Dissertation submitted to
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY
In partial fulfillment for the Degree of MASTER OF DENTAL SURGERY
BRANCH IV
CONSERVATIVE DENTISTRY AND ENDODONTICS APRIL 2016
Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, for his perseverance in motivating and supporting me throughout my study period.
My sincere thanks to Dr. R. Indira, M.D.S., Professor and HOD, Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, who helped me with her guidance, support and constant encouragement throughout my study period wherever and
whenever needed.
My sincere thanks to Dr. S. Ramachandran, M.D.S., Professor
& Principal, Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, who helped me with his advice and immense support throughout my post graduate curriculum.
I extend my sincere thanks to Dr. T.Sundararaj, M.Sc.,Ph.D., FABMS,(Former) Professor and HOD,Dept.of Microbiology,University of Madras presently Director of JERF who helped me with, without which my study would not have been possible.
Ragas Dental College and Hospital, for his guidance, and constant encouragement during the completion of my study.
My sincere thanks to Dr. C. S. Karumaran, M.D.S., Professor, Ragas Dental College and Hospital, for his encouragement, support and guidance all throughout my study period.
I extend my sincere thanks to Dr. M.Rajasekaran, M.D.S., Professor, for his constant encouragement throughout the completion of this work.
I would like to solemnly thank Dr. Veni Ashok, M.D.S., Dr. S.M.
Venkatesan, M.D.S., and Dr. Shankar Narayan, M.D.S., Readers, for all the help during my study period.
I would also like to thank Dr.Sabari, M.D.S, Dr.Aravind, M.D.S., Dr. B. Venkatesh , M.D.S., Senior lecturers for their friendly guidance and support.
I also wish to thank the management of Ragas Dental College and Hospital, Chennai for their help and support.
I would like to especially thank my parents, my brothers, my husband and my family for their love, understanding, support and encouragement throughout these years without which, I would not have reached so far.
My sincere thanks to Dr. Celena Bency, Mr. K. Thavamani and Miss. R. Sudha for their guidance and support in DTP and Binding works.
Above all, I am thankful to God, for blessing me with all the goodness and wonderful people in my life.
S. NO. INDEX PAGE.NO
1. INTRODUCTION 1
2. AIM & OBJECTIVES 8
3. REVIEW OF LITERATURE 9
4. MATERIALS AND METHODS 34
5. RESULTS 44
6. DISCUSSION 46
7. SUMMARY 66
8. CONCLUSION 68
9. BIBLIOGRAPHY 69
10. ANNEXURES ---
1 MIC and MBC values of Chlorhexidine,Alexidine and Sodium hypochlorite against E.Faecalis
2 Results of Time Kill Assay : Control 3 Results of Time Kill Assay : Alexidine 4 Results of Time Kill Assay : Chlorhexidine 5 Results of Time Kill Assay : Sodium hypochlorite 6 Results of Log values of Overall Time Kill Assay
LIST OF GRAPHS
GRAPH NO. TITLE
1 Time Kill Assay : Control
2 Time Kill Assay : Alexidine
3 Time Kill Assay : Chlorhexidine
4 Time Kill Assay : Sodium hypochlorite
5 Comparison Of Time Kill Assay With Different Irrigants
NO.
1 Alexidine Dihydrochloride - Sigma Aldrich U.S,A 2 Chlorhexidine – Hi Media,Bombay India
3 Sodium Hypochlorite – 5% Chenchems , Chennai, India 4 Brain Heart Infusion Broth (BHI)
5 Incubator
6 Hot Air Oven
7 Autoclave
8 Centrifuge- Table Top 9 Microcentrifuge- High Speed 10 Freezers
11 Laminar Flow Chamber 12 Nikon microscope
13 Microtitre Plates- 96 Well 14 Micro Pipettes
15 Micro Pipette Tips
16 Spectrophotometer- Semi Auto Analyser 17 Ph Meter
21 Time Kill Assay – 2 MIC Of Alexidine (CFU)
Introduction
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INTRODUCTION
The success of endodontic therapy depends on complete elimination of microbial contamination from the root canal system at the time of obturation . Bacteria have long been recognized as the primary etiologic factors in the development of pulp and periapical lesions⁽43,13,88⁾. The oral cavity harbours one of the highest accumulations of microorganisms in the body. Even though viruses, bacteria, fungi, and protozoa can be found as constituents of the oral microbiota, bacteria are by far the most dominant inhabitants of the oral activity. There are an estimated 10 billion bacterial cells in the oral cavity (Mims et al., 2005). However, only 150 microbial species have been isolated and cultured from root canals. The endodontium is a sterile cavity but whenever there is a breach in the structural integrity of enamel and dentin along with diminutive host response it leads to invasion of oral microbes to establish infection. (Bergenholtz, 1974). Although all the bacteria in the oral cavity can invade the root canal, only a few microbes have been identified in infected root canals (Miller, 1994; Sundqvist, 1994; Wilkins et al., 2003).
Endodontic infections can be classified according to anatomic location (intraradicular or extraradicular infection). Intraradicular infection is caused by microorganisms colonizing the root canal system and can be subdivided into three catagories according to the time microorganisms entered the root canal system: primary infection, caused by microorganisms that initially invade and colonize necrotic pulp tissue (initial or virgin infection); secondary
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infection, caused by microorganisms not present in the primary infection but introduced in the root canal at sometime after professional intervention(i.e secondary to intervention); and persistent infection, caused by microorganism that were members of a primary or secondary infection and in some way resisted intracanal antimicrobial procedures and were able to endure periods of nutrient deprivation in treated canals. Extraradicular infection is characterized by microbial invasion of the inflamed periradicular tissues and is a sequel to the intraradicular infection. . Extraradicular infections can be dependent or independent of the intraradicular infection.
Primary root canal infections are polymicrobial, typically dominated by obligatory anaerobic bacteria [92]. Obligatory anaerobic bacteria are strict anaerobes ie organisms that grow only in the absence of free oxygen. The most frequently isolated microorganisms before root canal treatment include Gram-positive anaerobic cocci,(Parvimonas) Gram-positive anaerobic and facultative rods (,Filifactor, Pseudoramibacter, Propionibacterium) Gram negative anaerobic bacteria (fusobacterium, prevotella, dialister, porphyromonas, tannerella, treponema, campylobacter and veillonela) Lactobacillus species, and Gram-positive facultative Streptococcus species[99]. The obligate anaerobes are rather easily eradicated during root canal treatment. On the other hand, facultative bacteria such as non mutans Streptococci, Enterococci, and Lactobacilli, once established, are more likely to survive chemomechanical instrumentation and root canal medication[20].
