i

Comparative Efficacy Of Endodontic

Medicaments Against Enterococcus Faecalis

Biofilms

A thesis submitted to the University of Adelaide in partial fulfilment of the

requirements for the Degree of Doctor of Clinical Dentistry (Endodontics)

December 2009

Barbara Plutzer BDS (Adel), FRACDS

School of Dentistry

University of Adelaide

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Contents

Contents ............................................................................................................................ ii

Abstract ............................................................................................................................ iv

Declaration ....................................................................................................................... vi

Acknowledgements ......................................................................................................... vii

1. Literature Review ....................................................................................................... 1

1.1 Introduction ........................................................................................................ 1

1.2 Microbiological goals of Endodontic Treatment ............................................... 1

1.3 Endodontic success and failure .......................................................................... 2

1.4 Enterococci ......................................................................................................... 3

1.5 Enterococci in the normal flora of the oral cavity .............................................. 4

1.5.1 Root canal sampling .................................................................................... 6

1.5.2 Enterococci in primary apical periodontitis ................................................ 7

1.5.3 Enterococci in the root canal after initiation of treatment .......................... 9

1.5.4 Enterococci in post-treatment apical periodontitis ................................... 10

1.5.5 Virulence Factors ...................................................................................... 16

1.6 The viable but non-culturable (VBNC) state ................................................... 20

1.7 The ‘biofilm’ concept ....................................................................................... 23

1.7.1 Biofilm formation as a survival strategy ................................................... 24

1.7.2 Evidence for biofilm structures in Endodontics ....................................... 25

1.7.3 Antimicrobial resistance of Biofilms ........................................................ 27

1.8 Antimicrobial agents ........................................................................................ 30

1.8.1 Sodium Hypochlorite ................................................................................ 30

1.8.2 Calcium Hydroxide ................................................................................... 35

1.8.3 Chlorhexidine ............................................................................................ 40

1.8.4 Antibiotics ................................................................................................. 47

1.9 Susceptibility of biofilms to antimicrobials ..................................................... 56

1.10 Rationale for experiment .................................................................................. 62

1.11 Aims ................................................................................................................. 66

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2. Materials and Methods ......................................................................................... 67

2.1 Organism .......................................................................................................... 67

2.2 Agar diffusion test ............................................................................................ 67

2.3 Dentine sample preparation .............................................................................. 67

2.4 Biofilm growth ................................................................................................. 68

2.5 Scanning electron microscopy (SEM) ............................................................. 70

2.6 Test Agents Used ............................................................................................. 71

2.7 Determining Antimicrobial Efficacy ................................................................ 72

2.7.1 Biofilm Harvesting ................................................................................... 72

2.7.2 Cellular viability and protein measurement .............................................. 73

3. Results ..................................................................................................................... 74

3.1 Biofilm Growth ................................................................................................ 74

3.2 Dentine surface prior to inoculation ................................................................. 75

3.3 Effective removal of bacterial biofilm ............................................................. 76

3.4 Sodium Hypochlorite ....................................................................................... 76

3.5 Crushed Samples .............................................................................................. 79

3.6 Ledermix and Odontopaste .............................................................................. 80

3.7 Calcium Hydroxide and 50:50 combinations of Calcium Hydroxide with either Ledermix or Odontopaste ............................................................................................ 83

3.8 Chlorhexidine gel ............................................................................................. 87

4. Discussion ............................................................................................................... 89

4.1 Antibiotic-containing medicaments ................................................................. 89

4.2 Calcium Hydroxide .......................................................................................... 91

4.3 Chlorhexidine gel ............................................................................................. 94

4.4 Sodium Hypochlorite ....................................................................................... 96

4.5 Limitations and future research direction ........................................................ 97

5. Conclusions ............................................................................................................ 99

6. References ............................................................................................................ 100

7. Appendix .............................................................................................................. 113

iv

Abstract

It is well established that bacteria cause pulpal and periradicular disease (Kakehashi et

al. 1965). Of the bacteria recovered from failing root canals, Enterococcus faecalis is

one of the most prevalent species (Molander et al. 1998; Sundqvist et al. 1998). Many

laboratory studies have investigated the effectiveness of root canal irrigants and

medicaments against E. faecalis. Most used planktonic cultures, which are not

representative of the in vivo growth conditions of an infected root canal system, where

bacteria grow as a biofilm adhering to the dentinal wall (Nair 1987). Organisation of

bacteria within biofilms confers a range of phenotypic properties that are not evident in

their planktonic counterparts, including a markedly reduced susceptibility to

antimicrobial killing (Wilson 1996).

Objectives: The aims of this study were: 1) To compare the efficacy of commonly used

endodontic medicaments against E. faecalis cultured as a biofilm. The medicaments

tested were Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine gel

and 50:50 combinations of Ledermix/calcium hydroxide and Odontopaste/calcium

hydroxide. 2) To compare the antimicrobial effect achieved through exposure to

endodontic medicaments with that achieved by exposure to a constant concentration of

sodium hypochlorite for varying times.

Methods: A biofilm was established using a continuous flow cell. E. faecalis inoculum

was introduced into the flow cell and allowed to establish on human dentine slices over

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4 weeks. Each test medicament was introduced into the flow cell for a period of 24 or

48 hours, while sodium hypochlorite was evaluated after 1, 10, 30 and 60 minutes.

Biofilms were harvested by sonication in sterile PBS. Cellular protein levels were

measured to quantitate the amount of biofilm harvested.

Cellular viability was determined using serial plating. The number of colony forming

units was then adjusted for cellular protein levels to allow treatment protocols to be

compared. Qualitative SEM analyses of the biofilm was performed following exposure

to each test agent.

Results: Sodium hypochlorite was the only agent that achieved total bacterial

elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis

biofilm, while calcium hydroxide and 50:50 combinations of calcium hydroxide with

either Ledermix or Odontopaste were able to reduce viability by > 99%.

Conclusion: When used in isolation, antibiotic containing medicaments had no

appreciable effect on the viability of Enterococcus faecalis. Sodium hypochlorite

remains the gold standard for bacterial elimination in root canal therapy.

vi

Declaration

This work contains no material which has been accepted for the award of any other

degree or diploma in any university or other tertiary institution, and, to the best of my

knowledge and belief, contains no material previously published or written by another

person, except where due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library,

being made available for loan and photocopying, subject to the provisions of the

Copyright Act 1968.

I also give permission for the digital version of my thesis to be made available on the

web, via the University’s digital research repository, the Library catalogue, the

Australaisan Digital Theses Program (ADTP) and also through the web search engines,

unless permission has been granted by the University to restrict access for a period of

time.

Signed by: ___________________

Barbara Plutzer

vii

Acknowledgements

I would like to extend my greatest thanks to my research supervisor, Associate

Professor Peter Cathro, for his vision, wisdom and support. I am forever grateful he

made the long journey from New Zealand. His approachability, optimism and fortitude

have been invaluable.

Many thanks to my research supervisor, Dr Peter Zilm, for his dedication and guidance.

His immense research experience and skill have been priceless. I appreciate the many

hours he has spent on my behalf and his thorough editing of my attempts at scientific

writing.

I am grateful to Professor Geoffrey Heithersay, who has been a source of inspiration

and encouragement throughout my post-graduate course. His wisdom, generosity and

friendship will not be forgotten.

I would like to recognise for their invaluable guidance Dr Tracy Fitzsimmons and Dr

Neville Gully. Thank you for your practical support and insight.

Thank you to the staff at the Adelaide microscopy centre for their expertise and

assistance.

viii

The support and friendship of my fellow D. Clin Dent candidates, Dr Aaron Seet and

Dr Mark Stenhouse made my time at the hospital a pleasure. I look forward to fulfilling

our promise of a Scandinavian sojourn.

Thank you to my mum, Kamila, for her unconditional love and moral support.

To my partner Andre, thank you for your support, love, and patience. Thank you for

putting my degree into perspective when it seemed all-consuming.

Finally, I would like to acknowledge that this research was financially supported by the

Australian Dental Research Foundation and the Australian Society of Endodontology.

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1. Literature Review

1.1 Introduction

In 1894 W.D. Miller described with fascination the micro-organisms seen in and

recovered from the infected root canal. He was perhaps the first to associate disease and

inflammation in the jaws with the infected pulp canal space (Miller 1894). Since that

time, the microbiological basis of both endodontic disease and of endodontic treatment

failure has become firmly established (Kakehashi et al. 1965; Sjögren 1997). Bacterial

penetration of the pulp is often asymptomatic but may lead to pain and discomfort, and

it is the sequel of infection that inspires greater concern and provides the strongest

impetus for intervention. Whereas the common, chronic forms of apical periodontitis

seldom pose medical problems of any magnitude, it is the smaller number of cases that

spread to cause inter-fascial infections and possibly serious complications at distant

sites that form a back-drop on which the concepts and rationales for treatment are based

(Ørstavik 2003).

1.2 Microbiological goals of Endodontic Treatment

Apical periodontitis is described as an infectious disease caused by microorganisms

colonizing the root canal system (Kakehashi et al. 1965; Sundqvist 1976; Möller et al.

1981). The endodontic treatment of teeth with irreversibly inflamed pulps is essentially

a prophylactic treatment from a microbiological perspective, because the radicular vital

pulp is usually free of infection; and the rationale is to prevent further infection of the

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root canal system and the emergence of apical periodontitis (Haapasalo 2005). In cases

of infected necrotic pulps or in root canal-treated teeth associated with apical

periodontitis of infective origin, an intraradicular infection is already established, and

the goal of endodontic procedures becomes the elimination of the infecting

microorganisms. Ideally, endodontic treatment procedures should sterilize the root

canal, but given the complex anatomy of the root canal system, it is widely recognized

that, with available instruments and techniques, fulfilling this goal is utopic for most

cases. The reachable goal is to reduce bacterial populations to a level below that

necessary to induce or sustain disease (Siqueira and Rôças 2008).

1.3 Endodontic success and failure

When endodontic treatment is performed under aseptic conditions and according to

accepted clinical principles, the success rate is generally high. Most follow up studies

on endodontic therapy report overall success rates of 85%-90% (Strindberg 1956;

Seltzer et al. 1963; Kerekes and Tronstad 1979; Molven and Halse 1988; Sjögren et al.

1990). Epidemiological studies, however, report success rates of only 60-75%,

identifying a surprisingly high prevalence of apical periodontitis associated with root

filled teeth (Eriksen 2002).

Although many failure cases are caused by technical problems during treatment, some

cases fail even when apparently well treated (Sundqvist et al. 1998). Studies

investigating the aetiology of endodontic treatment failures in cases where the initial

therapy was performed to a satisfactory standard have identified five factors that may

3

contribute to a persistent periapical radiolucency following treatment. The factors are:

intraradicular infection (Nair et al. 1990), extraradicular infection especially by bacteria

of the species Actinomyces israelii and Priopionibacterium propionicum (Nair and

Schroeder 1984; Sjögren et al. 1988) foreign body reactions (Yusuf 1982; Nair et al.

1990); cysts, especially when containing cholesterol crystals (Nair et al. 1993) and

fibrous scar tissue healing following conventional treatment (Nair et al. 1999). In most

cases, failure of endodontic treatment is believed to be due to microorganisms persisting

in the apical parts of the root canal system, even in seemingly well-treated teeth (Nair et

al. 1990).

1.4 Enterococci

Enterococci are gram-positive cocci that occur singly, in pairs, or as short chains. They

are facultative anaerobes, possessing the ability to grow in the presence or absence of

oxygen (Gilmore 2002). Enterococcus species live in vast quantities in the human

intestinal lumen and under most circumstances cause no harm to their host. They are

also present in human female genital tracts and the oral cavity in lesser numbers (Koch

et al. 2004). One of the characteristic features of enterococci is their ability to survive

very harsh environments including extreme pH levels (4.0-11.0) (Nakajo et al. 2006)

and salt concentrations. They resist bile salts, detergents, heavy metals, ethanol, azide

and desiccation (Gilmore 2002). They can grow in the range of 10oC to 45oC and

survive a temperature of 60oC for 30 minutes (Hartke et al. 1998).

4

Over the last few decades, enterococci have emerged as important nosocomial

pathogens. This importance is attributed primarily to the high degree of antibiotic

resistance that is exhibited by most enterococci. Of particular concern has been the

rapid spread of enterococci with resistance to vancomycin (VRE), creating therapeutic

problems for patients with serious infections such as endocarditis (Gilmore 2002).

Enterococcus faecalis is responsible for 80% of all infections caused by enterococci,

with E. faecium responsible for the remaining 20% (Ruoff et al. 1990).

1.5 Enterococci in the normal flora of the oral cavity

Enterococci form part of the normal gastrointestinal tract flora in animals and humans,

though only a few studies have focused on their prevalence in the oral cavity. In an

early report, Williams et al. collected saliva samples from 206 individuals and analysed

them for enterococci. Saliva was collected more than once from most donors. Overall,

enterococci were detected in the saliva of 21.8% of subjects on at least one occasion.

However, the carriage was not consistent, meaning that on one experimental day an

individual tested positive, while testing negative on a subsequent day (Williams et al.

1950). More recently, Sedgley et al. investigated the prevalence, phenotype and

genotype of oral enterococci. The study used microbiological culturing methods to

detect enterococcal isolates in the oral rinse samples of 11% of 100 patients receiving

endodontic treatment and 1% of 100 dental students with no history of endodontic

treatment. All enterococcal isolates were identified as E. faecalis (Sedgley et al. 2004).

In a follow-up study using quantitative real-time polymerase chain reaction (PCR) as

well as microbiological culturing, 30 oral rinse samples from endodontic patients were

analysed for E. faecalis. Quantitative PCR detected E. faecalis in 17% of the samples,

5

while parallel culture assays were less sensitive, detecting the microorganism in only

7% of samples. Proportionally, oral E. faecalis comprised less than 0.005% of the total

bacterial load (Sedgley et al. 2005).

