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
Keywords Abstract
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
Correspondence
(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
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. Introduction
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
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
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
Journal compilation 2007 Australian Society of Endodontology
Antibacterial Activity of Intracanal Drugs
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
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
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
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-
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,
Journal compilation 2007 Australian Society of Endodontology
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
†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
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
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
specific antibiotics in its composition: ciprofloxacin (14),which is an antibiotic specific for enterobcateria, such as
Discussion 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
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.
Journal compilation 2007 Australian Society of Endodontology
Antibacterial Activity of Intracanal Drugs
Furthermore, iodoform presented antibacterial activity
in a North American population. Oral Surg Oral Med
against all microorganisms tested. The MIC for B. fragilis
Oral Pathol Oral Radiol Endod 2001; 91: 579–86.
was the same found with CFC. Iodoform was expected to
7. Tronstad L, Barnett F, Cervone F. Periapical bacteria
be effective in anaerobic conditions owing to the higher
plaque refractory to endodontic treatment. Endod Dent
release of iodine (19). Iodoform was also effective against
S. aureus in a lower concentration than CH and IKI.
8. Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M.
According to other investigations, this drug is able to
Isolation of Enterococcus faecalis in previously root-filled
eliminate E. faecalis (16,19), but the MIC observed was
canals in a Lithuanian population. J Endod 2000; 26:593–5.
higher than those of the other drugs used. Similarly,
9. Nair PN, Henry S, Cano V, Vera J. Microbial status of
iodoform was effective against P. aeruginosa.
apical root canal system of human mandibular firstmolars with primary apical periodontitis after ‘one-visit’
Conclusion
endodontic treatment. Oral Surg Oral Med Oral PatholOral Radiol Endod 2005; 99: 231–52.
CFC showed the best results against the four bacterial
10. Haapasalo M. Bacteroides spp. In dental root canal infec-
species evaluated. The antibacterial action of CFC was
tion. Endod Dent Traumatol 1989; 5: 1–10.
similar to iodoform against S. aureus and B. fragilis, and
11. Sundquist G. Taxonomy, ecology, and pathogenicity of
similar to IKI against E. faecalis. CH showed a MIC value
the root canal flora. Oral Surg Oral Med Oral Pathol
for E. faecalis that was close to the one of iodoform and
presented the higher MIC values for all bacterial species,
12. Reader C, Boniface M, Bujanda-Wagner S. Refractive
except for S. aureus, against which IKI was ineffective.
endodontic lesion associated with Staphylococci aureus.
The results were achieved in vitro and cannot be
directly related to the clinical situation. This is especially
13. Ranta K, Haapasalo M, Ranta H. Monoinfection of root
so when drug mixtures (such as CFC) interact with
canal with Pseudomonas aeruginosa. Endod Dent Trauma-
mixed bacterial flora such as that infecting most root
14. Hoshino E, Kurihara-Ando N, Sato I et al. In vitro anti-
bacterial susceptibility of bacteria taken from infectedroot dentine to a mixture of ciprofloxacin, metronida-
Acknowledgement
zole and minocycline. Int Endod J 1996; 29: 125–30.
15. Ørstravik D, Haapasalo M. Disinfection by endodontic
The Authors thank Jarshen Lin, DDS, Department of
irrigants and dressings of experimentally infected
Endodontics, University of Harvard for reviewing this
dentinal tubules. Endod Dent Traumatol 1990; 6:
16. Cwikla SJ, Bélanger M, Giguère S, Progulske-Fox A,
References
Vertucci FJ. Dentinal tubule disinfection using threecalcium hydroxide. Formulations. J Endod 2005; 31:
1. Tronstad L, Barnett F, Riso K, Slots J. Extraradicular
endodontic infections. Endod Dent Traumatol 1987; 3:
17. Kishen A, George S, Kumar R. Enterococcus faecalis-
mediated biomineralized biofilm formation on root canal
2. Wayman BE, Murata SM, Almeida RJ, Fowler CB.
dentine in vitro. J Biomed Mater Res A 2006; 77: 406–
A bacteriological and histological evaluation of 58
periapical lesions. J Endod 1992; 18: 152–5.
18. Abdullah M, Ng Y-L, Gulavibala K, Moles DR, Spratt DA.
3. Leonardo MR, Rossi MA, Silva LA, Ito IY, Bonifacio KC.
Susceptibilities of two Enterococcus faecalis phenotypes to
EM evaluation of bacterial biofilm and microorganisms
root canal medications. J Endod 2005; 31: 30–6.
on the apical external root surface of human teeth.
19. Maisto OA. Medicación intracanal. In: Maisto OA, ed.
Endodoncia. 3rd edn. Buenos Aires: Mundi; 1975.
4. Noguchi N, Noiri Y, Narimatsu M, Ebisu S. Identification
and localization of extraradicular biofilm-forming bacte-
20. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial
ria associated with refractory endodontic pathogens.
activity of calcium hydroxide: a critical review. Int
Appl Environ Microbiol 2005; 71: 8738–43.
5. Wu MK, Dummer PM, Wesselink PR. Consequences of
21. DeMoor RJG, DeWitte MJC. Periapical Lesions acciden-
and strategies to deal with residual post-treatment root
tally filled with calcium hydroxide. Int Endod J 2002;
canal infection. Int Endod J 2006; 39: 343–56.
6. Hanock HH, Sigurdsson A, Trope M, Moiseiwitsch J.
22. Evans M, Davies JK, Sundquist G, Figdor D.
Bacteria isolated after unsuccessful endodontic treatment
Mechanisms involved in the resistance of Enterococcus
Journal compilation 2007 Australian Society of Endodontology
Antibacterial Activity of Intracanal Drugs
faecalis to calcium hydroxide. Int Endod J 2002; 35:
25. Manisali Y, Yücel T, Erisen R. Overfilling of the root – a
case repot. Oral Surg Oral Med Oral Pathol 1989; 68:
23. Haapasalo HK, Sirén EK, Waltimo TMT, Ørstavik D,
Haapasalo MPP. Inactivation of local root canal medica-
26. Andrews JM. Determination of minimum inhibitory
ments by dentine: an in vitro study. Int Endod J 2000;
concentrations. J Antimicrob Chemother 2001; 48
24. Lima KC, Fava LR, Siqueira JF. Susceptibilities of entero-
27. Woodford N, Ellington MJ. The emergence of antibiotic
coccus biofilms to some antimicrobial medications.
resistance by mutation. Clin Microbiol Infect 2007; 13:
Journal compilation 2007 Australian Society of Endodontology
ACCESS FLOOR TECHNICAL DOCUMENTATION CONTENTS AN INTRODUCTION TO ACCESS FLOORING INSTALLATION MANUAL ACCESS FLOOR COMPONENTS ACCESS FLOOR CLEANING MANUAL AN INTRODUCTION TO ACCESS FLOORING CONTENTS 1 BACKGROUND 2 THE CONCEPT OF FLEXIBILITY 3 ACCESS FLOORING AND ARCHITECTS 3.1 A NEW CREATIVE FREEDOM 3.2 NEW BUILDINGS 3.3 REDEVELOPING OLD BUILDINGS 4 ECONOM
AIDS InfoNet www.aidsinfonet.org Fact Sheet Number 611 PREGNANCY AND HIV HOW DO BABIES GET AIDS? Keep delivery time short: The risk of A pregnant woman should consider all of The virus that causes AIDS can be transmission increases with longer delivery transmitted from an infected mother to her newborn child. Without treatment, about viral load (see Fact Sheet 125) under