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Enterococcus faecalis an enterococci has gained attention in the endodontic literature, as it can frequently be isolated from root canals in cases of failed root canal treatments [29,38].
E.faecalis is a gram positive facultative anaerobe possessing the ability to grow in presence or absence of oxygen. It is typically recovered from root canals of teeth with post treatment disease the prevalence ranges from 24 % to 77%[90]. The difficulty in eliminating E.faecalis from the root canal is due to its ability to adapt to environmental changes while retaining its pathogenicity[90]. E.faecalis posseses virulence factors including lytic enzymes, cytolysis, aggregation substances pheromones (is a secreted or excreted chemical factor that triggers a social response in members of same species) and lipoteichonic acid. It has been shown to adhere to host cells, express proteins that allow to compete with other bacterial cells, and alter host responses. E.faecalis is able to suppress the action of lymphocytes, potentially contributing to endodontic failure. These virulence factors play key roles in E.faecalis survival in the harsh environment of root canal, enduring prolonged periods of nutritional deprivation. It binds to dentin and proficiently invade dentinal tubules. E.faecalis is a robust microorganism and therefore is often considered to be a model test organism.
The aim of endodontic therapy is to remove all vital or necrotic tissue, microorganisms, and microbial by products from the root canal system, This may be achieved through chemomechanical debridement of root canal. The
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root canal system is highly complex and variable and has limited the ability to clean and disinfect it predictably. Shaping of root canals is performed almost entirely by using hand and rotary instrumentation techniques [73]. Peters et al.
Using micro computed tomography scans before and after mechanical instrumentation found that, regardless of the instrumentation technique, 35%
or more of the root canal surfaces (including canal fins, isthmi and cul-de- sacs) remained uninstrumented. Consequently irrigation is an essential part of root canal debridement because it allows for cleaning beyond what might be achieved by root canal instrumentation alone. In addition to disinfection, irrigants having chelation property can also help remove the smear layer from the radicular wall.
Endodontic irrigants are classified as chemical agents and natural agents. Chemical agents are further classified as tissue dissolving agents(e.g.
NaOCl, ClO2), Antibacterial agents – Bactericidal(e.g. CHX) and Bacteriostatic(e.g. MTAD) and Chelating agents - Mild pH (e.g., HEBP) and Strong pH (e.g., EDTA). Natural agents are further classified as antibacterial agents (e.g., Green tea, Triphala)
Root canal irrigants ideally[99] should have broad antimicrobial spectrum, high efficacy against anaerobic and facultative microorganisms organized in biofilms,ability to dissolve necrotic pulp tissue remnants, inactivate endotoxin, to prevent the formation of a smear layer during instrumentation or to dissolve the latter once it has formed. Irrigant should be
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nontoxic when they come in contact with vital tissues, noncaustic to periodontal tissues, and with little potential to cause an anaphylactic reaction..
No single irrigant can perform all the desired actions , so a combination of irrigants are employed during root canal therapy. The most commonly used irrigants are sodium hypochlorite, an effective tissue solvent, EDTA, a chelating agent and chlorhexidine gluconate, a broad-spectrum antimicrobial agent.
Sodium hypochlorite (NaOCl) is the most commonly used irrigating solution because of its tissue-dissolving capability , its broad antimicrobial action and its ability to neutralize toxic products (99,83,17,72,66). NaOCl does not impart antimicrobial substantivity (94). Chlorhexidine gluconate (CHX) is a cationic bisbiguanide with antimicrobial efficacy against certain NaOCl-resistant bacteria and against its virulence factor (31,57,10,55). Unlike NaOCL , CHX has antimicrobial substantivity (94, 50), which is beneficial for more effective root canal disinfection. The effect of a root canal disinfection regimen with a combination of CHX and NaOCl was explored (54,12,16). It has been reported in a study conducted by Basrani et al that interaction between sodium hypochlorite and chlorhexidine gluconate results in the formation of a dense-brown precipitate, para-chloroaniline (PCA) (99, 12).
which is found to be carcinogenic . The precipitate formed is of clinical relevance with regard to staining the dentin, hampering the seal of obturation, and potentially leaching PCA into the periapex (16, 53).
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The disadvantages of existing irrigants paved way for research of newer irrigants with high antimicrobial efficacy, Alexidine (ALX) is a bisbiguanide similar to chlorhexidine differs chemically from chlorhexidine in possessing 2 hydrophobic ethyhexyl groups in its structure(28).Alexidine is more rapidly bactericidal and produces a significantly faster alteration in bactericidal permeability. Alexidine helps to inhibit the immune response of the major virulence factors (lipopolysaccharide and lipoteichoic acid) of bacteria (100) It has been previously used as a mouthwash solution (78) and contact lens solution (96). Alexidine has got an edge over chlorhexidine and more beneficial as the interaction of ALX and sodium hypochlorite did not form an insoluble precipitate known as para-chloroaniline, as with CHX. So, Combination of ALX and NaOCl can be safely used without any harmful interactions.[Kim et al]
To evaluate the antibacterial activities of root canal irrigants different methods like agar diffusion test, direct contact test, time kill assay (TKA) have been used. The agar diffusion method has been widely used to test the antimicrobial activity of dental materials and medications. This method allows direct comparisons of root canal irrigants against the test microorganisms. It is stated that this system could be affected by the diffusibility of the tested materials and the results obtained with this system do not reflect the true antimicrobial potential of the tested materials. The TKA is a method used in
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this study to assess the ability of a fixed concentration of an antimicrobial agent to destroy a bacterial isolate under controlled conditions
Aim of this study was to evaluate and compare the time depended antimicrobial efficiency of 1% Alexidine with 1 % Chlorhexidine, 5%
Sodium hypochlorite against E.faecalis using time kill assay. 1% alexidine solution was selected in this study because alexidine with a concentration higher than 1% caused moderate cytotoxicity against human gingival fibroblasts (Kim et al). Time-kill assays aids to assess the rate of bactericidal activity at varying concentrations and time. The hypothesis is Alexidine has potent bactericidal effect on E.faecalis within short time interval when
compared with sodium hypochlorite and chlorhexidine.
Aim and Objectives
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AIM AND OBJECTIVES AIM:
Aim of this study was to evaluate and compare the antimicrobial efficiency of 1% Alexidine 1 % Chlorhexidine and 5% Sodium hypochlorite against E.faecalis using time kill assay.
OBJECTIVE:
1. To evaluate the anti bacterial effectiveness of three different root canal irrigants sodium hypochlorite, chlorhexidine and alexidine against E.faecalis by in -vitro analysis by time kill assay method.