A significantly higher prevalence of oral enterococci was detected in a recent study

which used a multi-site oral sampling strategy in combination with sensitive detection

methods incorporating both culture and PCR. E. faecalis was detected in at least one

tongue, oral rinse, or gingival sulcus sample in 68% of patients and in the root canals of

5% of patients. The tongue was found to be the most prevalent detection site (Sedgley

et al. 2006). In other studies oral E. faecalis was detected at a higher prevalence in

periodontitis patients compared to controls (Sedgley et al. 2006; Souto and Colombo

2008). Evidence shows that E. faecalis is clearly a part of the human oral microbiota. In

healthy individuals not being treated with wide-spectrum antibiotics, the relative

amount of enterococci is very small, often below the detection levels of normal

sampling and culturing methods (Portenier 2003). Recent publications, however,

confirm former speculations that the occurrence of E. faecalis in the oral cavity is

higher than previously suggested.

A thought provoking recent review contends that enterococci are not part of the typical

commensal microbiota of the oral cavity, but rather a transient infective microorganism

sourced from food. Enterococci are ubiquitous in food products, such as cheese,

fermented sausages, minced beef, pork, and fish (Zehnder and Guggenheim 2009).

Razavi et al. investigated the clearance of E. faecalis from a highly contaminated

cheese among healthy volunteers. One minute after ingestion, a median of 5480 colony

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forming units were recovered. Bacterial counts were significantly reduced after 100

minutes, and below the limit of detection after 1 week (Razavi et al. 2007). These

findings may suggest that enterococci adhere to oral tissues of healthy subjects but fail

to become permanently established within the oral microbiota and are thus gradually

eliminated. This interpretation correlates well with Williams’ early findings of sporadic

sampling of E. faecalis in the saliva of the same individuals.

1.5.1 Root canal sampling

Absorbent paper points are typically used to sample the contents of the root canal for

bacterial culturing. (Möller 1966). Paper point sampling is clinically convenient.

However, establishing whether the microorganisms sampled are representative of those

involved in the infectious process is generally difficult. The technique also has other

limitations. A sample may show the presence of microorganisms although the root

canal is sterile – a false positive sample – or no microorganisms may be revealed

although the root canal system is infected – a false negative sample. False positive

samples are primarily caused by contamination from the oral cavity, a problem of

particular significance when PCR technology is used. The risk of obtaining a false

negative sample is equally as great. The root canal system is often a complex

arrangement of main, lateral and accessory canals, as well as ramifications, isthmuses

and cul de sacs (Vertucci 1984). These anatomical niches provide a protective

environment for bacterial survival, and they are precisely the regions that are difficult to

reach when taking a microbiological sample. False negative samples can also be due to

limitations in the cultivation methods available, combined with the fastidious nature of

the organisms. In fact, of the 620 predominant oral bacterial species, about 35% have

not yet been cultured in vitro (Paster & Dewhirst 2009).

7

Despite these inherent limitations, serial sampling of root canals at various stages of

root canal therapy showcases the changing microbiological environment, and provides a

means of evaluating the effectiveness of various stages of therapy. Various studies have

contributed to our understanding of the endodontic infection in untreated infected root

canals (Sundqvist 1976; Fabricius et al. 1982; Munson et al. 2002), in root canals where

therapy has been initiated (Siren et al. 1997), and in teeth with post treatment disease

(Möller 1966; Molander et al. 1998; Sundqvist et al. 1998; Hancock et al. 2001).

1.5.2 Enterococci in primary apical periodontitis

Although more than 700 species of bacteria have been isolated from the oral cavity,

only a limited number have been consistently isolated from endodontic infections

(Sundqvist 1994). The selective pressures operating in the root canal environment,

including a low redox potential and nutrients rich in peptides and low in carbohydrates,

favour the strong dominance of strictly anaerobic bacteria typical of primary apical

periodontitis. Together with some facultative anaerobic bacteria such as streptococci,

lactobacilli and Actinomyces, they can establish a wide variety of bacterial

combinations. Usually three to six species can be isolated from a single tooth using

conventional culturing techniques (Sundqvist 1976; Fabricius et al. 1982). Modern

molecular methods are able to detect a significantly higher number and greater diversity

of species, including numerous as-yet uncultivable organisms (Munson et al. 2002).

Munson et al. recovered a mean number of 20 bacterial taxa from each infected root

canal sample using combined culture and molecular methods (Munson et al. 2002).

8

Enterococcal species have traditionally been regarded as atypical isolates in primary

endodontic infections. Culturing methods of detection have isolated enterococci in 5-

12% of sampled root canals (Engström 1964; Möller 1966; Sundqvist et al. 1989).

More recently, Siqueira et al. used molecular techniques to examine the prevalence of

Actinomyces spp., streptococci and E. faecalis in primary root canal infections.

Samples were obtained from 53 infected teeth, of which 27 had acute periradicular

abscesses. The checkerboard DNA-DNA hybridization assay detected streptococci in

22.6% of the samples, actinomyces spp. in 9.4%, and E. faecalis in 7.5%. The

occurrence of E. faecalis was significantly lower in acute infections (3.7%) than in

asymptomatic teeth (11.5%) (Siqueira et al. 2002). Rôças et al. similarly concluded that

E. faecalis was more frequently detected in asymptomatic cases. Using the nested PCR

method, E. faecalis was detected in 33% of root canals associated with asymptomatic

chronic periradicular lesions, in 10% of root canals associated with acute apical

periodontitis, and in 5% of the pus samples aspirated from acute periradicular abscesses

(Rôças et al. 2004).

The proportions of E. faecalis as part of the total microbial load were studied by

examining samples from both primary infection and retreatment cases. The samples

were divided into two equal aliquots that were independently analysed by investigators

using culturing and real-time PCR methods. E. faecalis was detected in a surprisingly

high 67.5% of 40 primary infection samples and 89.6% of the 48 retreatment samples.

The high prevalence was attributed by the authors to the sensitivity of the molecular

method used and the patient selection which was based on referrals of teeth with

suspected persistent infection. The median proportion of E. faecalis in the samples was

2.8% of the total bacteria in the primary infection samples and 0.98% in the retreatment

9

cases (Sedgley et al. 2006). It is obvious that while the prevalence of E. faecalis is high,

particularly in retreatment cases, proportionally this microorganism makes up only a

very small part of the total bacterial flora. This may imply that other organisms present

did not reach detection levels, or had more fastidious growth requirements that could

not be mimicked in the laboratory environment.

1.5.3 Enterococci in the root canal after initiation of treatment

Reports of culture reversals in longitudinal studies, characterized by the sudden positive

sampling of enterococci after initiation of treatment, have led to speculations of coronal

leakage through the temporary restoration (Sjögren et al. 1991; Sundqvist et al. 1998).

Siren et al., studied the correlation between several clinical parameters and the

occurrence of enterococci in teeth where treatment did not result in healing. The clinical

treatment history of 40 Enterococcus-positive and 40 Enterococcus-negative teeth was

compared. A higher proportion of E. faecalis was found in teeth whose canals lacked an

adequate seal for a period during the treatment or teeth that were treated over 10 or

more visits (Siren et al. 1997). Compromised asepsis during endodontic treatment thus

appears to be an important causative factor for contamination of the root canal by E.

faecalis.

The possibility of E. faecalis from food products causing a transient oral infection of

sufficient magnitude to infect the root canal was recently tested by Kampfer et al. An

artificial oral environment was established using a masticator device perfused with

artificial saliva in which test teeth, temporarily restored with Cavit, were exposed to

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controlled loads of mastication over a period of 1 week. A cheese containing viable E.

faecalis cells was placed between the occlusal surfaces of the test teeth. Of the 16

specimens restored with 2mm of Cavit, 6 showed leakage, while 1 of the 16 specimens

with a 4mm cavit restoration had cultivable E. faecalis cells detected. These results go

some way to giving credence to the suspicion that food derived microbiota could enter

the root canal system via microleakage (Kampfer et al. 2007).

1.5.4 Enterococci in post-treatment apical periodontitis

The microbial flora of root-filled canals with persisting periapical lesions differs

markedly from that of untreated necrotic dental pulps, with a very limited assortment of

microorganisms able to survive. Generally, only one or a small number of species are

recovered, with a predominance of gram-positive microorganisms and facultative

anaerobes (Möller 1966; Molander et al. 1998; Sundqvist et al. 1998; Hancock et al.

2001). E. faecalis is the most commonly recovered species in these teeth (Siqueira and

Rôças 2004; Sundqvist et al. 1998; Molander et al. 1998; Pinheiro et al. 2003; Gomes

et al. 2008), with the likelihood of detection in failing root canals 9 times higher than in

primary endodontic infections (Rôças et al. 2004).

As far back as 1964, Engström examined 223 teeth for the presence of enterococci. In

teeth undergoing primary endodontic treatment, enterococci were isolated in 12% of

culture positive cases, while for previously root-filled teeth, the recovery rate was 21%

(Engström 1964).

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Sundqvist retreated 54 teeth with post-treatment disease. Microbial growth was

recovered in 24 of the canals (45%) and E. faecalis was the most frequent isolate,

detected in 38% of culture-positive canals. On each occasion, E. faecalis was isolated in

pure culture. The success rate for the teeth from which E. faecalis was isolated after

removal of the earlier root filling was somewhat lower (66%) than the overall average

(74%) (Sundqvist et al. 1998).

Molander examined the microbiological status of 100 root-filled teeth with apical

periodontitis. The presence of intracanal microbiota was demonstrated in 68% of teeth,

most of which contained one or two strains of microorganisms. E. faecalis was the most

frequently isolated species, recovered in 32 teeth, or 47% of the culture-positive root

canals. A further 20 teeth without signs of periapical pathosis were similarly cultured

for microbial presence. Mostly sparse growth of intracanal bacteria was detected in 9

teeth (45%), while 11 were deemed bacteria-free (Molander et al. 1998).

Pinheiro et al. studied the microbial flora of 60 root-filled teeth with persisting

periapical lesions. Microorganisms were recovered from 51 teeth (85%); mostly one or

two strains were present per canal. Of the species isolated, 57% were facultative and

43% obligate anaerobes. E. faecalis was the most frequently recovered species, detected

in 53% of culture-positive canals, 18 times in pure culture. Significant clinical

associations were observed between polymicrobial or anaerobic infections and pain:

Prevotella intermedia/Prevotella nigrescens and tenderness to percussion;

Streptococcus spp./Actinomyces spp. and sinus formation; and Streptococcus spp./

Candida spp. with coronally unsealed teeth (Pinheiro et al. 2003).

12

Siqueira & Rôças identified microorganisms associated with post-treatment disease

using PCR. DNA was extracted from 22 teeth selected for endodontic re-treatment, and

analysed for the presence of 19 target species. All samples were positive for at least one

of the microorganisms, with E. faecalis being the most prevalent species and detected in

77% of teeth. The mean number of species in canals filled 0-2mm short of the

radiographic apex was 3 (range 1-5), whereas canals filled shorter than 2mm from the

apex yielded a mean of 5 species (range 2-11) (Siqueira and Rôças 2004). These

findings support the notion that the microflora of poorly obturated root canals resembles

more closely that of a primary endodontic infection.

Gomes et al. recently investigated the presence of selected bacterial species in 45 teeth

with post-treatment disease using PCR analysis. The bacterial isolates detected in each

canal were correlated with the clinical features of each case. The 9 target species were

detected in 39 of 45 cases (87%). E. faecalis was the most frequently recovered species,

detected in 78% of the study teeth, followed by Peptostreptococcus micros (51%),

Porphyromonas gingivalis (36%), Filifactor alocis (27%), Treponema denticola (24%)

Porphyromonas endodontalis (22%), Prevotella intermedia (11%) and Prevotella

nigrescens (11%). T. denticola and P. micros were statistically associated with

tenderness to percussion. P. nigrescens was associated with the presence of

spontaneous pain and abscess; P. endodontalis and P. nigrescens were associated with

purulent exudates (Gomes et al. 2008).

In summary, enterococci have been identified in a considerable proportion of teeth with

persisting periapical lesions that had endodontic therapy completed to a technically

13

satisfactory level (Sundqvist et al. 1998), as well as in counterparts that had insufficient

root fillings (Peciuliene et al. 2000). They are detected frequently in studies from

different regions irrespective of treatment protocol; in root filled teeth both in regions

where calcium hydroxide is commonly used as an interim dressing (Molander et al.

1998) and in countries where this topical antiseptic is usually not applied (Hancock et

al. 2001).

It is apparent that prevalence data for E. faecalis are quite variable, and very much

influenced by the method of detection used in each investigation. Cultivation is time-

consuming, requires controlled conditions during sampling and transport to ensure

microorganism viability, and can lead to variable results based on the experience of the

microbiologist (Siqueira and Rôças 2005). In contrast, PCR-based detection methods

enable rapid identification of specific DNA sequences with a high degree of sensitivity.

This higher sensitivity, however, can be at least partly attributed to the detection of free

floating DNA, and DNA from nonviable, culturable viable cells and viable but non-

culturable cells (Sedgley et al. 2006). Consequently, the number of intact and viable

microorganisms cannot be readily established. In addition, molecular techniques

analyse total DNA extracted from the sample and in the process destroy the

microorganism. Thereafter, being no longer culturable, it is not possible to study

phenotypic characteristics and potential pathogenicity associated with individual strains

recovered from the root canal (Sedgley et al. 2005).

A further factor affecting the validity of many investigations is the problem of

contamination from saliva or plaque from the outer tooth surface. Engström highlighted

14

that many teeth harboring enterococci in the root canal system also show positive

enterococcal growth on the outer tooth surfaces (Engström 1964). False positive results

are more likely to occur when PCR technology is used (Nair 2007). Hence, a

meticulous sampling technique which includes disinfection of the tooth and the access

cavity with associated sterility checks of both sites is a prerequisite to yielding

meaningful results (Zehnder and Guggenheim 2009). Unfortunately, relatively few

studies comply with the current decontamination protocols which recommend that root

canal sampling for polymerase chain reaction be preceded by sodium hypochlorite

(NaOCl) rather than iodine surface disinfection (Ng et al. 2003).

