully as a medi- percentage of cases (6,8,9). Bacterial flora in this region is cament and filling paste for many years (16,19,25). Iodo- normally mixed, with the predominance of anaerobic and form seems to stimulate immunological response and to facultative species (4,6–8,11–14).
interact with bacterial contamination by promoting the In these situations, the use of irrigating agents and an growth of granulation tissue, and thus accelerating the intracanal medication with an effective antibacterial healing process (19).
action is recommended to achieve an adequate decon- There are also specific antiseptics, such as iodine potas- tamination of this system (15–18). These medications sium iodide (IKI), an iodide compound which presents 2007 The Authors Journal compilation 2007 Australian Society of Endodontology Antibacterial Activity of Intracanal Drugs R. C. Pallotta et al. excellent biocompatibility and good bactericidal action containing sterile medium until the desired turbidity in vitro. However, clinical evaluations do not show the pattern was reached (approximately 104–105 bacteria same antibacterial activity (15,16,23).
mL-1). One millilitre of this combination (bacteria + Considering the challenge of decontaminating the medium with no medication) was transferred to an periapical tissues, the authors decided to evaluate the empty tube as positive control.
action of four medicaments prescribed in endodontics Treated groups consisted of test tubes containing (iodoform, CH, IKI, and CFC) to kill four distinct bacteria 0.9 mL of this initial bacteria + medium combination, and (Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus 0.1 mL of the chosen medicament in every concentration.
faecalis and Bacteroides fragilis) by determining their Using the above protocol, the experiment was repeated in minimum inhibitory concentration (MIC). These bacteria 20 tubes for each of the 10 medicament concentrations.
are characterised by great resistance to antibacterial For S. aureus and P. aeruginosa, and E. faecalis, the drugs, either by their natural resistance or by the devel- medium used was brain heart infusion during the entire opment of different defence mechanisms against the experiment, and they were incubated at 37°C (98°F) for host response.
24 h. For B. fragilis, the test tubes containing the bacteriaand medicament combinations in RCM were stored in Materials and methods
anaerobiosis jars, which were kept sealed up for 48 h. Atthe end of the incubation period, the bacterial growth in Of the four selected bacterial species, the two aerobic the test tubes was checked.
bacteria (P. aeruginosa – ATCC 27853 and S. aureus – ATCC The MICs of the drugs for each bacterial species were 6538) and the facultative one (E. faecalis – ATCC 29212) determined. The MIC was the lowest concentration of the were in a solid culture medium of nutrient agar and drug at which bacterial growth could not be observed. For stored in a test tube. B. fragilis (ATCC 25285), had to be the aerobe or facultative strains, the results were con- reactivated by filling an ampoule of the lyophilised firmed by transferring samples, by means of an inoculat- bacteria with Reinforced Clostridium Medium (RCM) and ing loop, from tubes with no bacterial growth into sterile then inserted in anaerobic jars. All inoculums were trans- nutrient agar plates. For B. fragilis, tubes where no growth ferred to a dry-heat oven at 37°C (98°F) where they could be observed after 48 h had a sample of 0.1 mL remained for 24–48 h for initial growth, according to placed in test tubes containing sterile RCM. All these new samples were once again incubated and the presence of At the end of the initial growth phase, the experimen- bacteria checked.
tal groups to be studied were stratified, as follows: (i) Data were compiled into tables and individual values according to the bacteria: E. faecalis, S. aureus, P. aeruginosa and mean values were statistically analysed, with a level and B. fragilis; (ii) according to the medicament to be of significance of 5%. Comparisons were made among tested: iodoform, CH, CFC and IKI; and (iii) according to the different drugs in terms of MIC required to kill the the drug concentration. The drugs were diluted in same microorganism. These comparisons were then sub- glycerin to reach the concentrations of 0.125 mg mL-1, mitted to statistical analysis using the ANOVA test.
4 mg mL-1, 8 mg mL-1, 16 mg mL-1, 32 mg mL-1 and Iodoform and CH were directly diluted in glycerin up MICs of the drugs required to kill the four specific bacte- to the desired dilution. The three CFC compounds, rial species are shown in Table 1 and Figure 1. The evalu- (CH, ciprofloxacin and metronidazole) were mixed in the ation showed that IKI was not active against S. aureus and commonly used proportion (2:1:1 respectively), and then P. aeruginosa, even in the highest concentration used in this mixture was diluted in glycerin. Regarding IKI, this is this study (64.0 mg mL-1), and CH was not active against the association of 2% iodine and 4% potassium iodide. In this study, IKI was prepared in the proportion of 1:2 of At the concentrations used, all drugs were found to be iodine and potassium iodide, respectively, and diluted in able to eliminate E. faecalis and B. fragilis. For E. faecalis, glycerin up to the desired concentration.