2. To evaluate the antimicrobial efficacy of the irrigants with its Minimum Inhibitory Concentration in varying time period - 30, 60, 90 and 120 seconds.
Review of Literature
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REVIEW OF LITERATURE
Parsons GJ et al (1980)74 treated bovine pulp and dentin specimens were with either a 0.02 or 1.00 percent solution of chlorhexidine for either 20 or 40 minutes. Culture determination of the acquisition of antibacterial properties by the treated specimens immediately and 1 week after the treatment was evaluated using the test organism Streptococcus faecalis. It was concluded that chlorhexidine is a potent antibacterial agent under the test conditions and its use as an endodontic irrigating solution should be further evaluated
Byström A et al (1981)18 studied the presence of bacteria in 17 single- rooted teeth, with periapical lesions, throughout a whole period of treatment.
The root canals were irrigated with physiologic saline solution during instrumentation. No antibacterial solutions or dressings were used. Bacteria were found in all initial specimens from the teeth (median number of bacterial cells 4 x 10(5), range 10(2) - 10(7)) and the number of strains in the specimens ranged from 1 to 10.88% of the strains were anaerobic. Mechanical instrumentation reduced the number of bacteria considerably. Specimens taken at the beginning of each appointment usually contained 10(4) - 10(6) bacterial cells and at the end 10 (2)-10 (3) fewer. Bacteria were eliminated from the root canals of eight teeth during treatment. Teeth where the infection persisted
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despite being treated five times were those with a high number of bacteria in the initial sample.
Roberts WRet al (1981)78 carried out a blind crossover trial was to compare the effects of a 0.2% chlorhexidine gluconate mouthrinse and a 0.035% alexidine mouthrinse on plaque accumulation and salivary bacteria in a group of volunteers. Plaque scores were recorded at the end of each 10-day period. Significantly more plaque accumulated in subjects rinsing with alexidine when compared with chlorhexidine. Significant and comparable reductions in salivary bacterial counts were observed with both chlorhexidine and alexidine on day 4 and day 10 when compared with pre-rinse counts.
Although at the concentrations used alexidine was less effective than chlorhexidine, it may be of value as a short-term adjunct to oral hygiene.
Greenstein G et al (1986 )36 determined Chlorhexidine as an effective antimicrobial agent. Its application can enhance periodontal therapy. The pharmacology of chlorhexidine and suggestions for its use were outlined.
Baker PJ et al ( 1987 )8 assayed chlorhexidine and a series of its analogues, in which the chlorophenyl terminal substituents were replaced with alkyl chains, for their in vitro antimicrobial activity against the Gram-negative and Gram-positive oral bacteria. Changes in antimicrobial activity were correlated with changes in agent structure for identification of structural
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criteria which may be important in the optimization of agent activity.
Chlorhexidine showed substantial antimicrobial activity against the Gram- negative as well as the Gram-positive oral bacteria. The alkyl agents were comparable with chlorhexidine in their activity against Bacteroides gingivalis and Bacteroides intermedius, black-pigmented Gram-negative obligate anaerobes associated with periodontal disease in adults. Alkyl agents alexidine, heptihexidine (1,6-bis-n-heptylbiguanidohexane), hexoctidine (1,8- bis-n-hexylbiguanidoctane), and hexhexidine (1,6-bis-n-hexyl biguanidohexane) , as well as chlorhexidine, were active against Actinobacillus actinomycetemcomitans, a Gram-negative organism associated with localized juvenile periodontitis. Hexidecidine (1,10-bis-n- hexylbiguanidodecane) and heptoctidine (1,8-bis-n-heptylbiguanidooctane) were more active, and hexhexidine was as active as chlorhexidine against Fusobacterium nucleatum, also associated with periodontal disease. Seven of the agents were more active than chlorhexidine against Actinomyces species.
All test agents were active against Streptococcus mutans, a Gram-positive coccus associated with dental caries. Hexidecidine had activity equal to that of chlorhexidine when evaluated against the entire battery of organisms. Analysis of structure-activity relationships revealed that alkyl chains could replace chlorophenyl groups with retention or improvement of antimicrobial activity.
Kontakiotis E et al (1995)51 evaluated in vitro a possible mechanism involved in the antimicrobial action of calcium hydroxide, namely absorption
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of carbon dioxide from the root canal. Twenty obligate and 20 facultative anaerobic bacteria isolated from infected root canals and identified to species level were used. One experimental and one control group were studied: the experimental group included one plate with the bacterial species as well as one open plate containing 32 g calcium hydroxide paste at a mixing ratio of 6:4.
Both plates were incubated in an anaerobic chamber for 72 h. The control group included only one plate containing the same bacterial species and was incubated under the same conditions. After a 72-h incubation, the number of the recovered bacteria were counted in both groups. Statistical analysis showed that the number of bacteria recovered from the control group was significantly lower than that of the experimental group, but no particular resistance of any bacterial species to calcium hydroxide could be detected.
This finding strongly suggests that the ability of calcium hydroxide to absorb carbon dioxide may contribute to its antibacterial activity
Siren EK et al (1997)86 investigated the relationship between bacteriological findings and clinical treatment procedures in root canal treatment cases that were selected for bacteriological investigation by general dental practitioners in Finland. Two groups of teeth were selected based on the type of infection present in the root canal system. The 'enteric bacteria' group consisted of 40 sequential cases where Enterococcus faecalis and/or other facultative enteric bacteria or Pseudomonas sp. The group 'non-enteric bacteria' consisted of 40 sequential cases where only non-enteric bacteria were
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found. The dentists who had sent the bacteriological samples received a questionnaire where they were asked about the treatment protocol and procedures. If the root canals had been unsealed at some point during the treatment, enteric bacteria were found more frequently than in canals with an adequate seal between the appointments. Of cases with enteric bacteria 55%
had been open during the treatment, while in the group where only non-enteric bacteria were found 30% had been open. Enteric bacteria were also more frequently isolated in cases with a high number of appointments before sampling. In the enteric bacteria group 35% of the samples were taken at the 10th visit or later, while the corresponding percentage in the non-enteric group was 3%. In addition, the number of retreatment cases was significantly higher, 12 out of 34, in the enteric bacteria group than in non-enteric bacteria group, which was five out of 36. The results emphasize the importance of controlled asepsis throughout the root canal treatment.