The presence of microleakage, through defective coronal restorations, old temporary

restorative materials, or nonrestored teeth is also likely to influence microbial findings.

In the study by Pinheiro et al. the authors acknowledge that microleakage was detected

in the majority of teeth (45/60) being re-treated (Pinheiro et al. 2003), as was the case

with the investigation by Gomes, where 35 of the 45 teeth had defective coronal seals

(Gomes et al. 2008). Consequently, proper decontamination of the access for PCR

would have been exceptionally difficult if not impossible to achieve, and enterococcal

DNA could have originated from sources outside the root canal.

While there has been considerable focus in the literature on the prevalence of E.

faecalis at various stages of endodontic infection, the clinical features of an

enterococcal infection have not been elucidated. Neither Pinheiro nor Gomes, who

attempted to correlate the presence of the bacterial species in root canals with the

clinical features of each case, could associate E. faecalis with any acute symptoms in

15

teeth with post-treatment disease (Pinheiro et al. 2003; Gomes et al. 2008). This is

consistent with Siqueira’s findings that E. faecalis was detected more often in

symptom-free teeth with primary apical periodontitis than in teeth with acute symptoms

(Siqueira et al. 2002). Kaufman et al. investigated the association between E. faecalis

and endodontically treated teeth with and without periradicular lesions. The latter group

of teeth were retreated because of technically inadequate root canal therapy or

suspected coronal leakage. The study found teeth with a periradicular lesion were

significantly more likely to harbor bacteria, but enterococci were detected more

frequently in teeth with a normal periapex (Kaufman et al. 2005).

It has been suggested that enterococci may be selected in root canals undergoing

standard endodontic treatment because of low sensitivity to antimicrobial agents

(Dahlén et al. 2000). Furthermore, Love postulated that the proficiency with which this

bacterium invades dentinal tubules provides it protection from chemo-mechanical root

canal preparation and intracanal dressings (Love 2001). Subsequently, when the

opportunity arises, E. faecalis could be released from the tubules into the root canal

space and act as a source of re-infection (Sedgley et al. 2005). Even bacteria entombed

at the time of root filling could provide a long-term nidus for subsequent reinfection. In

a study evaluating the survival of entombed E. faecalis, root canals were inoculated

with one of two strains of this microorganism following chemomechanical preparation.

After 48 hours of incubation, the canals were obturated with gutta percha, a zinc-oxide

eugenol sealer and restored with glass ionomer cement (GIC). Culture, PCR and

histological methods were used to recover E. faecalis after 6 and 12 months. All root

filled teeth demonstrated viable E. faecalis cells (Sedgley et al. 2005).

16

An alternative possibility is that enterococci are opportunistic coronal invaders of the

improperly sealed necrotic or filled root canal system, gaining entry during or after

treatment (Zehnder and Guggenheim 2009). Whether E. faecalis is a major pathogen

involved with the aetiology of endodontic failures or merely a colonizer that takes

advantage of the environmental conditions within obturated root canals has yet to be

fully appreciated (Rôças et al. 2004).

1.5.5 Virulence Factors

An appreciation of the potential virulence factors of E. faecalis is useful in our attempts

to decipher the role of this microbe in endodontic infections. enterococci possess a

number of virulence factors that permit adherence to host cells and extracellular matrix,

facilitate tissue invasion, effect immune-modulation and cause toxin-mediated damage

(Portenier 2003). These factors are discussed below.

1.5.5.1 Aggregation Substance

This is a surface localized protein, which mediates the cell-to-cell contact that enables

the exchange of plasmids between recipient and donor strains. Plasmids are

autonomous, covalently closed circular, double-stranded, supercoiled DNA elements,

which frequently confer traits that facilitate growth and survival under atypical

conditions, such as resistance to antibiotics (Sedgley et al. 2005). Aggregation

substance may serve as a virulence determinant by assisting the dissemination of

plasmid-encoded virulence factors and facilitating attachment to leukocytes, connective

extracellular matrix and possibly dentine (Hubble et al. 2003). E. faecalis strains

17

expressing aggregation substance were able to bind to type I collagen twice as

effectively as aggregation substance-negative strains (Rozdzinski et al. 2001). This may

be of particular importance with respect to endodontic infections, since collagen is the

main organic component of dentine (Kayaoglu and Ørstavik 2004). Hence, an

expression of aggregation substance could enable the E. faecalis cells to attach and

colonise the dentine surface and possibly be a factor in their invasion of dentinal

tubules.

Aggregation substance may also act as a protective element, favouring bacterial

survival by assisting in efforts to resist host defence mechanisms in endodontic

infections. For example, aggregation substance has been reported to facilitate the

intracellular survival of phagocytosed E. faecalis cells within human macrophages

(Kayaoglu and Ørstavik 2004). Similarly, E. faecalis cells expressing aggregation

substance have been found to be more resistant to killing by human neutrophils (Rakita

et al. 1999). Aggregation substance has also been identified as being capable of

inducing inflammation through the stimulation of T-lymphocytes, followed by a

massive release of inflammatory cytokines, and leading to host tissue damage

(Kayaoglu and Ørstavik 2004). Sedgley et al. identified aggregation substance in all

endodontic enterococcal strains tested (Sedgley et al. 2005).

1.5.5.2 Enteroccus surface protein (Esp)

This is a large chromosome-encoded surface protein, whose role in virulence is yet to

be fully elucidated. It is speculated, however, that Esp has a role in allowing enteroccal

cells to evade the host immune system. Toledo-Arana et al. demonstrated a relationship

18

between the presence of Esp and the biofilm formation capacity of E. faecalis, with

none of the esp-defective E. faecalis strains able to establish biofilm (Toledo-Arana et

al. 2001).

1.5.5.3 Gelatinase

Gelatinase is an extracellular zinc-containing metalloproteinase capable of hydrolyzing

gelatin, collagen and its degradation products, effectively providing the organism with

nutrients. However, it is also possible for proteases to cause direct or indirect damage to

the host tissues, and thus be classified as virulence factors (Portenier 2003). E. faecalis

isolated from hospitalized patients and patients with endocarditis have been found to

produce increased levels of gelatinase compared with community isolates (Coque et al.

1995). In relation to endodontic infection, gelatinase activity was expressed in 70% of

the 33 endodontic enterococcal isolates tested (Sedgley et al. 2005). Expression of the

gelatinase gene was associated with increased adhesion of E. faecalis to dentine in vitro

(Hubble et al. 2003) and enhanced biofilm formation in microtiter plates (Kristich et al.

2004). Gelatinase activity may also play a role in the long-term survival of E. faecalis

in obturated root canals, as evidenced by higher viable counts of gelatinase-producing

compared to gelatinase-negative variants of the microbe in an ex vivo model (Sedgley

2007).

1.5.5.4 Cytolysin

This is a plasmid-encoded toxin produced by beta-hemolytic E. faecalis isolates. It lyses

erythrocytes, polymorphonuclear leukocytes and macrophages, kills bacterial cells and

may lead to reduced phagocytosis (Portenier 2003). Epidemiological investigations

19

partly support a role for cytolysin in disease occurrence. Ike et al. reported that

approximately 60% of E. faecalis clinical isolates derived from various sources were

haemolytic, compared to only 17% of E. faecalis isolates derived from the faecal

specimens of healthy individuals (Ike et al. 1987).

1.5.5.5 Extracellular superoxide

Superoxide anion is a highly reactive oxygen radical involved in cell and tissue damage.

Extracellular superoxide production has been reported to be a common trait in strains of

E. faecalis, with 87 out of a total of 91 clinical and community isolates found to

produce detectable extracellular superoxide anion levels (Huycke et al. 1996). Isolates

associated with bacteremia or endocarditis produced significantly higher extracellular

superoxide levels than those from the stool of healthy subjects (Huycke et al. 1996),

indicating its potential role as a virulence factor. In relation to an endodontic infection,

production of superoxide by persisting E. faecalis cells within the root canal system

could translate into continuing tissue damage at the periapex and an absence of healing.

An imbalance between oxygen radical production in periapical lesions and its

elimination has been suggested to contribute to periapical damage and bone loss in

chronic apical periodontitis (Marton et al. 1993).

1.5.5.6 Pheromones

Pheromones are chromosomally encoded, small hydrophobic peptides which have a

signalling function between E. faecalis cells. The transfer frequency of certain plasmids

in E. faecalis is increased several-fold by the action of pheromones (Kayaoglu and

Ørstavik 2004). Briefly, the latter phenomenon occurs as follows. The recipient strain

20

secretes pheromones corresponding to the plasmid which it does not carry. In response,

the donor strain produces aggregation substance that provides tight contact between the

recipient and donor strain, facilitating the conjugative transfer of the replicated plasmid.

Once a copy of the plasmid is acquired, the recipient shuts off the production of that

pheromone, but continues to secrete pheromones specific for other plasmids that it does

not carry. Antibiotic resistance and other virulence traits, such as cytolysin production,

can be disseminated among strains of E. faecalis via the pheromone system (Kayaoglu

and Ørstavik 2004). Response to pheromones was identified in 48% of endodontic

enterococcal isolates, suggesting the potential of these cells to transfer plasmid DNA

between strains.

1.5.5.7 Virulence factors and Pathogenicity

In conclusion, for a bacterium to be pathogenic, it must be able to adhere to, grow on,

and invade the host. It must then survive host defence mechanisms, compete with other

bacteria, and produce pathological changes. With the virulence factors described above,

E. faecalis appears to possess the requisites to establish an endodontic infection and

maintain an inflammatory response potentially detrimental to the host (Kayaoglu and

Ørstavik 2004). Which, if any of these factors play a role in the pathogenesis of

periradicular diseases remains to be elucidated (Rôças et al. 2004).

1.6 The viable but non-culturable (VBNC) state

It has been observed that after an extended period of starvation, the number of

culturable cells as determined by plate counts declines, while the total number of cells

21

present, as determined by a direct count method, remains unchanged (Bogosian et al.

1998). While one explanation is that the non-culturable cells are dead, an alternative

explanation is that the non-culturable cells have entered a state in which they are still

viable but cannot be cultured by standard microbiological techniques (Lleo et al. 1998;

Lleo et al. 2001). The viable but non-culturable state is believed to constitute a survival

strategy adopted by bacteria when exposed to environmental stress (Barer and Harwood

1999). The hostile environmental conditions that have been described as inducing the

activation of the VBNC state include low nutrient concentrations, low or high

temperatures, high salinity and extreme pH. When in the VBNC state, bacteria are no

longer culturable on conventional growth media, but the cells display active

metabolism, respiration, membrane integrity, and gene transcription (Lleo et al. 2001).

To date, there are no adequate techniques to prove the viability of cells in the VBNC

physiological state (Portenier 2003). Recovery of culturable cells from a population of

non-culturable cells would provide convincing support for the VBNC hypothesis. The

appearance of large numbers of culturable cells after the addition of nutrients to

populations of non-culturable cells has been reported to occur via a process termed

resuscitation. However, such recovery studies can be confounded by the presence of

small numbers of culturable cells, which can grow in response to the addition of

nutrients and give the illusion of resuscitation. Bogosian et al. prepared samples that

contained only non-culturable cells of various enteric bacteria, and subjected them to

either a temperature shift or a nutrient addition in order to determine their resuscitation

potential. They did not yield any culturable cells (Bogosian et al. 1998).

22

It has also been suggested that the presence of culturable cells is required for the

resuscitation of non-culturable cells. This possibility was tested by using a mixed

culture recovery method, which evaluated a mixture of easily distinguishable culturable

and non-culturable cells to determine which group of cells responded to various

resuscitation techniques. The results showed that the only culturable cells yielded from

the mixtures were from the initial culturable cells, indicating that the culturable cells

were not capable of resuscitating the non-culturable cells (Bogosian et al. 1998). The

authors concluded that the non-culturable cells were dead, questioning the existence of

the VBNC state for enterococcal bacteria.

In an attempt to develop methods capable of detecting non-culturable bacteria and

establishing their viability, numerous researchers have used a modification of the

conventional PCR method by using RNA as the amplification template (Lleo et al.

2000; Williams et al. 2006). This method is referred to as reverse transcription-PCR or

RT-PCR. Messenger RNA is a short-lived molecule which serves as a marker of

viability and active replication. Lleo et al. reported that E. faecalis conserves its

viability in the VBNC state by limiting protein synthesis to a few key proteins, such as

the penicillin binding protein (pbp5). Thus RT-PCR amplification of the pbp5-

encoding regions of E. faecalis mRNA should allow the detection of VBNC E. faecalis

and therefore, enable us to determine whether endodontic infections harbor the

bacterium in this state (Lleo et al. 2000). Williams et al. detected E. faecalis in 7 out of

87 intra-canal samples that were negative by cultivation, suggesting that even when

undetected by cultivation, E. faecalis remains viable and may return to an active,

pathogenic state. However, every sample that was positive by the RNA-based RT-PCR

assay was also positive by the DNA-based qualitative PCR assay, indicating that viable

23

bacteria may simply have been present in numbers small enough to be detected by the

more sensitive molecular methods but not by cultivation (Williams et al. 2006). Some

caution must therefore be exercised when evaluating recent studies that purport to show

E. faecalis in the VBNC state and studies claiming to show cellular resuscitation

(Portenier 2003).

1.7 The ‘biofilm’ concept

Although the concept of a microbial biofilm is not a new one, it has not been until

recent years that its importance in human disease has been recognized. A biofilm can be

defined as a microbial community characterized by cells that are attached to a

substratum, encased in a matrix of extracellular polymeric substance, and exhibiting

altered growth phenotypes (Donlan and Costerton 2002). A complex system of cell-to-

cell communication underlies this process. Bacteria themselves account for a variable

fraction of the total biofilm volume, typically 5-35%. The remainder of the volume is

comprised of extracellular matrix (del Pozo and Patel 2007)

The diversity of biofilm-associated infections is rising, with estimates indicating their

involvement in more than 65% of all bacterial infections (Lewis 2001). A number of

human bacterial diseases produce lesions characterized by the presence of biofilm.