the MICs were 32.0 mg mL-1 for iodoform, 16.0 mg mL-1 Initially, the experimental groups were prepared in test for CH, 0.125 mg mL-1 for CFC and 2.0 mg mL-1 for IKI.
tubes containing pre-sterilised medium. One millilitre For B. fragilis, MIC values were 0.25 mg mL-1 for iodo- was removed from each of these tubes and incubated to form and CFC, 4.0 mg mL-1 for IKI and 16.0 mg mL-1 be used as negative control. The experiment itself con- sisted of removing 1-mL aliquots from tubes with bacte- MIC values for S. aureus were 0.5 mg mL-1 for CFC, rial growth, and repeatedly inoculating them into tubes 2.0 mg mL-1 for iodoform and 16.0 mg mL-1 for CH, 2007 The Authors Journal compilation 2007 Australian Society of Endodontology R. C. Pallotta et al. Antibacterial Activity of Intracanal Drugs Table 1 Minimum inhibitory concentration (MIC) of iodoform, calcium hydroxide, CFC (ciprofloxacin, Flagyl (metronidazole) and calcium hydroxide) and
iodine potassium iodine (IKI) required to kill Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa and Bacteroides fragilis
E. faecalis† S. aureus† P. aeruginosa† B. fragilis† Calcium hydroxide †Statistically significant difference (P < 0.05) between the MIC of the drugs.
The bacteria evaluated in this study are representative of the different groups normally found in cases oftherapy-resistant lesions in root canals and have sig- E. faecalis is usually associated with refractory lesions (8,9,17,18,22,24), owing to its capacity to survive for long periods without nutrients (15,22,23). B. fragilis is a gram- negative anaerobic bacilli and is the most common bac- teria found in endodontic infections (2,4,9–11). Owing to E. faecalis P. aeruginosa B. fragilis its great capacity for adaptation and resistance, S. aureus can be found in both pulp and periapical infections (12).
In endodontics, P. aeruginosa is found in teeth with peri- Figure 1 Minimum inhibitory concentration of iodoform, calcium hydrox-
apical lesions exposed to the oral cavity and this bacte- ide, CFC (ciprofloxacin, Flagyl (metronidazole) and calcium hydroxide) and rium is usually related to monoinfections (2,13).
iodine potassium iodine (IKI) required to kill Enterococcus faecalis, Staphy- CFC was shown to be the most effective drug in this lococcus aureus, Pseudomonas aeruginosa and Bacteroides fragilis.
investigation. CFC demonstrated the ability to eliminate CH, calcium hydroxide.
all the bacterial strains at the lowest concentration used,that is, 0.125 mg mL-1, except for S. aureus. The great whereas, for P. aeruginosa, MICs were 0.125 mg mL-1 for antibacterial action of CFC is due to the presence of two CFC and 64.0 mg mL-1 for iodoform.
specific antibiotics in its composition: ciprofloxacin (14),which is an antibiotic specific for enterobcateria, such as E. faecalis and P. aeruginosa; and metronidazole, which isable to eliminate anaerobic bacteria, such as B. fragilis Incomplete decontamination of infected root canals may lead to failure of the endodontic therapy and the devel- The evaluation of the action of IKI showed that this opment of periapical lesions (1–3). Clinically, the pres- medication was not able to kill S. aureus and P. aeruginosa, ence of anatomic variations as well as the high number even at the highest concentration used. However, IKI was and great variety of microorganisms make it difficult to effective against E. faecalis in a low concentration, which completely eliminate microorganisms from the canal is consistent with other studies (15,16). IKI was able to (5,6,8,9). Also, microorganisms can organise themselves kill B. fragilis as well.
in a biofilm, and conventional endodontic therapy tends CH is the drug most commonly used as intracanal to fail in a higher percentage of these cases (3,4,6,7,9,17).
medication. The MIC of CH for B. fragilis and S. aureus was In order to eliminate as many remaining bacteria as 16.0 mg mL-1, the highest value found for these bacteria.
possible following debridement, intracanal medication is The MIC of CH against E. faecalis was 16.0 mg mL-1 as highly recommended (14–16,18,19,21,24).
well; however, the MIC of iodoform was higher, although The determination of the MICs is used by diagnostic without statistical difference. This drug was not able to laboratories mainly to confirm resistance, but most often eliminate P. aeruginosa in this investigation. Therefore, CH as a research tool to determine the in vitro activity of new showed the higher MIC values. The direct antibacterial antimicrobials (26). This study was carried out to deter- action of CH on E. faecalis (15,16,18,19), B. fragilis and mine the MIC of iodoform, CH, IKI, and an association of S. aureus (2,12) observed in this study was expected based CH, metronidazole and ciprofloxacin, the CFC.
on other investigations.
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