Sjögren U et al (1997)88 investigated the role of infection on the prognosis of endodontic therapy by following-up teeth that had had their canals cleaned and obturated during a single appointment. The root canals of 55 single-rooted teeth with apical periodontitis were thoroughly instrumented and irrigated with sodium hypochlorite solution. Using advanced anaerobic bacteriological techniques, post-instrumentation samples were taken and the teeth were then root-filled during the same appointment. All teeth were initially infected; after instrumentation low numbers of bacteria were detected
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in 22 of 55 root canals. Periapical healing was followed-up for 5 years.
Complete periapical healing occurred in 94% of cases that yielded a negative culture. Where the samples were positive prior to root filling, the success rate of treatment was just 68%--a statistically significant difference. Further investigation of three failures revealed the presence of Actinomyces species in each case; no other specific bacteria were implicated in failure cases. These findings emphasize the importance of completely eliminating bacteria from the root canal system before obturation. This objective cannot be reliably achieved in a one-visit treatment because it is not possible to eradicate all infection from the root canal without the support of an inter-appointment antimicrobial dressing.
Wright TL et al (1997)95 evaluated the effects of metronidazole on porphyromonas gingivalis biofilms. Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system. MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces. The treated biofilms were examined for growth and studied by scanning electron microscopy. A broth assay resulted in an MIC of 0.125 microgram/ml for metronidazole against P. gingivalis, P. gingivalis biofilms exhibited growth after treatment with 20 micrograms/ml metronidazole, which was 160 times the MIC for
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planktonic organisms. The results of this study indicated that biofilm- associated P. gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.
Das JR et al (1998)26 studied Changes in the biocide susceptibility of Staphylococcus epidermidis and Escherichia coli cells associated with rapid attachment to plastic surfaces. Differences in opacity between wells of a microtitre plate containing different volumes of inoculated growth medium reflected planktonic growth without any contribution from cells attached at the well surface. In this manner, minimum inhibitory concentrations (MICs) were determined at various stages of growth (0-20 h), both for cells growing attached to the bases of the plate wells and, simultaneously, for cells growing in suspension above them. Biocides included cetrimide, polyhexamethylene biguanide, peracetic acid, phenoxyethanol and chloroxylenol. Results, expressed as planktonic:biofilm MIC ratios, showed susceptibility to change, not only as a function ofattachment and biofilm formation, but also with respect to the nature of the chemical agent. In some instances, changes in susceptibility greater than twofold occurred immediately on attachment and could occur in the presence of biocide concentrations which exceeded the MIC.
Adams J.L. et al (1999)2 determined Impact of rpoS Deletion on Escherichia coli Biofilms. Slow growth has been hypothesized to be an
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essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (ΔlacZ rpoS+) and its isogenic ΔrpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h−1) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P< 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h−1. Overall, these studies suggest that rpoS is important for biofilm physiology.
Gomes BP et al (2001)34 assessed, in vitro, the effectiveness of several concentrations of NaOCl (0.5%, 1%, 2.5%, 4% and 5.25%) and two forms of chlorhexidine gluconate (gel and liquid) in three concentrations (0.2%, 1% and 2%) in the elimination of E. faecalis.A broth dilution test using 24-well cell culture plates was performed and the time taken for the irrigants to kill bacterial cells was recorded. Isolated 24 h colonies of pure cultures of E.
faecalis grown on 10% sheep blood plus Brain Heart Infusion (BHI) agar plates were suspended in sterile 0.85% NaCI solution. The cell suspension was
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adjusted spectrophotometrically to match the turbidity of a McFarland 0.5 scale. One mL of each tested substance was placed on the bottom of wells of 24-well cell culture plates (Corning, NY), including the control group (sterile saline). Six wells were used for each time period and irrigant concentration.
Two mL of the bacterial suspension were ultrasonically mixed for 10 s with the irrigants and placed in contact with them for 10, 30, and 45 s; 1, 3, 5, 10, 20, and 30 min; and 1 and 2 h. After each period of time, 1 mL from each well was transferred to tubes containing 2 mL of freshly prepared BHI + neutralizers in order to prevent a residual action of the irrigants. All tubes were incubated at 37 degrees C for 7 days. The tubes considered to have positive growth were those which presented medium turbidity during the incubation period. Data were analysed statistically by the Kruskal-Wallis test. with the level of significance set at P < 0.05. All irrigants were effective in killing E.
faecalis. but at different times. Chlorhexidine in the liquid form at all concentrations tested (0.2%, 1% and 2%) and NaOCI (5.25%) were the most effective irrigants. However, the time required by 0.2% chlorhexidine liquid and 2% chlorhexidine gel to promote negative cultures was only 30 s and 1 min, respectively. Even though all tested irrigants possessed antibacterial activity, the time required to eliminate E. faecalis depended on the concentration and type of irrigant used.
Lima KC et al (2001)58 evaluated the effectiveness of chlorhexidine- or antibiotics-based medications in eliminating E. faecalis biofilms. One-day
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and three-day biofilms of E. faecalis were induced on cellulose nitrate membrane filters. Each biofilm-containing membrane was thoroughly covered with 1 ml of the test medications and incubated for 1 day at 37 degrees C.
Treated biofilms were then aseptically transferred to vials containing a neutralizing agent in saline solution and vortexed. Suspensions were 10-fold diluted, seeded onto Mitis salivarius agar plates, and the colony-forming units counted after 48 h of incubation. There were significant differences between the formulations tested. The association of clindamycin with metronidazole significantly reduced the number of cells in 1-day biofilms. However of all medications tested, only 2% chlorhexidine-containing medications were able to thoroughly eliminate most of both 1-day and 3-day E. faecalis biofilms.
Luppens SB et al (2002)61 Developed a standard test to assess the resistance of Staphylococcus aureus biofilm cells .Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and
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hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Results showed that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.
Distel JW et al (2002)28 tested the hypothesis that Enterococcus faecalis resists common intracanal medications by forming biofilms. E.
faecalis colonization of 46 extracted, medicated roots was observed with scanning electron microscopy (SEM) and scanning confocal laser microscopy.
SEM detected colonization of root canals medicated with calcium hydroxide points and the positive control within 2 days. SEM detected biofilms in canals medicated with calcium hydroxide paste in an average of 77 days. Scanning confocal laser microscopy analysis of two calcium hydroxide paste medicated roots showed viable colonies forming in a root canal infected for 86 days, whereas in a canal infected for 160 days, a mushroom-shape typical of a biofilm was observed. These observations support potential E. faecalis biofilm formation in vivo in medicated root canals
Basrani Bet al (2002)12 assessed the substantive antimicrobial activity in chlorhexidine treated human root dentin. Canals of 98 roots were enlarged to standard size and medicated for 7 days with the following: (1) 2%
chlorhexidine (CHX) gel, (2) 0.2% CHX gel, (3) 2% CHX solution, (4)
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Ca(OH)(2), (5) Ca(OH)(2)+ 0.2% CHX gel, (6) 2% CHX solution + a 25%
CHX-containing controlled-release device, (7) saline, and (8) gel vehicle.