Examples include infections of the oral soft tissues and teeth, as well as the middle ear,

gastrointestinal and urogenital tract. Biofilms have been observed on invasive medical

devices, such as indwelling catheters, cardiac implants, and tracheal and ventilator

tubing (Costerton et al. 1999).

24

Biofilms grow comparatively slowly, and infections are often slow to produce overt

symptoms. Sessile bacterial cells release antigens and stimulate the production of

antibodies, but these are ineffective in killing biofilm bacteria, and can in fact cause

immune complex damage to surrounding tissues (Costerton et al. 1999). Even in

individuals with excellent cellular and humoral immune reactions, biofilm infections

are rarely resolved by the host defence mechanisms. Antibiotic therapy typically

suppresses symptoms of infection by killing free-floating bacteria shed from the

attached population, but fails to eradicate those cells that are still embedded in the

biofilm. When antimicrobial chemotherapy stops, the biofilm can act as a nidus for the

recurrence of infection (Stewart and Costerton 2001).

1.7.1 Biofilm formation as a survival strategy

A major advantage to its colonizing species is the protection afforded by the biofilm

structure from competing microorganisms, from environmental factors such as host

defence mechanisms, and from potentially toxic substances in the environment such as

lethal chemicals and antibiotics (Socransky and Haffajee 2002). Biofilms also facilitate

the processing and uptake of large, complex nutrient molecules that could not be

efficiently degraded by an individual bacterium. The breakdown of these nutrients

requires the sequential action of a range of complementary extracellular enzymatic

profiles (Svensater 2004). Other benefits of a community microbial lifestyle are cross-

feeding (one species providing nutrients for another), the removal of potentially harmful

metabolic products, and the development of an appropriate physicochemical

environment to facilitate microbial survival (Socransky and Haffajee 2002).

25

In many aspects the biofilm mode of growth is a survival strategy, and may therefore be

favoured by bacteria colonizing the harsh environmental conditions of the root canal.

This aspect is supported by the finding that clinically isolated E. faecalis species

possess many of the characteristics of a biofilm style of growth, including increased

adherence capacity, increased virulence factors and increased resistance to

antimicrobials (George et al. 2005).

1.7.2 Evidence for biofilm structures in Endodontics

Currently, limited information is available on the development or the physiology of

biofilms in the root canal (Figdor and Sundqvist 2007). An accurate depiction of the

ultrastructural features of the biofilm of infected root canals was first reported by Nair,

who observed ‘dense aggregates sticking to the canal walls’ and forming layers of

bacterial condensations. Amorphous material filled the interbacterial spaces and was

interpreted as an extracellular matrix of bacterial origin (Nair 1987).

Noiri et al. examined the surfaces of extracted teeth, root tips and extruded gutta-percha

points acquired from teeth with refractory periapical periodontitis and noted bacterial

biofilms in 9 of the 11 samples. The gutta-percha surface was almost completely

covered with a glycocalyx-like structure, with filaments, long rods, and spirochete-

shaped bacteria predominating. Planktonic cells were observed at a distance from the

glycocalyx structure in some areas. The findings suggested that bacterial biofilms

formed in extra-radicular areas were related to refractory periapical periodontitis (Noiri

et al. 2002). A follow up in vitro investigation concluded that E. faecalis, Strep.

26

sanguis, Strep intermedius, Strep. pyogenes and Staph. aureus produced single-species

biofilms on the surfaces of gutta-percha points. High serum concentrations were

required for the biofilm to establish (Takemura et al. 2004).

Distel et al. noted the ability of pure cultures of E. faecalis to form biofilm structures on

the canal walls of both calcium hydroxide medicated and non-medicated root canals.

Colonies of E. faecalis consisted of bacteria embedded in branching networks of

filamentous material representing the extracellular polysaccharide matrix. The authors

suggested that biofilm formation may be a mechanism that allows this organism to

resist treatment (Distel et al. 2002).

Nair et al. assessed the intracanal microbial status of the apical root canal system of the

mesial roots of human mandibular first molars with primary apical periodontitis.

Periradicular surgery was performed on 16 teeth immediately after the completion of

one-visit endodontic treatment. Fourteen of the sixteen mandibular molars examined

showed residual infection of the mesial roots after instrumentation, irrigation with

NaOCl, and obturation were completed. The infectious agents were mostly located in

the uninstrumented recesses of the main canals, the isthmus communicating between

them, and in accessory canals. They were mostly organized as biofilms (Nair et al.

2005).

27

1.7.3 Antimicrobial resistance of Biofilms

Susceptibility tests of in vitro biofilm models have shown the survival of bacterial

biofilms after treatment with antibiotics at concentrations hundreds or even a thousand

times the minimum inhibitory concentration of the bacteria measured in a suspension

culture (Stewart and Costerton 2001). Abdullah et al. demonstrated that E. faecalis

grown as a biofilm was more resistant to chlorhexidine and povidone iodine than the

same strain grown in planktonic suspension (Abdullah et al. 2005).

It is likely that biofilms evade antimicrobial challenges by multiple mechanisms. One

mechanism of biofilm resistance is the failure of an agent to penetrate the full depth of

the biofilm. The biofilm matrix is known to retard the diffusion of some antibiotics and

solutes in general (Costerton et al. 1999). The negatively charged polymers within the

matrix may neutralize strong oxidizing agents, such as sodium hypochlorite, making it

difficult for them to penetrate and kill microorganisms (Stewart et al. 2001).

The second mechanism involves the physiological state of biofilm microorganisms.

Bacterial cells residing within a biofilm grow more slowly than planktonic cells, and

consequently take up antimicrobial agents at a reduced rate. Furthermore, the depletion

of nutrients can force bacteria into a dormant growth phase in which they are protected

from killing (Portenier et al. 2005).

The third suggested mechanism responsible for antimicrobial tolerance is that

microorganisms within the biofilm experience metabolic heterogeneity. The physical

28

conditions available to support bacterial growth, such as pH, ion concentration, nutrient

availability, and oxygen supply, vary throughout the biofilm (Distel et al. 2002). Most

antibiotics are not active in a variety of physical environments. Aminoglycosides for

instance are more effective against bacteria growing in aerobic conditions than the same

microorganism growing anaerobically (Costerton et al. 1999); therefore not all cells

within the biofilm will be affected in the same way.

It is evident that oral microorganisms have the capacity to respond and adapt to

changing environmental conditions (Bowden and Hamilton 1998). Individual

microorganisms are able to sense and process the chemical information from the

environment and adjust their phenotypic properties accordingly. Quorum sensing refers

to the bacterial cell-to-cell communication mechanism for controlling cellular functions

within biofilm structures. The signalling is mediated by diffusible molecules which,

when present in sufficient concentrations, serve to modify gene expression in

neighbouring microorganisms. Quorum sensing is known to be involved in the

regulation of several microbial properties, including virulence and the ability to form

biofilms, incorporate extracellular DNA and cope with environmental stress

(Cvitkovitch et al. 2003).

The proximity of individual bacteria in biofilms increases the opportunity for gene

transfer, making it possible to convert a previously avirulent organism into a highly

virulent pathogen, or a bacterium that is susceptible to antimicrobials into a resistant

one (Potera 1999). This potential for gene transfer within biofilms is particularly

significant in the case of E. faecalis, because a number of its virulence factors are

29

encoded on transmissible plasmids. These include collagenase, gelatinase and adhesins,

all having the potential to contribute to the survival of this species in the root canal

(Distel et al. 2002).

A recent study demonstrated that over 80% of the bacteria isolated from acute

endodontic abscesses or cellulitis had the ability to coaggregate with genetically distinct

bacteria from the same site (Khemaleelakul et al. 2006). The significance of this is that

coaggregated bacteria in biofilms might be able to transfer genetic material. Evidence

for interspecies gene transfer was produced recently when it was shown that horizontal

exchange of antibiotic resistance can occur between different bacterial species within

root canals. Transfer of a conjugative plasmid carrying erythromycin resistance

between 2 endodontic infection-associated species, S. gordonii and E. facealis in an ex

vivo tooth model was observed (Sedgley et al. 2008).

Finally, it has been speculated that a sub-population of microorganisms exists within a

biofilm which are known as ‘persisters’. These constitute a small percentage of the

original population and are thought to represent a highly resistant phenotypic state that

is resistant to killing by antimicrobial agents (Stewart and Costerton 2001). This

hypothesis is supported by studies indicating that immature or newly formed biofilms

are significantly less susceptible to antibiotics than corresponding planktonic cells, even

though the formed layer is too thin to pose a barrier to penetration to either medication

or metabolic substrates (Lima et al. 2001; Spratt et al. 2001).

30

1.8 Antimicrobial agents

Antimicrobial agents have generally been developed and optimized for their activity

against fast growing, dispersed populations containing a single micro-organism.

Unfortunately the effectiveness of such agents does not translate to the in vivo

conditions present on the tooth surface, where bacteria grow as biofilms (Svensäter

2004). The biofilm structure may go some way to explain the frequent discrepancy

observed in studies testing the antimicrobial effectiveness of endodontic irrigants and

medicaments clinically, and in vitro.

1.8.1 Sodium Hypochlorite

Sodium hypochlorite is the most widely used irrigating solution. It has broad spectrum

antimicrobial activity, rapidly killing vegetative and spore-forming bacteria, fungi,

protozoa, and viruses (Siqueira et al. 2007). It exerts its antibacterial effect through its

ability to oxidise and hydrolyse cell proteins and to some extent, osmotically draw

fluids out of cells due to its hypertonicity (Pashley et al. 1985). When hypochlorite

contacts tissue proteins, nitrogen, formaldehyde, and acetaldehyde are formed within a

short time and peptide links are broken, resulting in protein dissolution. Sodium

hypochlorite is thus a potent antimicrobial agent and additionally possesses the unique

ability to dissolve necrotic tissue and organic components of the smear layer (Haapasalo

2005).

In vitro studies unequivocally support the strong antibacterial activity of sodium

hypochlorite. Siqueira et al. reported that 4% NaOCl was significantly more effective

31

than saline solution in disinfecting root canals inoculated with E. faecalis (Siqueira et

al. 1997). In an agar diffusion study, NaOCl at concentrations of 2.5% and 4% was

effective against four black-pigmented obligately anaerobic bacteria and four

facultative bacteria (Siqueira et al. 1998). Vianna et al. tested three gram-negative

anaerobes typically isolated from primary apical periodontitis: Porphyromonas

gingivalis, Porphyromonas endodontalis and Prevotella intermedia. All three species

were effectively killed within 15 seconds when exposed in a broth suspension to NaOCl

at concentrations ranging from 0.5 to 5% (Vianna et al. 2004). Berber et al. assessed the

efficacy of 0.5%, 2.5% and 5.25% NaOCl irrigation in human extracted teeth infected

for 21 days with E. faecalis. The study protocol involved comparing various preparation

techniques and either hand or rotary instrumentation. Following preparation, root canals

were sampled and dentine chips obtained to assess dentine tubule disinfection. For all

preparation techniques and at all depths of dentine tested, 5.25% NaOCl was shown to

be the most effective irrigant solution tested, followed by 2.5% (Berber et al. 2006).

The antifungal efficacy of sodium hypochlorite was investigated by Waltimo et al. Both

5% and 0.5% NaOCl achieved effective killing of Candida albicans after a 30 second

exposure. Sodium hypochlorite concentrations of 0.05% and 0.005% were too weak to

kill the yeast, even after 24 hours of incubation (Waltimo et al. 1999). The antifungal

properties of NaOCl were less impressive when Sen et al. used root sections of teeth

inoculated with C. albicans and exposed them to 1% NaOCl, 5% NaOCl and 0.12%

chlorhexidine for variable times. The smear layer had an inhibitive effect on the

antifungal properties of the test agents. In its presence, antifungal activity was observed

only in the 1 hour treatment groups for all solutions. When the smear layer was absent,

5% NaOCl was the first to display antifungal activity after 30 minutes (Sen et al. 1999).

32

Radcliffe et al. observed that E. faecalis was more resistant to the antimicrobial activity

of hypochlorite compared to the yeast C. albicans (Radcliffe et al. 2004).

Byström and Sundqvist evaluated the effect of 0.5% NaOCl on fifteen single-rooted

teeth in vivo. Each tooth was treated at five appointments, and the presence of bacteria

in the root canal was studied on each occasion. No antibacterial intracanal dressings

were used between appointments. When 0.5% NaOCl was used, no bacteria could be

recovered from twelve of fifteen root canals at the fifth appointment, compared with

eight of fifteen control root canals which had no recoverable bacteria after the use of

saline irrigation. The results were statistically significant in favour of NaOCl, but the

study highlighted that not all root canals could be rendered bacteria free through

mechanical debridement and NaOCl irrigation, even after several appointments

(Byström and Sundqvist 1983; Byström and Sundqvist 1985).

More recent clinical investigations have confirmed the ability of NaOCl to greatly

reduce microbial counts within a treated root canal, and even render a proportion of root

canals free of cultivable microorganisms. Yet, independent of the mechanical technique

used for preparation, the size of the apical preparation or the concentration of NaOCl

irrigation used, about half the root canals will continue to harbor cultivable bacteria

(Shuping et al. 2000; McGurkin-Smith et al. 2005; Siqueira et al. 2007).

Peculiene et al. studied the effect of instrumentation and NaOCl irrigation in previously

root-filled teeth with apical periodontitis. Retreatment was carried out under aseptic

33

conditions using rubber dam. The root fillings were removed with hand instruments,

and without the use of chloroform. After the first microbiological sample was taken, the

canals were cleaned and shaped with reamers and Hedstroem files to a size 40 or larger,

using 2.5% NaOCl and 17% EDTA. A second microbiological sample was then taken.

Bacteria were initially isolated in 33 out of 40 teeth, with E. faecalis present in 21 teeth,

C. albicans in 6 teeth, gram-negative enteric rods in 3 teeth and other microbes in 17

teeth. Following instrumentation and irrigation no enteric gram-negative rods or yeasts

were found in the second sample. In contrast, E. faecalis persisted in 6 root canals, and

other microbes were found in 5 canals (Peciuliene et al. 2001). This finding

corroborates the results of the in vitro studies, indicating the increased resistance of E.

faecalis to NaOCl when compared with C. albicans and other gram-negative rods.

The root canal milieu is a complex mixture of a variety of organic and inorganic

compounds. The relative importance of these compounds in the inactivation of root

canal disinfectants has been evaluated in an in vitro model. Dentine powder was shown

to exert an inhibitory effect on the antibacterial effectiveness of 1% sodium

hypochlorite. When hypochlorite was pre-incubated with dentine for 24 hours, the

killing of E. faecalis required 24 hours (Haapasalo et al. 2000).

Evaluations of antibacterial efficacy as a function of sodium hypochlorite concentration

reveal conflicting results. In vitro studies, particularly agar diffusion tests, generally

report reduced antibacterial effectiveness of NaOCl following dilution (Yesilsoy et al.

1995; Siqueira et al. 1998) Gomes et al. tested in vitro the effect of various

concentrations against E. faecalis using cell culture plates. The microbe was killed in

34

less than 30 seconds by the 5.25% solution, while it took 10 and 30 minutes for

complete killing of the bacteria by 2.5% and 0.5% solutions, respectively (Gomes et al.

2001).

In a more clinically applicable ex vivo model, Siqueira et al. evaluated the impact of

hypochlorite concentration using extracted human teeth that were inoculated with E.

faecalis prior to instrumentation and irrigation with either 1%, 2.5%, or 5.25% NaOCl.

No significant difference was noted between the three NaOCl solutions tested (Siqueira

et al. 2000). Numerous clinical studies have similarly found no significant differences

in antibacterial efficiency between 0.5% and 5% NaOCl solutions (Cvek et al. 1976;

Byström and Sundqvist 1985). This suggests clinical factors such as the regular

exchange and large volumes of irrigant may maintain the antibacterial effectiveness of

the NaOCl solution, compensating for a reduced concentration.

One of the fundamental properties of sodium hypochlorite is its ability to dissolve

organic matter inside the root canal system. This dissolution is dependent of three

factors: frequency of agitation, amount of organic matter in relation to the amount of

irrigant and the surface area of the tissue to undergo dissolution (Mohammadi 2008).

Okino et al. evaluated the tissue dissolving capacity of sodium hypochlorite and

chlorhexidine by placing weighed bovine pulp fragments in contact with 20mls of

NaOCl or chlorhexidine within a centrifuge, until total dissolution took place. Sodium

hypochlorite was the only agent tested that effectively dissolved the organic tissue. The

dissolution speed was affected by concentration, with 2.5% NaOCl dissolving pulpal

tissue faster than 1% and 0.5% NaOCl (Okino et al. 2004). These findings were

35

confirmed by Naenni et al., who used standardized samples of necrotic tissue obtained

from pig palates for this dissolution study (Naenni et al. 2004).

The main disadvantage of using sodium hypochlorite as an endodontic irrigant is that it

is highly toxic at high concentrations, and tends to induce tissue irritation on contact

(Hauman and Love 2003). Zhang et al. evaluated the cytotoxicity of four concentrations

of NaOCl (5.25%, 2.63%, 1.31%, and 0.66%) on fibroblasts grown on cell culture

plates. The results showed that toxicity of NaOCl was dose-dependent (Zhang et al.

2003). Barnhart et al. measured the cytotoxicity of several endodontic agents on

cultured gingival fibroblasts, and showed that iodine potassium-iodide and calcium

hydroxide were significantly less cytotoxic than NaOCl (Barnhart et al. 2005).

1.8.2 Calcium Hydroxide

The efficacy of calcium hydroxide in endodontic therapy stems mainly from its

bactericidal effects, its ability to stimulate the formation of calcified tissue and its

capacity to denature protein, aiding the dissolution of pulpal remnants. Cvek

popularised the use of calcium hydroxide in the endodontic treatment of incompletely

developed tooth roots, exploiting its ability to produce sterile necrosis and subsequent

calcification (Cvek, 1976). Currently, calcium hydroxide is recognised as one of the

most effective antimicrobial dressings available for the purposes of endodontic

treatment (Siqueira and Lopes 1999). Its antimicrobial activity is due to the release and

diffusion of hydroxyl ions, which produce a highly alkaline environment incompatible

with microbial survival. Hydroxyl ions are indiscriminately reactive with a range of

36

biomolecules, exerting their lethal effects on bacterial cells by damaging their

cytoplasmic membranes and denaturing proteins and DNA (Siqueira and Lopes 1999).

Byström et al. examined the anti-bacterial effects of different medicaments and found a

4-week application of calcium hydroxide to be more effective than camphorated

paramonochlorophenol or camphorated phenol. Calcium hydroxide was capable of

rendering 97% of the canals culture-negative; while this was achieved in only 66% of

the canals treated with paramonochlorophenol or camphorated phenol (Byström et al.

1985). Sjögren et al. found that dressing canals with calcium hydroxide for 7 days was

sufficient to reduce canal bacteria to a level that produced a negative culture (Sjögren et

al. 1991). The effectiveness of calcium hydroxide as an anti-bacterial agent was further

confirmed in the study by Shuping et al., in which the addition of calcium hydroxide

medicament after chemomechanical preparation with rotary Ni-Ti instrumentation and

NaOCl irrigation increased the number of culture-negative canals from 61.9% to 92.5%

(Shuping et al. 2000). These reports contrast with findings of little difference in

treatment outcome between single and multiple-visit treatment using a calcium

hydroxide dressing (Trope et al. 1999; Weiger et al. 2000; Peters and Wesselink 2002).

For calcium hydroxide to act effectively as an intracanal dressing, the hydroxyl ions

must be able to diffuse through dentine and pulpal tissue remnants. Studies have

demonstrated that hydroxyl ions derived from a calcium hydroxide medication do

diffuse through root dentine, but the pH values decrease as a function of distance from

the main canal. Tronstad et al. showed that the pH in the root canal was greater than

12.2, whereas in the most peripheral dentine, the pH ranged from 7.4 to 9.6 (Tronstad et

37

al. 1981). Similar results were reported by Nerwich et al., who also showed that there

was a difference in the rate and amount of diffusion, with cervical dentine increasing in

pH much more rapidly than apical dentine. This was attributed to an increased

concentration and diameter of dentinal tubules in the cervical compared to the apical

third of the root. It was also found that a time period of 1-7 days was required for the

hydroxyl ions to reach the outer root dentine in this in vitro model, and 3-4 weeks was

needed to reach peak pH levels peripherally (Nerwich et al. 1993). This has

implications for establishing appropriate time-frames for dressing changes.

In order to have antibacterial effects within dentinal tubules, the ionic diffusion of

calcium hydroxide needs to exceed the dentine’s buffering capacity, reaching pH levels

sufficient to destroy bacteria (Siqueira and Lopes 1999). Haapasalo clearly showed that

dentine powder abolished the killing of E. faecalis by calcium hydroxide. Without the

dentine, the test organism was eliminated within minutes, whereas with the dentine

powder added, no reduction in the bacterial colony-forming units could be measured,

even after 24 hours of incubation (Haapasalo et al. 2000). The strong impact of dentine

on the antibacterial action of a saturated calcium hydroxide solution has been attributed

to the buffering action of dentine against alkali, with proton donors such as H2PO4-,

H2CO3 and HCO3- found in the mineralized hydroxyapatite of dentine (Haapasalo et al.

2007). With a modification of the dentine powder model, Portenier et al. showed that

organic material may also be capable of buffering calcium hydroxide because killing of

E. faecalis by calcium hydroxide was completely prevented by 18% bovine serum

albumin. This protein is used to simulate the high protein content of an inflammatory

exudate which may be present within the root canal (Portenier et al. 2001). These

findings must be treated with a degree of caution, however, as they are in vitro studies

38

not necessarily representative of the clinical situation. In particular, the agents tested in

these experiments are exposed to a much larger surface area of the dentine powder than

would be the case clinically, a fact that is likely to have increased the overall buffering

effect.

In vitro studies have demonstrated the capacity of calcium hydroxide to dissolve

necrotic tissue, using porcine and bovine muscle tissue (Hasselgren et al. 1988; Turkun

and Cengiz 1997). In addition, these studies found sodium hypochlorite and calcium

hydroxide had an additive effect, with calcium hydroxide pre-treatment of root canals

increasing the effectiveness of 0.5% NaOCl in achieving clean root canal walls as

assessed by SEM (Turkun and Cengiz 1997). Calcium hydroxide also makes a

significant contribution to the inactivation of lipopolysaccharides. Bacterial

lipopolysaccharide (LPS) has a major role in the development of periapical bone

resorption (Spångberg 2002). Treatment with an alkali such as calcium hydroxide

affects the hydrolization of the lipid moiety of bacterial LPS, resulting in the release of

free hydroxyl fatty acids. Thus calcium hydroxide mediated degradation of LPS may be

an additional important reason for the beneficial effects obtained with calcium

hydroxide use in clinical endodontics (Spångberg 2002).

E. faecalis is well recognized for its capacity to resist the antibacterial effect of calcium

hydroxide (Byström et al. 1985; Haapasalo and Ørstavik 1987; Ørstavik and Haapasalo

1990; Safavi et al. 1990; Siqueira and de Uzeda 1996). In fact its ability to withstand

high pH levels is an identifying characteristic of this microorganism. Byström et al.

showed that the pH needs to reach levels of 11.5 or higher to exert a bactericidal effect

39

on E. faecalis (Byström et al. 1985). Investigations of the mechanisms involved in the

resistance of E. faecalis to calcium hydroxide identified that its survival at high pH was

related to the effective function of its proton pump. When cells were exposed to

calcium hydroxide at pH 11.1 for 30 minutes, there was a 20-fold reduction in cell

survival in the presence of the proton pump inhibitor CCCP (Evans et al. 2002).

Furthermore, the buffering effects of dentine may not allow a sufficiently high pH to be

achieved in the dentinal tubules (Haapasalo et al. 2000). The biofilm forming ability of

E. faecalis constitutes another important survival strategy (Distel et al. 2002), while its

proficiency at invading dentinal tubules affords the organism physical protection from

chemo-mechanical root canal preparation and intracanal dressings (Love 2001).

Haapasalo and Ørstavik reported that a calcium hydroxide paste failed to eliminate,

even superficially, E. faecalis within the dentinal tubules (Haapasalo and Ørstavik

1987), a finding later supported by Heling et al. (Heling et al. 1992). Currently,

investigations are focusing on identifying specific ‘stress’ genes that become

upregulated following prolonged exposure to alkaline pH levels (Appelbe and Sedgley

2007).

Calcium hydroxide has great value in endodontics, but it is not a panacea. A limited

antibacterial spectrum means that calcium hydroxide does not affect all members of the

endodontic microbiota. The frequent isolation of enterococci in cases of endodontic

failure has led some to question its routine use as an intracanal medicament, especially

in re-treatment cases (Molander et al. 1998). In a study of retreatments, 11% of the root

canals still contained bacteria after two consecutive dressings with calcium hydroxide.

Two-thirds of these re-treatments failed (Sundqvist et al. 1998).

40

1.8.3 Chlorhexidine

This synthetic cationic bisbiguanide is highly efficacious against several gram-positive

and gram-negative oral bacterial species, as well as yeasts. The positive charge of the

chlorhexidine molecule interacts with the negatively charged phosphate group on

microbial cell walls, thereby altering the cells’ osmotic equilibrium (Gomes et al.

2003). This increases the permeability of the cell wall, allowing the chlorhexidine

molecule to penetrate into the bacteria. At low concentrations (0.2%) chlorhexidine

exerts a bacteriostatic effect, by causing low molecular weight elements such as

potassium and phosphorous to leak out of the cell. At higher concentrations (2%)

chlorhexidine is bactericidal as precipitation of the cytoplasmic contents results in cell

death (Gomes et al. 2003). Despite claims of biocompatibility, chlorhexidine is

cytotoxic when in direct contact with human cells. A comparative study using

fluorescence assays on human PDL cells showed similar cytotoxicity between 0.4%

NaOCl and 0.1% chlorhexidine (Chang et al. 2001). Boyce et al. found chlorhexidine

0.05% to be uniformly toxic to both cultured human cells and microorganisms (Boyce

et al. 1995).

Most comparative studies evaluating the effectiveness of chlorhexidine and sodium

hypochlorite find their antibacterial effects in vitro and ex vivo to be similar at

comparable concentrations. Oncag et al. evaluated the antibacterial properties of 5.25%

sodium hypochlorite, 2% chlorhexidine and 2% chlorhexidine with 2.2% cetrimide. The

root canals of extracted teeth were infected with E. faecalis and the antibacterial effects

of the irrigants were examined after 5 minutes and 48 hours. Two percent chlorhexidine

with cetrimide was significantly more effective against E. faecalis than 5.25% NaOCl at

41

5 minutes, although no significant differences could be observed when the same

specimens were sampled at 48 hours (Oncag et al. 2003).

Two in vitro studies investigated the effectiveness of several concentrations of NaOCl

(0.5%, 1%, 2.5%, 4% and 5.25%) and three concentrations of chlorhexidine (0.2%, 1%,

and 2%) in both liquid and gel forms, against various endodontic pathogens. All

irrigants were effective in killing E. faecalis, but required vastly different times of

exposure. Chlorhexidine in liquid form at all concentrations tested, and NaOCl at

5.25% were the most effective irrigants, eliminating the test organism within 30

seconds. The 0.2% chlorhexidine gel, on the other hand, required 2 hours contact time

to produce a negative culture of E. faecalis (Gomes et al. 2001; Vianna et al. 2004).