After medication, canals were inoculated with Enterococcus faecalis for 21 days. Dentin samples were collected with Gates-Glidden burs into brain heart infusion broth, and bacterial growth was assessed with spectrophotometric analysis of optical density after 72 hours of incubation.Mean optical densities were significantly lower for groups with 2% CHX (1, 3, and 6) when compared *with those of the controls (P < .05, analysis of variance with the Tukey test). Other groups did not differ significantly from the controls.Concluded Canal dressing for 1 week with 2% CHX may provide residual antimicrobial activity against E faecalis.
Portenier. I et al (2003)76 addressed Enterococcus faecalis– as the root canal survivor and ‘star’ in post-treatment disease. A recognized pathogen in post-treatment endodontic infections, Eradication of E. faecalis from the root canal with chemomechanical preparation and using disinfecting irrigants and antibacterial dressings is difficult. Summarized the different factors that make E. faecalis a potential problem in medicine and dentistry with special focus on the role of E. faecalis in post-treatment endodontic disease.
Naenni N et al (2004)69 assessed the necrotic tissue dissolution capacity of some popular and some potential root-canal irrigants: 1% (wt/vol) sodium hypochlorite (NaOCl), 10% chlorhexidine, 3% and 30% hydrogen
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peroxide, 10% peracetic acid, 5% dichloroisocyanurate (NaDCC), and 10%
citric acid. Standardized necrotic tissue samples obtained from pig palates were incubated in these solutions, and their weight loss was measured over time. None of the test solutions except sodium hypochlorite had any substantial tissue dissolution capacity. It was concluded that this might be important when considering the use of irrigants other than NaOCl.
Sedgley CM et al (2005)84 carried out tests to provide evidence for Survival of Enterococcus faecalis in root canals ex vivo.The hypotheses tested in this study were that: (i) Enterococcus faecalis can survive long-term entombment in root filled teeth without additional nutrients, (ii) initial cell density influences the survival of E. faecalis in instrumented root canals and (iii) gelatinase-production capacity influences the survival of E.faecalis in root canal. The root canals of 150 extracted single canal teeth were instrumented to apical size 60 and divided into six groups of 25. Within each group 10 canals were inoculated with either gelatinase-producing E.faecalis OG1-S and the other 10 with its gelatinase-defective mutant E. faecalis OG1-X. Five canals per group were kept as uninoculated controls. The root canals in groups 1 and 2 were inoculated with 10(6) bacteria, incubated for 48 h at 37 degrees C then filled with gutta-percha and zinc-oxide eugenol sealer. Root canals were inoculated with 10(6), 10(5), 10(4) and 10(3) bacteria in groups 3-6, respectively, and left unfilled. All teeth were sealed coronally with glass- ionomer cement. After 6- (groups 1, 3-6) and 12-month (group 2) incubation
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at 37 degrees C in 100% humidity, root fragments were analysed for presence of E. faecalis, using culture, polymerase chain reaction and histological methods.Viable E. faecalis was recovered from all root filled teeth and from 95-100% of unfilled inoculated teeth. Initial cell density and gelatinase production did not influence the recovery of viable E. faecalis (P > 0.05; chi- square test). Enterococcus faecalis 16S rRNA gene products were present in all inoculated teeth and absent in all noninoculated controls. Dentinal tubule infection was evident under light microscopy in sections from inoculated teeth after 48-h, 6- and 12-month incubation.nterococcus faecalis inoculated into root canals maintained viability for 12-months ex vivo. The clinical implications are that viable E. faecalis entombed at the time of root filling could provide a long-term nidus for subsequent infection
Zehnder M et al (2006)99 reviewed 0the specifics of the pulpal microenvironment and the resulting requirements for irrigating solutions are spelled out. Sodium hypochlorite solutions are recommended as the main irrigants.Chemical and toxicological concerns related to their use are discussed, including different approaches to enhance local efficacy without increasing the caustic potential. In addition, chelating solutions are recommended as adjunct irrigants to prevent the formation of a smear layer and/or remove it before filling the root canal system. Based on the actions and interactions of currently available solutions, a clinical irrigating regimen is
23
proposed. Furthermore, some technical aspects of irrigating the root canal system are discussed, and recent trends are critically inspected.
Yang SE et al (2006)97 determined the effects of a smear layer and chlorhexidine (CHX) treatment on the adhesion of Enterococcus faecalis to bovine dentin. Forty dentin blocks from bovine incisors were prepared and randomly divided into four groups of 10 each. The blocks in group 1 were placed in sterile saline for 5 minutes, while those in group 2 were treated with 17% EDTA for 5 minutes. The blocks in group 3 were placed in 2% CHX for 7 days. The blocks in group 4 were treated with 17% EDTA for 5 minutes, and then placed in 2% CHX for 7 days. All the blocks were immersed in a suspension of E. faecalis for 3 hours. The bacteria adhering to the dentin surface were counted by examination using a scanning electron microscope.
The most significant amount of bacteria was retained on the samples from group 1 (p < 0.05) and the smallest amount of bacteria adhered to the samples from group 4. These results suggested that a smear layer enhances the adherence of E. faecalis to the dentin, and CHX is effective in reducing the adherence of microorganisms.
Luciano Giardino et al (2007 )30 compared the antimicrobial efficacy of 5.25% NaOCl, BioPure MTAD (DentsplyTulsa Dental, Johnson City, TN), and Tetraclean (OgnaLaboratori Farmaceutici, Milano, Italy) against Enterococcus faecalis biofilm generated on cellulose nitrate membrane filters.
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After incubation, the membrane filters were transferred into tubes containing 5 mL of the selected antimicrobial solution test agent or NaCl 0.9%(positive control) and incubated for 5, 30, and 60minutes at 20°C. After each period of time, the test agents were vortexed for 60 seconds to resuspend the microorganisms. Ten-fold serial dilutions were generated in reduced transport fluid. Each dilution was plated onto a brain heart infusion plates. The plateswere then incubated for 48 hours in an aerobic atmosphere at 37°C and colony-forming units per membrane was calculated. Statistical analysis showed that only 5.25% NaOCl can disgregate and remove the biofilm at every time; however, treatment with Tetraclean caused a high degree of biofilm disgregation in every considered time intervals as compared with MTAD .