Siqueira et al. tested the antibacterial effect of several endodontic irrigants against four

black-pigmented gram-negative anaerobes and four facultative anaerobes by means of

an agar diffusion test. The 4% and 2.5% concentrations of NaOCl were found to have a

more pronounced antibacterial effect than 2% and 0.2% chlorhexidine. No significant

difference was observed between the zones of inhibition recorded for the two

concentrations of chlorhexidine (Siqueira et al. 1998).

Numerous studies use the agar diffusion method to compare the antibacterial efficacy of

chlorhexidine, calcium hydroxide and their combination, usually against E. faecalis.

The results consistently show chlorhexidine to be more effective than calcium

hydroxide paste in eliminating the test organism, and generally chlorhexidine alone is

42

more successful than its combination with calcium hydroxide (Gomes et al. 2006;

Ballal et al. 2007; Neelakantan et al. 2007; de Souza-Filho et al. 2008). The results of

the agar diffusion method depend upon the molecular size, solubility and diffusion of

the materials through the aqueous agar medium, the bacterial species and the number of

bacteria inoculated, pH of the substrates in plates, agar viscosity, storage conditions of

the agar plates, incubation time and the metabolic activity of the microorganisms

(Gomes et al. 2006). Therefore, the inhibition zones may be more related to the

materials’ solubility and diffusibility in agar than to their actual efficacy against the

microorganisms. The antimicrobial activity of calcium hydroxide is related to its high

pH, which causes the medicament to precipitate on agar, preventing its diffusion. This

may explain the poor performance of calcium hydroxide when the agar diffusion

method is used (Gomes et al. 2006).

A more clinically applicable model than the agar diffusion test uses extracted teeth

which are instrumented, inoculated with a test organism, usually E. faecalis, and

exposed to the antimicrobial agent. Investigations comparing the efficacy of

chlorhexidine and calcium hydroxide at eliminating E. faecalis from infected dentinal

tubules found chlorhexidine to be significantly more effective than calcium hydroxide

paste or a mixture of the two (Schafer and Bossmann 2005; Ercan et al. 2006).

Chlorhexidine achieves its optimal antimicrobial activity within a pH range of 5.5-7.0.

It is likely that increasing the pH above 7.0 by adding calcium hydroxide will lead to

the precipitation of chlorhexidine molecules and a reduction in its effectiveness

(Mohammadi and Abbott 2009). Therefore the usefulness of mixing calcium hydroxide

and chlorhexidine remains unclear and controversial (Athanassiadis et al. 2007).

43

As alluded to already, many in vitro studies use extracted bovine or human teeth mono-

infected with E. faecalis. Chlorhexidine is known to be less effective against gram-

negative bacteria than gram-positive taxa. Its efficacy against gram-positive taxa such

as E. faecalis in laboratory experiments may therefore cause an over-estimation of its

clinical usefulness (Zehnder 2006). This could at least partly explain why the potential

benefits of chlorhexidine indicated by in vitro studies have not been fully realised in

vivo. In a randomised clinical trial on the reduction of intracanal microbiota by either

2.5% NaOCl or 0.2% chlorhexidine irrigation, hypochlorite was found to be

significantly more efficient than chlorhexidine in obtaining negative cultures (Ringel et

al. 1982).

In another randomised clinical trial by Zamany et al., a 2% chlorhexidine solution, used

as a final irrigant, significantly decreased bacterial loads in root canals that had been

irrigated with sodium hypochlorite during canal preparation. Cultivable bacteria were

retrieved at the conclusion of the first visit in 1 out of 12 chlorhexidine-treated teeth, in

contrast to the control group in which 7 out of 12 teeth showed growth (Zamany et al.

2003). A shortcoming of this otherwise well conducted study was that the final

chlorhexidine rinse was compared to an identical procedure using sterile saline, thus not

clarifying whether this regimen is superior to the use of hypochlorite as a final rinse.

When chlorhexidine is used as an irrigating solution, it has a relatively short effective

exposure time in the root canal, which does not allow its full antibacterial action to be

expressed. Chlorhexidine has the unique feature of imparting antibacterial substantivity

to dentine. The positively charged molecules of chlorhexidine can adsorb onto dentine,

44

and prevent microbial colonisation for some time after medication. In order to achieve

this substantivity, prolonged interaction is required to allow saturation of the dentine

with chlorhexidine molecules. It has been suggested that this necessitates the placement

of chlorhexidine as an intracanal medicament between appointments for at least seven

days, rather than using it only as an irrigant (Athanassiadis et al. 2007).

Lin et al. placed a slow release device containing 5% chlorhexidine in cylindrical

bovine root specimens infected with E. faecalis for seven days. Subsequently, no

bacteria were recovered up to 500µm into the dentinal tubules. This compared to a

significant reduction but a failure to eliminate viable bacteria when using 10ml of 0.2%

chlorhexidine as an irrigant (Lin et al. 2003).

The antibacterial efficacy of 2% chlorhexidine gel, calcium hydroxide and a

combination of both was assessed in teeth with chronic apical periodontitis. This

randomised clinical trial by Manzur concluded that, when used as intracanal dressings,

the antimicrobial efficacy of chlorhexidine gel and calcium hydroxide was comparable

(Manzur et al. 2007). In the limited sample of thirty-three teeth, a one-week calcium

hydroxide dressing reduced the bacteria-positive canals from 3 to 2, while medicating

with 2% chlorhexidine gel showed a reversal of culture for one tooth, increasing the

number of bacteria-positive canals from 4 to 5. Wang et al. evaluated the additional

antibacterial effect of a 2 week intracanal dressing with a mixture of 2% chlorhexidine

gel and calcium hydroxide. No significant difference could be detected in the

percentage of positive samples before and after the placement of the medicament,

45

suggesting that the intracanal dressing did not significantly improve bacterial reduction

(Wang et al. 2007).

Paquette et al. evaluated the antimicrobial effectiveness of 2% chlorhexidine gluconate

liquid applied in vivo as an intracanal medicament for 7-15 days. No increase in the

proportion of teeth with negative cultures or reduction in bacterial counts beyond that

achieved after chemo-mechanical preparation could be found (Paquette et al. 2007). In

fact, a significant increase in bacteria positive samples was reported, from 32% before

to 54% after medication with chlorhexidine liquid. The use of a liquid form of

chlorhexidine was suggested as a possible reason for its poorer-than-expected

performance, with the authors indicating that its consistency may not be well suited for

use as an intracanal medicament.

In another in vivo study, root canals that yielded positive cultures a week after complete

chemo-mechanical preparation and camphorated paramonochlorophenol dressing were

medicated with either calcium hydroxide, chlorhexidine or camphorated

paramonochlorophenol for one week. There were no statistically significant differences

in the percentage of negative cultures obtained with each medicament (Barbosa et al.

1997).

One of the major disadvantages of chlorhexidine is that it has no tissue solvent activity.

It was proposed that this shortcoming could be compensated for by producing a gel

formulation which increased its mechanical removal. This hypothesis has not been

46

validated by the literature. Okino et al. evaluated the tissue dissolving ability of 0.5%,

1% and 2.5% sodium hypochlorite, 2% chlorhexidine digluconate liquid, 2%

chlorhexidine digluconate gel and distilled water. Distilled water and both solutions of

chlorhexidine did not dissolve the pulp tissue within 6 hours (Okino et al. 2004). Unlike

calcium hydroxide, chlorhexidine does not inactivate bacterial LPS (Tanomaru et al.

2003).

The prolonged antibacterial activity of chlorhexidine resulting from its substantivity,

has been the focus of numerous investigations. Khademi et al. found that a 5 minute

application of 2% chlorhexidine solution in a bovine root dentine model, induced

substantivity for up to 4 weeks (Khademi et al. 2006). Rosenthal et al., using bovine

root blocks, measured the substantivity of chlorhexidine in dentine after root filling for

up to 3 months. After instrumentation and irrigation with NaOCl and EDTA, the test

canals were irrigated with 2% chlorhexidine for 10 minutes before root filling. No

chlorhexidine was used in the control group. Residual chlorhexidine activity was

measured within the dentine at various time periods after filling, and antibacterial

activity was clearly present even 3 months post-obturation (Rosenthal et al. 2004).

Another potential weakness of chlorhexidine is its susceptibility to the presence of

organic matter. Haapasalo et al. showed the effect of 0.05% chlorhexidine against E.

faecalis is reduced, but not totally eliminated, by the presence of dentine (Haapasalo et

al. 2000), while no inhibition could be measured when a 0.5% solution was used. The

strongest inhibitor of chlorhexidine was bovine serum albumin, with more than 10% of

E. faecalis cells still viable after 24 hours of incubation with the medicament (Portenier

47

et al. 2001). Inflammatory exudate, rich in proteins such as albumin, may thus weaken

the antibacterial effects of chlorhexidine more so than the dentine will (Haapasalo

2005).

A suggested clinical protocol by Zehnder consisted of irrigation with sodium

hypochlorite to dissolve the organic components, irrigation with EDTA to eliminate the

smear layer and irrigation with chlorhexidine to increase the anti-microbial spectrum of

activity and impart substantivity (Zehnder 2006). Although such a combination of

irrigants may enhance the overall antimicrobial effectiveness, the possible chemical

interactions of the irrigants must be considered (Mohammadi and Abbott 2009). The

combination of chlorhexidine and sodium hypochlorite has been shown to result in the

formation of a precipitate which has been found to occlude dentinal tubules (Basrani et

al. 2007; Bui et al. 2008). This may interfere with the seal of the root filling and the

combination should therefore be avoided.

1.8.4 Antibiotics

In light of the limitations of calcium hydroxide and chlorhexidine as intracanal

medicaments, preparations containing antibiotics represent another approach to treating

persisting endodontic infection.

The two most common antibiotic-containing commercial preparations currently

available are Ledermix paste and Septomixine Forte. Septomixine Forte contains two

48

antibiotics – neomycin and polymyxin B sulphate. Neither of these can be considered

suitable for use against the commonly reported endodontic bacteria, because of their

inappropriate spectra of activity (Abbott et al. 1990). In addition, although the anti-

inflammatory agent in Septomixine Forte, dexamethasone, is clinically effective, the

triamcinolone component of Ledermix paste is considered to have less systemic side

effects (Abbott et al. 1990).

1.8.4.1 Ledermix

Ledermix paste was developed by Schroeder and Triadan in 1960, primarily as a

corticosteroid vehicle aimed at controlling pain and inflammation in vital pulp therapy.

The antimicrobial properties were initially catered for by a formalin-based paste, while

the inclusion of an antibiotic component was to compensate for what was perceived to

be a possible corticoid-induced reduction in the host immune response (Schroeder

1981). Today, Ledermix paste consists of a combination of the tetracycline antibiotic

demeclocycline HCl, at a concentration of 3.2%, and a corticosteroid, triamcinolone

acetonide at a concentration of 1%, in a polyethylene glycol base (Athanassiadis et al.

2007).

Heling and Pecht evaluated the efficacy of Ledermix paste in the disinfection of

dentinal tubules. Ledermix was effective in reducing the number of viable

Staphylococcus aureus bacteria in dentinal tubules after 7 days of incubation and after

recontamination (Heling and Pecht 1991). However, its antibacterial effect is limited to

a small spectrum of bacteria. Lin et al. found that exposure to Ledermix for 7 days had

no significant effect on the amount of viable Streptococcus sanguis bacteria in bovine

49

dentinal tubules (Lin et al, 2003). A further limitation of Ledermix paste is that

increasing numbers of bacterial species have become resistant to tetracycline (Goodson

and Tanner 1992). For instance, numerous in vitro antimicrobial susceptibility studies

have revealed a significant proportion of enterococcal isolates to be resistant to

tetracyclines (Dahlén et al. 2000; Geijersstam et al. 2006). Dahlén et al. found 4 out of

29 strains to be highly resistant to tetracycline, while Geijersstam et al. identified 17

tetracycline resistant isolates out of 59 (Geijersstam et al. 2006). The latter study

observed antibiotic susceptibility patterns of E. faecalis isolated from endodontic

infections in Finland and Lithuania to be similar, despite the differing antibiotic usage

in these countries (Geijersstam et al. 2006).

The concentration of demeclocycline achieved within the dentinal tubules and

periapical tissues varies as a function of time and location – the further from the canal,

the lower the concentration achieved and this concentration decreases with time (Abbott

et al. 1990). Abbott et al. showed dentinal tubules are the major supply route of the

active components to the periradicular tissues, while the apical foramen is not as

effective as a supply route (Abbott et al. 1988). Demeclocycline within the root canal

itself is present in sufficiently high concentrations to be effective against susceptible

species of bacteria. In the periradicular tissues and peripheral parts of the dentine,

however, the concentration achieved through diffusion is insufficient to inactivate

bacteria (Abbott et al. 1990).

Chu et al. conducted an in vivo study comparing the efficacy of disinfection of

Ledermix, Septomixine and Calasept - a calcium hydroxide paste. Each tooth was

50

prepared using hand instrumentation and repeatedly irrigated with 0.5% sodium

hypochlorite. Each medicament was spiralled into the root canal and left for seven days.

Bacteriological samples were taken before and after the two-visit endodontic treatment.

After seven days, the percentages of canals with positive growth after dressing with

Ledermix, Septomixine or Calasept, were 48% (13 of 27), 31% (8 of 26) and 31% (11

of 35), respectively. These differences were not statistically significant (Chu et al.

2006). There was a marked similarity in microbiological outcomes achieved despite the

use of different inter-appointment dressings.

Trope et al. evaluated the relationship of intracanal medicaments to endodontic flare

ups. Formocresol, Ledermix, and calcium hydroxide were used to dress 474 teeth in

strict sequence irrespective of the presence or absence of symptoms or radiographic

signs of apical periodontitis. No significant difference in the flare-up rate among the

three intracanal medicaments was detected (Trope 1990). Ehrmann et al. showed that

greater postoperative pain relief was achieved when teeth were medicated with

Ledermix paste compared to calcium hydroxide (Ehrmann et al. 2003). Dentine acts as

a slow release mechanism for triamcinolone to the periodontal tissues, which is the

probable basis of its long-acting therapeutic effect (Schroeder 1981).

One of the drawbacks of using Ledermix as an intracanal medicament is its potential to

cause tooth discolouration. It is well known that uptake of tetracyclines into hard tissues

can produce colour changes. Tetracycline uptake was thought to be limited to periods of

active calcification (Lambrou et al. 1977), but it is now recognized that even fully

mineralized teeth can undergo colour changes resulting from topical exposure to

51

tetracycline (Bjorvatn et al. 1985). Kim et al. found that after 12 weeks, teeth dressed

with Ledermix paste and exposed to sunlight, acquired a dark grey-brown staining. This

did not occur when the teeth were kept in the dark. More severe staining was noted

when Ledermix paste filled the pulp chamber than when the paste was restricted to

below the cemento-enamel junction (Kim et al. 2000). In a similar study, immature

teeth became more stained than their mature counterparts undergoing identical

treatment. This was attributed to a greater number and wider diameter of dentinal

tubules in immature teeth, allowing greater diffusion to take place (Kim et al. 2000).

In addition to its antimicrobial properties, Ledermix paste has important applications in

the prevention and treatment of inflammatory root resorption following dental trauma.

Triamcinolone is a highly active steroid, known to suppress inflammation and have an

inhibitory effect on osteoclastic activity (Neuberger 1975). It was demonstrated to have

a direct inhibitory effect on dentinoclasts (Pierce et al. 1988). Tetracycline has been

shown to possess antimicrobial activity and have antiresorptive properties (Chang et al.

1994; Keller and Carano 1995). Abbott et al. demonstrated that both the antibiotic and

corticosteroid components of Ledermix paste penetrate the dentinal tubules and the

cementum and are readily available at the root surface. The rate of diffusion is

significantly greater when the cementum is absent from the root surface, as may be the

case in the root surfaces of traumatised teeth (Abbott et al. 1988).

Pierce and Lindskog reported that root canal treatment with intracanal Ledermix

introduced 3 weeks after 1-hour delayed replantation of extracted teeth was effective

against inflammatory root resorption (Pierce and Lindskog 1987). More recent studies

52

have evaluated the effect of immediate placement of intracanal Ledermix on root

resorption of delayed replanted teeth in monkey and canine models. Immediate

placement of intracanal Ledermix paste in replanted monkey and dog teeth resulted in

more complete healing and less unfavourable healing including inflammatory root

resorption and replacement resorption compared to root-filled replanted teeth (Wong

and Sae-Lim 2002; Chen et al. 2008). When the individual influence of corticosteroid

and tetracycline on external root resorption was assessed, there was no statistically

significant difference between the Ledermix group and the triamcinolone group in the

degree of favourable healing or the remaining root mass, while the tetracycline group

showed less favourable healing and reduced root mass (Chen et al. 2008). The steroid

component therefore appears largely responsible for the anti-resorptive effect of

Ledermix paste, although the antimicrobial contribution of tetracycline may play an

important adjunctive role. Ledermix paste treated canine roots had significantly more

healing and less resorption than roots treated with calcium hydroxide, and consequently

maintained significantly more root mass (Bryson et al. 2002). These observations

suggest that Ledermix is highly effective in treating resorption and promoting

favourable periodontal healing in delayed replantation cases.

In conclusion, the range of activity of demeclocycline in Ledermix, and the

concentrations of this antibiotic established within the root canal system are not

sufficient to achieve predictable antibiosis for an adequate length of time. Nevertheless,

Ledermix is an effective intracanal medicament for the control of postoperative pain

associated with acute apical periodontitis (Ehrmann et al. 2003) and vital pulp therapy

(Negm 2001). It also has important applications in the prevention and treatment of

inflammatory root resorption (Pierce and Lindskog 1987). These benefits need to be

53

balanced with the clinical concern of possible discolouration as a result of its use (Kim

et al. 2000; Kim et al. 2000).

1.8.4.2 Odontopaste

Odontopaste was released in February 2008 by Australian Dental Manufacturing. It

contains the antibiotic clindamycin hydrochloride 5% and triamcinolone acetonide 1%

and now also comes premixed with 1-2% calcium hydroxide. The manufacturers of

Odontopaste claim that this formulation will not cause dentinal discolouration as seen

with Ledermix. At this stage there is no published data available to validate

Odontopaste as an effective intracanal medicament.

The key antimicrobial component of Odontopaste is clindamycin, which has proven

effectiveness against a comprehensive spectrum of microbes associated with endodontic

infections, including Actinomyces, Eubacterium, Fusobacterium, Propionibacterium,

microaerophilic streptococci, Peptococcus, Peptostreptococcus, Veillonella, Prevotella,

and Porphyromonas (Pharmacists 1998). Only a small number of reports, however,

describe the use of clindamycin as an intracanal medicament.

Gilad et al. found clindamycin to be an effective antimicrobial agent in extracted human

teeth. In an initial experiment using a pure culture of Prevotella intermedia, all seven

experimental teeth treated with clindamycin fibers demonstrated complete elimination

of this organism when growth was assessed by either paper point or crushed tooth

sampling. When the effect of clindamycin fibers on a mixed inoculum was assessed, a

54

significant reduction in Fusobacterium nucleatum and Prevotella intermedia was noted.

In contrast, the clindamycin fibers failed to significantly reduce the levels of

Peptostreptococcus micros. Total bacterial elimination failed to be achieved in any of

the specimens inoculated with a mixed culture made up of F. nucleatum, P. micros and

P. intermedia (Gilad et al. 1999).

Lin et al. sought to evaluate and compare the antibacterial effect of clindamycin and

tetracycline in bovine dentinal tubules infected with Streptococcus sanguis. Following a

1 week exposure, the amount of viable bacteria in the dentinal tubules was significantly

reduced through the use of clindamycin, while tetracycline yielded results similar to

those of controls. Neither tetracycline nor clindamycin rendered any dentine samples

bacteria-free (Lin et al. 2003).

Molander et al. evaluated the effects of clindamycin when used as an intracanal

dressing on 25 teeth with necrotic pulps and periapical radiolucencies. A 150 mg

clindamycin capsule was mixed with sterile saline to a paste consistency and placed

into the infected root canals. The presence or absence of bacteria was determined in

samples taken immediately after removal of the dressing and after a period of 7 days,

during which the canals were filled with sampling fluid. Bacteria were recovered from 4

and 6 teeth respectively. Enterococci constituted the dominant flora in the teeth with

persisting infection. Although there was an absence of direct comparison, these results

indicated that clindamycin was not superior to calcium hydroxide in the eradication of

bacteria from the root canal and its use was therefore not recommended (Molander et

al. 1990).

55

E. faecalis generally displays intrinsic resistance to clindamycin (Singh et al. 2002)

although Geijersstam et al. noted that 2 of the 59 endodontic isolates they tested did not

display the classic LSA phenotype which confers intrinsic resistance to streptogramin A

and lincosamides such as clindamycin. These aberrant isolates were thus unusually

susceptible to clindamycin and a further 3 isolates had reduced resistance to the

antibiotic (Geijersstam et al. 2006). Similarly, Dahlén et al. found 25 out of the 26 E.

feacalis species isolated from root canals to be resistant to clindamycin (Dahlén et al.

2000).

1.8.4.3 Combinations of antibiotics and calcium hydroxide

In order to compensate for its limited antibacterial efficacy and produce a dressing that

has both antimicrobial and anti-inflammatory activity, Ledermix paste can be combined

with calcium hydroxide in a 50:50 mixture. In vitro studies investigating this

combination have generated contradictory results. Taylor et al. found that combining

the two preparations marginally increased the anti-bacterial activity of Ledermix paste

against the microorganisms L. Casei and S. mutans (Taylor et al. 1989). Seow,

however, showed that for S. sanguis and S. aureus, the addition of only 25% of calcium

hydroxide to Ledermix, converted the original zone of complete inhibition to one of

only partial inhibition, thus countering claims of any synergistic activity of this mixture

(Seow 1990). It has been shown that the 50:50 mixture results in slower release and

diffusion of the active components of Ledermix paste (Abbott et al. 1989). Abbott

hypothesized that an equal parts mixture of Ledermix and calcium hydroxide paste is

likely to have more potent and longer lasting effects than when Ledermix is used alone,

and less frequent changing of the dressings will be necessary (Abbott et al. 1989).

56

Molander and Dahlén evaluated the intracanal antibacterial potential of two

antibiotic/calcium hydroxide mixtures against enterococci in vivo. Fifty-five teeth in

which enterococci were present were dressed for one month with either tetracycline or

erythromycin, each mixed with calcium hydroxide. The tetracycline mixture was

effective against enterococci in 22 teeth (79%). In 7 teeth, other microorganisms were

recovered, resulting in a total antimicrobial effect of 54%. The overall antimicrobial

effect of this combination was deemed to be relatively weak (Molander and Dahlén

2003). The corresponding results for erythromycin were 96% and 56% respectively.

Limitations of this study include the 3 week interval between the pre-intervention

microbiological sampling of the canals to determine the presence of enterococci and the

experimental dressing placement. During this time the teeth were dressed with calcium

hydroxide. We are thus unable to conclude whether the treatment outcome was the

result of the antibiotic mixture or the effects of calcium hydroxide alone. The high

prevalence of microorganisms detected in the second sample which were absent in the

initial sampling suggests the high likelihood of contamination, possibly due to

ineffective temporization. Möller highlighted the specific concern of bacterial

contamination in endodontic microbial studies, and identified certain microbial species

which should be suspected to be contaminants – these include Staphylococcus

epidermidis and lactobacilli (Möller 1966), which were identified on numerous

occasions in the post-treatment samples in this study.

1.9 Susceptibility of biofilms to antimicrobials

Spratt et al. used a simple biofilm model to evaluate the effectiveness of a range of

commonly recommended antimicrobial irrigants against monocultures of five root canal

57

isolates: Prevotella intermedia, Peptostreptococcus micros, Streptococcus intermedius,

Fusobacterium nucleatum, and Enterococcus faecalis. Single species biofilms were

generated on membrane filter discs and incubated with colloidal silver, 2.25% sodium

hypochlorite, 0.2% chlorhexidine, 10% iodine or the phosphate buffered saline control.

The effectiveness of an agent was dependent on the organism in the biofilm, and on the

contact time. Iodine and NaOCl were more effective than chlorhexidine except against

P. micros and P. intermedia where they were all 100% effective. Iodine and NaOCl

elicited a 100% kill after 1 h incubation for all strains used. None of the agents were

effective against F. nucleatum after 15 min but NaOCl, iodine and chlorhexidine were

all effective after 1 h. Colloidal silver was generally ineffective.Sodium hypochlorite

was the most effective antimicrobial overall, successfully achieving 100% killing of E.

faecalis at both 15 and 60 minute time intervals. Iodine was only 100% effective after

60 minutes of exposure. Although there was clear evidence of a reduction in bacterial

counts after 15 minutes with both chlorhexidine and iodine, both agents left in excess of

107 colony forming units (Spratt et al. 2001). The results of this in vitro study are

limited by the absence of dentine in the experimental model, which is known to have an

inhibitive effect on antibacterial agents (Haapasalo et al. 2000). In addition, there is no

accounting for the complexities of root canal anatomy nor the polymicrobial nature of

root canal infection, both factors that may complicate disinfection.

The susceptibility of E. faecalis biofilms to antibiotic and chlorhexidine-based

medicaments was evaluated by Lima et al. (Lima et al. 2001). One and three day

biofilms were grown on cellulose nitrate membranes and exposed to the test agents for

24 hours. The formulations containing 2% clindamycin as the active antimicrobial did

not achieve any reduction in the bacterial biofilm, with one formulation actually

58

generating bacterial overgrowth. The association of clindamycin with metronidazole

significantly reduced the number of cells in 1-day biofilm compared to the control.

Chlorhexidine-containing formulations were able to render a portion of the membranes

bacteria-free, and were more effective than the antibiotic formulations tested (Lima et

al. 2001).

Abdullah et al. investigated the variable efficacy of selected endodontic antimicrobials

on a clinical isolate of E. faecalis grown as either a biofilm on cellulose nitrate

membrane filters or as a planktonic suspension. Each bacterial presentation was

exposed to calcium hydroxide, 0.2% chlorhexidine, EDTA, 10% povidone iodine or 3%

sodium hypochlorite. Sodium hypochlorite was very effective against E. faecalis

regardless of the growth conditions, achieving 100% killing after 2 minutes contact

time. Calcium hydroxide was the only other agent equally effective against bacteria in a

planktonic and biofilm presentation. While it could not achieve 100% kill, a greater

than 4 minute exposure resulted in a bacterial reduction of 99.9%. Both 0.2%

chlorhexidine and 10% povidone iodine were significantly less effective against the

biofilm phenotype. The authors concluded that due to their capacity to dissolve organic

tissue, sodium hypochlorite and calcium hydroxide were less inhibited in their

antimicrobial activity by the exopolysaccharide matrix of the biofilm (Abdullah et al.

2005).

A 7 day polymicrobial biofilm was developed on the hemisections of root apices by

Clegg et al. Each biofilm was separately immersed in 6% NaOCl, 3% NaOCl, 1%

NaOCl, 2% chlorhexidine, 1% NaOCl followed by BioPure MTAD or sterile phosphate

59

buffered saline (PBS) for 15 minutes. Only 6% NaOCl was capable of both rendering

bacteria nonviable and physically removing the biofilm. One percent and three percent

NaOCl showed disruption and physical removal of bacteria when viewed under the

SEM; however, both gave rise to bacterial colonies when dentinal shavings were

cultured, indicating that bacteria had escaped the effects of the irrigants, probably by

invading the dentinal tubules. No cultivable organisms were found after dentine

sections were placed in 2% chlorhexidine, but when the samples were viewed under a

scanning electron microscope, the biofilm was virtually intact. This was attributed by

the authors to potential fixation of cells by chlorhexidine (Clegg et al. 2006).