Lee Y et al (2008)56 assessed the antimicrobial efficacy of a novel polymeric chlorhexidine-controlled release device as an intracanal medicament. One hundred cylindrical dentin blocks prepared from human single-rooted teeth were inoculated with Enterococcus faecalis for 3 weeks.
The intracanal medicaments tested were calcium hydroxide, a polymeric chlorhexidine-controlled release device (PCRD), a polymeric controlled release device without chlorhexidine (CHx), 0.2% CHx solution, and sterile saline. Dentin samples (at 200-mum and 400-mum depths) were collected from the medicated canal lumens after 1 week of medication with sterile LightSpeed files and placed in growth medium. Bacterial growth was assessed
25
spectrophotometrically by analysis of optical density (OD) after 24 hours of incubation. The OD values at both depths were significantly lower in the PCRD group than in the other experimental groups (P < .001). These results indicate that a PCRD can be an effective intracanal medicament against E.
faecalis.
Chivatxaranukul et al (2008)23 investigated dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured ortho dentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2-2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 micro m(2)The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (ANOVA, P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P =
26
0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate-buffered saline (ANOVA, P < 0.001).Although E.
faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.
Zorko M et al (2008)100 provided evidence alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid and prevent cell activation by antibiotics.Many antibiotics used to treat infections cause release of immunostimulatory cell wall components from bacteria. Therefore, a combination of antimicrobial and endotoxin-neutralizing activity is desired to prevent inflammation induced by destroyed bacteria. Chlorhexidine and alexidine are amphipathic bisbiguanides and could neutralize bacterial membrane components as stimulators of Toll-like receptors (TLRs)Binding of chlorhexidine and alexidine to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) was determined by fluorescence displacement assay and isothermal calorimetric titration. Neutralization of the biological effect of LPS and LTA on TLR-activated cellular activation was determined by NF-kappaB reporter luciferase activation on cells transfected with specific TLRs and NO production of murine macrophages in the presence of isolated agonists and antibiotic-treated bacteria. Alexidine and chlorhexidine bind not only to LPS but also to LTA from Gram-positive bacteria. Alexidine has a higher affinity than chlorhexidine for both compounds. Calorimetric titration shows an initial
27
endothermic contribution indicating participation of hydrophobic interactions in LPS binding, while binding to LTA displayed initial exothermic contribution. Both compounds prevent cell activation of TLR4 and TLR2 by LPS and LTA, respectively. The addition of both compounds suppressed NO production by macrophages in the presence of bacteria treated with different types of antibiotics.Chlorhexidine and alexidine suppress bacterial membrane- induced cell activation at concentrations two orders of magnitude lower than that used in topical applications.
Lee JK et al (2009)55 determined Chlorhexidine gluconate attenuates the ability of lipoteichoic acid from Enterococcus faecalis to stimulate toll-like receptor 2. if CHX attenuates the inflammatory activity of Enterococcus faecalis and its major virulence factor, lipoteichoic acid (LTA). An enzyme- linked immunosorbent assay showed that CHX-killed E. faecalis was less potent than heat-killed E. faecalis in the production of tumor necrosis factor alpha (TNF-alpha) by a murine macrophage cell line, RAW 264.7 (p < 0.05).
Interestingly, pretreatment of LTA with 2% CHX for 6 hours or with 0.2%
CHX for 24 hours almost eliminated the TNF-alpha inducibility (p < 0.05).
Furthermore, CHX abrogated the ability of LTA to stimulate Toll-like receptor 2, resulting in the attenuated induction of TNF-alpha expression. Collectively, our results suggest that CHX can inactivate LTA of E. faecalis leading to the alleviation of inflammatory responses induced by E. faecalis and its LTA.
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Mohammadi Z et al (2009)65 disc discussed the properties and applications of chlorhexidine in endodontics.Microorganisms and their by- products are considered to be the major cause of pulp and periradicular pathosis. Hence, a major objective in root canal treatment is to disinfect the entire root canal system, which requires that all contents of the root canal system be eliminated as possible sources of infection. This goal may be accomplished using mechanical instrumentation and chemical irrigation, in conjunction with medication of the root canal system between treatment sessions. To reduce or eliminate bacteria, various irrigation solutions have been advocated. Chlorhexidine is a cationic molecule, which can be used during treatment. It has a wide range of antimicrobial activity. Its cationic structure provides a unique property named substantivity. The purpose of this paper is to review the structure and mechanism of action of CHX, its antibacterial and antifungal activity, its effect on biofilm, its substantivity (residual antibacterial activity), its tissue solvent ability, its interaction with calcium hydroxide and sodium hypochlorite, its anticollagenolytic activity, its effect on coronal and apical leakage of bacteria, its toxicity and allergenicity and the modulating effect of dentine and root canal components on its antimicrobial activity.
Arias-Moliz MT et al (2009)4 evaluated the minimal biofilm eradication concentration (MBEC) of sodium hypochlorite (NaOCl), chlorhexidine (CHX), EDTA, and citric and phosphoric acids after 1, 5, and
29
10 minutes of exposure to biofilms of Enterococcus faecalis. The biofilms grew in the MBEC high-throughput device for 24 hours at 37 degrees C and were exposed to 10 serial two-fold dilutions of each irrigating solution. The viable cell counts were log(10) transformed, and a concentration of an irrigant was considered to eradicate the biofilms when it produced a reduction of > or
= 5 logarithmic units. NaOCl was the most effective agent, capable of eradicating the biofilms after 1 minute at a concentration of 0.00625%. CHX eradicated biofilm after 5 minutes at 2%. EDTA and citric and phosphoric acid solutions were not effective against the biofilms at any concentration or time tested.
Rucucci D et al (2010)82 evaluated the prevalence of bacterial biofilms in untreated and treated root canals of teeth evincing apical periodontitis. The associations of biofilms with clinical conditions, radiographic size, and the histopathologic type of apical periodontitis were also investigated.The material comprised biopsy specimens from 106 (64 untreated and 42 treated) roots of teeth with apical periodontitis. Specimens were obtained by apical surgery or extraction and were processed for histopathologic and histobacteriologic techniques. Bacteria were found in all but one specimen.
Overall, intraradicular biofilm arrangements were observed in the apical segment of 77% of the root canals (untreated canals: 80%; treated canals:
74%). Biofilms were also seen covering the walls of ramifications and isthmuses. Bacterial biofilms were visualized in 62% and 82% of the root
30
canals of teeth with small and large radiographic lesions, respectively. All canals with very large lesions harbored intraradicular biofilms. Biofilms were significantly associated with epithelialized lesions (cysts and epithelialized granulomas or abscesses) (p < 0.001). The overall prevalence of biofilms in cysts, abscesses, and granulomas was 95%, 83%, and 69.5%, respectively. No correlation was found between biofilms and clinical symptoms or sinus tract presence (p > 0.05). Extraradicular biofilms were observed in only 6% of the cases.
Arias-Moliz MT et al (2010 )4 assessed the efficacy of cetrimide and chlorhexidine (CHX), alone and in association, in combined and alternating form, in eradicating biofilms of E. faecalis. Biofilms grown in the MBEC- high-throughput device for 24 hours were exposed to irrigating solutions for 30 seconds and 1 and 2 minutes. Eradication was defined as 100% kill of biofilm bacteria. The Student t test was used to compare the efficacy of the associations of the 2 irrigants. Cetrimide eradicated E. faecalis biofilms at concentrations of 0.5%, 0.0312%, and 0.0078% at 30 seconds and 1 and 2 minutes of contact time, respectively. CHX did not eradicate the biofilms at any of the concentrations (4% initial concentration) or times assayed. The association of 0.1% and 0.05% cetrimide with any concentration of CHX, whether in combined or alternating application, effectively eradicated E.
faecalis biofilms at all the contact times tested. Eradication was also achieved with 0.02% and 0.01% cetrimide at 2 minutes. Statistical analysis revealed
31
significantly better results with alternating rather than combined use of cetrimide and CHX (P < .05).The associated use of cetrimide and CHX provided better results than their applications as single agents against E.
faecalis biofilms, and the alternating application was significantly more effective than the combined mode of application.
Persoon IF et al (2011)75 explored the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains. Twenty-four-hour biofilms of E.
faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts.The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05).VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing.
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Kim HS et al (2012)49 reported the color change and formation of precipitates containing para-chloroaniline (PCA) after a reaction of sodium hypochlorite (NaOCl) and chlorhexidine (CHX). Alexidine (ALX), a biguanide disinfectant similar to CHX, has greater affinity for bacterial virulence factors than CHX. This study determined by electrospray ionization mass spectrometry (ESI-MS) and scanning electron microscopy (SEM) whether the chemical interaction between ALX and NaOCl results in PCA or precipitates. ESI-MS was performed on 4 different concentrations of ALX (1%, 0.5%, 0.25%, and 0.125%) with 4% NaOCl to detect the presence of PCA. As control groups, 1% ALX, 0.5% PCA, and a mixture of 2% CHX and 4% NaOCl were analyzed. The formation of precipitates on the dentinal surfaces of premolar root canals treated with the solutions of ALX and NaOCl (AN) or CHX and NaOCl (CN) was observed by SEM and the color change in the reaction solutions was also analyzed. ESI-MS showed that the peak (mass/charge ratio = 128.026) in the PCA spectrum was not detected in any of the 4 AN solutions, whereas the peak was found in the CN solution. SEM revealed precipitates covering dentinal surfaces in the CN solution. The AN solutions produced no precipitate. The AN solutions changed in color from light yellow to transparent with decreasing ALX concentration, whereas peach-brown discoloration was observed The interaction of ALX and NaOCl did not produce PCA or precipitates, and the color of the reacted solution changed transparent with decreasing ALX concentration.
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Baca P et al (2012)7 evaluated the antimicrobial substantivity against Enterococcus faecalis of a dentin-volumetric unit exposed for 1 minute to chlorhexidine (CHX) and cetrimide (CTR).Standardized coronal dentin blocks of human molars, with and without collagen, were treated for 1 minute with
0.2% and 2% CHX and 0.2% CTR. Afterwards, they were exposed to E. faecalis suspension to determine the antimicrobial substantivity over a
period of 60 days. Results were analyzed by means of Kaplan-Meier survival analysis (P < .05).A direct relationship was seen between CHX concentration and survival time, and the most statistically significant results were obtained in specimens with collagen. CTR showed intermediate survival values close to those of 2% CHX. The present study shows that 2% CHX used for 1 minute provides the longest substantivity followed by 0.2% CTR when applied to a dentin-volumetric model.
Materials and Methods
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MATERIALS
Alexidine Dihydrochloride - A8986 - Sigma Aldrich U.S,A – Fig 1
Chlorhexidine – Hi Media,Bombay India – Fig 2
Sodium hypochlorite – 5% Chenchems , Chennai, India – Fig 3
E.faecalis (ATCC 29212)
Brain Heart Infusion Broth (BHI) – Fig 4
Blood Agar .
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ARMAMENTARIUM
Incubator – Fig 5
Hot air oven - Fig 6
Autoclave – Fig 7
Centrifuge- Table top – Fig 8
Microcentrifuge- high speed – Fig 9
Freezers - Fig 10
Laminar flow chamber - Fig 11
Nikon Microscope – Fig 12
Culture dishes
Microtitre plates- 96 well - Fig 13
Micro pipettes-m1-20, 20-200, 100-1000 microliter - Fig 14
Micropipette tips- yellow , blue, micro tips – Fig 15
Spectrophotometer- semi auto analyser – Fig 16
pH meter – Fig 17
Electronic balance – Fig 18
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PREPARATION & CALCULATION OF CONCENTRATION OF 1% SOLUTION
A Concentration of 1% solution of Alexidine was prepared by dissolving 1mg of alexidine powder in 100 microlitre of diluent. Similarly 1%
Chlorhexidine was prepared by dissolving 1mg of chlorhexidine powder in 100 microlitre of diluent.
1% solution = 1 gm in 100 ml 1000 mg in 100 ml 10mg in 1ml
10mg in 1000 microlitre 1mg in 100 microlitre = 1%
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METHODOLOGY
MIC AND MBC DETERMINATION OF ALEXIDINE,
CHLORHEXIDINE AND NaOCl AGAINST E.FAECALIS.
A concentration of 1% {1mg in 100 microlitre} solution of alexidine was prepared. It was serially diluted to get 0,00095% equivalent to 9.7 microgram per ml,likewise chlorhexidine was also prepared. Similarly, NaOCl (5%) was diluted in BHI broth to the potency of 500,250,125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, 0.97, and 0.48 micro grams/ml. Into the U-bottomed microtitre wells 100 µl of different agents were taken. To these dilutions, equal quantity of E.faecalis suspension (0.5 Mcfarland opacity) was added and incubated at 37⁰C for 24 hours. Growth was observed in these dilution and compared with the control which received no agent. Last well showing no growth as indicated by no turbidity or button formation at the bottom of the well was taken as Minimum Inhibitory concentration (MIC) .Subcultures were made from these dilutions on to Blood agar plates and the plates were incubated at 37⁰C for 24 to 48 hours. Growth or no growth against the dilutions was noted. Last dilution showing no growth was taken as Minimum Bactericidal Concentration (MBC).The MIC and MBC values of the irrigants were found and results obtained were tabulated. From MIC other concentrations of antimicrobial agent (ie) 2 times the MIC was determined for performing the Time Kill Assay.
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DILUTION OF THE IRRIGANT AND THE CONCENTRATION
The preparation protocol for Alexidine and chlorhexidine for MIC/MBC is shown in the above fig .
TIME KILL ASSAY:
Principle:
Rate at which concentration of an antimicrobial agent kills a bacterial isolate.
Concentration and time at which it kills.
Standardised inoculums was added to broth with various concentrations of root canal irrigants and control. The number of viable colony forming units CFU is determined at initial time of inoculation and various time interval of 30seconds,60 and 90 seconds thereafter by serial dilution bacterial colony counts on a sample from each test vial. Results for control and each reagent concentration are plotted versus time. Generally a 3-log 10 cfu/ml decrease in bacterial counts in anti microbial solution compared with counts for the
39
growth control indicates an adequate bactericidal response .Prior to performing time kill assay the MIC of the specific anti microbial agent for E.faecalis by a standard MIC procedure preferably by a broth macro dilution method was determined. From MIC other concentrations of antimicrobial agent (i.e) 2 times the MIC was determined.
TIME KILL ASSAY: ALEXIDINE 2MIC
A 1%{1mg in 100 microlitre } solution of alexidine was prepared and was labelled as stock1. From this equal volumes of stock 1 and diluent (BHI broth) were mixed and it was labelled as stock2. To 10 microliter of stock 2, 117 mic.lit of broth was added to get 39 microgram/ml [working concentrations to get 2MIC] of the agent . From this 20 µl was taken in an ependroff tube and mixed with 20 µl of E.faecalis culture (0.5 Macfarland standard) and incubated at various lengths of time viz. 30, 60, 90, and 120 seconds. After this a viable count was performed to get the colony forming units (CFU) so that the reduction of CFU could be detected. It was done by a serial 10 fold dilutions prepared (from 10-1,10-2,10-3,10-4 and 10-5 etc). From each dilutions 5ul was placed on the surface of BHI agar plates and after incubation, the number of colonies counted after 24-48 hours and multiplied with the dilution factor. Control received no irrigant.
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Time Kill Assay: Preparation Protocol For Alexidine is shown in above fig.
TIME KILL ASSAY: CHLORHEXIDINE 2MIC
A 1% solution of Chlorhexidine {1mg in 100 microlitre } was prepared and was labelled as stock 1.From this 10 micolitre was mixed with 10mic.lit of diluents (BHI broth) so this 20 mic,lit contains 5000micro gm labelled as stock 2.To 10 micro.lit of stock 2 taken 5 micro lit of diluent was added to get stock 3 which contains 166microgm/1microlitre. To 10microlit of stock3;11micro. lit of diluent was added to get 21 micro.lit which contains 1660 micro gram in 79 microgm/ml [working concentration to get 2MIC)
41
labelled as stock 4. For the test , 1 micolitre of stock 4 was taken to that 20 micolitre culture was added and incubated at various lengths of time viz. 30, 60, 90, and 120 seconds. . After this a viable count was performed to get the colony forming units (CFU) so that the reduction of CFU could be detected. It was done by a serial 10 fold dilutions prepared (from 10-1,10-2,10-3,10-4 and 10-5 etc). From each dilutions 5ul was placed on the surface of BHI agar plates and after incubation, the number of colonies counted after 24-48 hours and multiplied with the dilution factor. Controls received no agent.
Time Kill Assay: Preparation Protocol For CHX is shown in above fig.
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TIME KILL ASSAY:
Sodium hypochlorite 2MIC
MIC of Sodium hypochlorite determined was more than 1% so 2.5% was tested. To 2.5ml of 5.5% NaOCl 3ml of diluent was added to get 2.5%. From this 20 µl was taken in an ependroff tube and mixed with 20 µl of E.faecalis culture (0.5 Macfarland standard) and incubated at various lengths of time viz.
30, 60, 90, and 120 seconds. At the end of this time, serial 10 fold dilutions were made and 5 µl placed on a BHI agar plate and the number of colonies counted after 24-48 hours.
Time Kill Assay: Preparation Protocol For NaOCl is shown in above fig.
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Results of Time Kill Assay of irrigants were tabulated and compared with each other and control. Graph is drawn with results for control and each irrigant concentrations were plotted versus time. A 3-log10 CFU/ml decrease in bacterial counts in irrigants compared with counts for the growth control indicates an adequate bactericidal response.
On further testing with increasing concentration, NaOCl showed MIC and MBC at 1.25% concentration.
prepared prepared
Serially diluted in BHI broth to get 0.00095% equivalent to 9.7 microgram per ml..ie diluted in broth to a potency of
5000,2500,1250,625,312.5,156.2,78.1,39,19.5 and 9.7 µgm/ml [microgram/ml]
Diluted in BHI broth to the potency of
500,250,125,62.5,31.25,15.6,7 7.8,3.9,1.9,.97 and 0.48 µgm/ml [microgram/ml.]
Into the U - bottomed microtitre wells 100 microlitre of these agents taken
To these dilutions equal quantity of E faecalis suspension (0.5 McFarland opacity )was added and incubated at 37 degrees C for 24 hours.
Last well showing no growth indicating no turbidity or button formation at bottom of the well taken as MIC[Minimum Inhibitory Concentration]
Subcultures made from these dilutions and on to the Blood agar plate..
Plates incubated at 37 ° C for 24 to 48 hours.
Growth or no growth against the dilutions noted .
Last dilution showing no growth was taken as MBC (minimum bactericidal concentration )
MIC and MBC values for the respective irrigants was noted and results were tabulated.
Broth with various concentration of Alexidine. (2MIC)
Broth + various concentrations of Chlorhexidine. Broth with
various
concentrations of NaOCI 2MIC
Time Interval 5,30,60,90 and 120 seconds
The number of viable colony forming units (cfu)determined . Count done at initial time of inoculation and various time interval thereafter by serial dilution bacterial colony counts on a sample from each test vial
Reduction in CFU noted
Results for control and each irrigant concentration were plotted versus time.
Control without any agent Broth with various
concentrations of Chlorhexidine 2MIC
3-log 10 cfu/ml decrease in bacterial counts in antimicrobial solution compared with counts for growth control indicates adequate bactericidal response