The superiority of NaOCl was also reported by Dunavant et al., who found that both

6% and 1% NaOCl were efficient in eliminating E. faecalis, achieving >99.99% and

99.78% biofilm elimination, respectively. In comparison, 2% chlorhexidine achieved

only a 60.5% bacterial reduction after a 5 minute exposure. This study used a novel in

vitro testing system, in which a 24-hour biofilm was established on ceramic coupons in

a flow cell system (Dunavant et al. 2006). Fluid flow is considered a principal

determinant of the biofilm structure (Purevdorj et al. 2002). Smooth, dense and stable

biofilms are formed at relatively high shear stress (Liu and Tay 2001). In addition to

influencing structure, hydrodynamic conditions also influence biofilm density and

strength, which in turn influences the diffusion of nutrients and signals through the

biofilm (Purevdorj et al. 2002) and affects the dispersal of cells from the biofilm

surface (Stoodley et al. 2002). The fluid flow in a root canal system is generated by the

inflammatory exudate that forms subsequent to pulpal necrosis. Studies have

demonstrated that occlusal forces can induce retrograde fluid movement into the apical

portion of the root canal (Kishen 2005). This cyclic influx of tissue fluid into the apical

60

portion of the root canal may promote the persistence of bacterial biofilms in this region

by providing a source of nutrients and exerting a shear force on the biofilm structure.

Single-species biofilms of E. faecalis, S. aureus, C. albicans, P. intermedia, P.

gingivalis, P. endodontalis and F. nucleatum were used to test the antimicrobial activity

of sodium hypochlorite and chlorhexidine (Sena et al. 2006). Ten day old biofilms were

generated on cellusose nitrate membranes and immersed in either sodium hypochlorite

or chlorhexidine for variable periods, with or without mechanical agitation. P.

intermedia, P. gingivalis, P. endodontalis and F. nucleatum were eliminated in 30

seconds by all antimicrobial agents, while S. aureus and E. faecalis were the most

resistant. Under conditions of mechanical agitation, E. faecalis was eliminated in 30

seconds by exposure to 5.25% sodium hypochlorite, 2.5% sodium hypochlorite or 2%

chlorhexidine liquid, while 2% chlorhexidine gel needed 5 minutes to achieve microbial

elimination. When the irrigants were not mechanically agitated, the effectiveness of the

antimicrobial agents was reduced. E. faecalis was only eliminated after a 20 minute

exposure to 2% chlorhexidine gel, while 2.5% NaOCl did not achieve total disinfection

within the time limits of the experiment (60minutes). It was hypothesized by the authors

that the agitation improved the contact between the antimicrobial agent and the test

organism in the biofilm, resulting in more efficient microbial elimination (Sena et al.

2006).

Williamson et al. subjected a 48-hour E. faecalis biofilm established on a sterile glass

microscope slide to either 6% NaOCl or 2% chlorhexidine, in formultations with or

without surface modifiers (Chlor-XTRA and CHX-Plus). The time of exposure was

61

either 1, 3 or 5 minutes. It was found that NaOCl with or without surface modifiers

reduced bacterial numbers within the biofilm significantly better than chlorhexidine,

although none achieved total bacterial elimination within the experimental time of

exposure (Williamson et al. 2009).

Mixed-species biofilms of F. nucleatum and P. micros showed time-dependent synergy

in growth and resistance to NaOCl. As the age of the biofilms increased, so did their

resistance to NaOCl. Exposure of the 24-hour mixed-species biofilm to 0.005% NaOCl

resulted in no recoverable bacteria, whereas this concentration of NaOCl had no

significant effect on the viability of the 96-hour biofilm (Ozok et al. 2007). This finding

is in accordance with earlier reports suggesting that as the age of the biofilm increases,

its susceptibility to antimicrobials decreases (Stewart et al. 2001). The increased

resistance is at least partly the result of increased numbers of bacterial cells and more

exopolymeric substance. The extremely reactive NaOCl is chemically consumed on its

way into the deeper layers of the biofilm, reducing its effectiveness (Stewart et al.

2001).

Chai et al. investigated the antimicrobial efficacy of 6 groups of antibiotics and calcium

hydroxide against a two-day E. faecalis biofilm established on membrane filter discs.

After a one hour exposure, the antimicrobial activity was neutralized by washing each

disc five times in PBS, and then the colony forming units of the remaining viable

bacteria on each disc were counted. It was concluded that erythromycin, oxytetracycline

and calcium hydroxide were 100% effective in eliminating E. faecalis biofilm, whereas

62

ampicillin, co-trimoxazole, vancomycin and vancomycin followed by gentamicin were

ineffective (Chai et al. 2007).

A recent study observed the qualitative effects of various antibiotics on endodontic

bacteria cultured from necrotic pulps and grown as biofilms on dentine slices. The

biofilms were grown for a period of 12 days, and exposed to 5 antibiotics (amoxicillin,

clindamycin, metronidazole, doxycycline and azithromycin) at their respective

minimum inhibitory concentrations. Scanning electron microscope images of biofilms

exposed to each antibiotic for 3 and 8 days showed that none of the antibiotics had any

observable effects on the biofilms (Norrington et al. 2008). As alluded to earlier, cells

growing in biofilms have been shown to be up to 1000 times more resistant to

antimicrobial agents than their planktonically growing counterparts (Ceri et al. 1999),

so the ineffectiveness of antibiotics used at minimum inhibitory concentrations (MIC) is

not surprising. This was also highlighted by Rossi-Fedele and Roberts who identified

the MIC of plate-grown E. faecalis to tetracycline was 32 μg ml -1. In the root canal

model, where the microbe had presumably established a biofilm, cells survived

irrigation with a tetracycline concentration of 30 mg ml -1, almost 1000 times more than

the concentration effective in the plate culture (Rossi-Fedele and Roberts 2007).

1.10 Rationale for experiment

Biofilm studies at this time constitute a considerably simplified microenvironment to

that of the root canal system, in which complex anatomy, inhibitive actions of dentine

and organic matter and a polymicrobial infection exponentially complicate the

63

disinfective process. Interestingly, in the field of endodontics, the evidence obtained

from biofilm research appears more consistent with the findings of clinical research,

compared to bench top studies. This is particularly so in relation to chlorhexidine,

which has gained popularity as a result of its excellent performance in vitro, (Gomes et

al. 2006; Ballal et al. 2007; Neelakantan et al. 2007; Filho et al. 2008) yet failed to

fully realize its promise in vivo (Ringel et al. 1982). Perhaps this is an indication of the

importance of the biofilms in the endodontic milieu and highlights the validity of using

the biofilm model when testing the efficacy of endodontic disinfection agents in vitro.

The most commonly used endodontic medicaments in Australia are calcium hydroxide

and the antibiotic/steroid combination of Ledermix paste (Heithersay 1984). Recently a

new product, Odontopaste, has entered the market and been promoted as a non-staining

alternative to Ledermix. In practice, many dentists and endodontists empirically

combine Ledermix and calcium hydroxide in a 50:50 mixture. Presumably, similar

practices will be adopted by practitioners who substitute Ledermix with Odontopaste.

No research has been conducted on the antimicrobial efficacy of Ledermix or

Odontopaste in the context of a microbial biofilm. Combinations of these medicaments

with calcium hydroxide are also yet to be tested in a biofilm model, while calcium

hydroxide on its own has only been evaluated in a handful of biofilm studies (Abdullah

et al. 2005; Chai et al. 2007).

An in vitro study was thus devised to test the antimicrobial efficacy of some commonly

used endodontic medicaments. E. faecalis was selected as the test microorganism

because of its apparent association with persistent disease (Molander et al. 1998;

64

Sundqvist et al. 1998; Pinheiro et al. 2003; Siqueira and Rôças 2004; Gomes et al.

2008), and as a means of comparison to other single species biofilm studies (Lima et al.

2001; Spratt et al. 2001; Abdullah et al. 2005; Dunavant et al. 2006; Chai et al. 2007;

Williamson et al. 2009). A dense biofilm was generated to evenly cover the substrate

surface. A mature biofilm represents a highly organized structure which is less

susceptible to antimicrobial effects (Stewart et al. 2001), and may therefore present a

greater and more realistic challenge to microbial killing.

Biofilm was developed on the root canal walls of hemi-sectioned teeth. It is established

that the biofilm forming capacity and its structural organization are influenced by the

chemical nature of the substrate. Biofilm experiments conducted on polycarbonate or

glass substrate will not provide a true indication of the bacteria-substrate interaction on

root canal walls (Kishen et al. 2006). In addition, exposure to dentine has been shown

to have a powerful inhibitive effect on the antimicrobial effectiveness of several key

disinfectants (Haapasalo et al. 2000). The downside of using dentine as a substrate is

that the complexity and variability of the root canal system can represent an

uncontrolled variable when evaluating antimicrobial agents. Results based on such

models reflect a combination of the antimicrobial efficacy of the agent and its access to

the biofilm (Spratt et al. 2001). To minimise the variability introduced through the

complexity of the root canal system, the roots of extracted teeth were sectioned

longitudinally to generate two flat slices incorporating half the periphery of the root

canal in each section, increasing access of the test agent to the microbial load.

Incorporation of fluid flow was a further feature designed to increase the clinical

relevance of this in vitro model.

65

The period of growth of the biofilm was selected to achieve a dense and mature

structure which evenly covered the substrate surface. Recent reports indicate that as the

age of a biofilm increases, its susceptibility to antimicrobials decreases. Ozok et al.

found that a 100-fold higher NaOCl concentration was needed to create a similar killing

effect on aged biofilms than younger ones (Ozok et al. 2007). This increased resistance

of aged biofilms can partly be explained by differences in biomass, with increased

numbers of bacterial cells and more exopolymeric substance (Stewart et al. 2001).

Recent investigations of the topography and ultrastructure of biofilms of varying

maturity indicated a time-dependant tendency for bacteria-induced corrosion of the

dentine surface, production of a mineral precipitate and potentiation of biofilm

mineralization (Kishen et al. 2006). The capacity of E. faecalis to form distinct calcified

biofilms may be another mechanism that allows this microbe to persist in

endodontically treated teeth. The maturity of the biofilm therefore appears to be a

variable with significant clinical implications, warranting in vitro efforts to replicate its

structure.

66

1.11 Aims

The aims of this study were:

1) To compare the efficacy of commonly used endodontic medicaments against E.

faecalis cultured as a biofilm on dentine substrate. The medicaments tested were

Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine and 50:50

combinations of Ledermix/calcium hydroxide and Odontopaste/calcium

hydroxide.

2) To compare the antimicrobial effect achieved through exposure to endodontic

medicaments with that achieved by exposure to a constant concentration of

sodium hypochlorite for varying times.

67

2. Materials and Methods

2.1 Organism

E. faecalis was stored as frozen stock cultures in 40% v/v glycerol at – 80oC. 100uL

aliquots were used to inoculate 20mls of Todd Hewitt Broth (Oxoid Aust.). After

overnight growth, the broth was used to inoculate the flow cells. Two strains of E.

faecalis were used: a tetracycline resistant strain ATCC 51299, and a tetracycline

sensitive strain ATCC 29212. Purity of cultures was monitored by periodic plating onto

Todd Hewitt agar (Oxoid Aust.).

2.2 Agar diffusion test

Todd Hewitt Broth agar plates were streaked with the two strains of E. faeacalis. Wells

were punched out in the agar and filled with Ledermix paste. Zones of inhibition were

evaluated after incubation for 48 hours.

2.3 Dentine sample preparation

One hundred dentine samples were produced from extracted teeth stored in thymol,

which were decoronated and sectioned longitudinally to expose the inner walls of the

pulp chamber and canals. They were then sectioned further to a size which

approximately corresponded to the dimensions of the stainless steel coupons in the flow

cell. Following sectioning, the smear layer and any pulpal tissue remnants were

removed by treating the specimens with 17% EDTA, followed by 1% sodium

hypochlorite for 4 minutes each. This was a modified disinfection protocol described by

Haapasalo and Ørstavik, who used an ultrasonic bath and a 5.25% concentration of

NaOCl (Haapasalo and Ørstavik 1987). The dentine slices were then allowed to air dry

68

for 10 minutes, before being attached to the stainless steel coupons using Araldite

adhesive.

Materials and Methods

Nutrient

Reservoir

Peristaltic

Pump

Flow Cell

Waste

Vessel

Figure 1. Experimental set-up consisting of a nutrient reservoir, peristaltic pump, flow

cell, and a waste vessel, all connected by sterile silicone tubing.

2.4 Biofilm growth

A biofilm was established using a continuous flow cell model based on that described

by Dunavant et al. (Dunavant et al. 2006). The system consisted of a nutrient reservoir,

a single channel flow cell, a peristaltic pump, and a waste vessel, all connected with

silicone tubing. The flow cell temperature was maintained at 370C using a heating bed.

69

The flow channel design incorporated circular recesses which allowed for the

placement of 9 stainless steel coupons. Each coupon had a human dentine sample

attached as described previously.

The flow cell, with coupons and dentine slices in situ, was then sterilised using ethylene

oxide. The nutrient reservoir, silicone tubing, and waste vessel were sterilised by

autoclaving at 1210C for 20 minutes and the system was carefully assembled

aseptically.

Using the in-line peristaltic pump, the flow cell was filled with Todd Hewitt broth

(Oxoid Aust.). The pump was shut off and the system was allowed to remain static for

48 hours to confirm sterility. After confirmation of sterility, 10 mls of an overnight

broth culture was introduced into the flow cell through a syringe injection port upstream

of the flow cell. The culture volume remained in the flow cell for 24 hours to allow for

bacterial growth. Laminar flow was then established at a flow rate of 20ml/h. The

volume of the flow cell was 50ml, making the dilution co-efficient 0.4 h-1.

Preliminary experiments were conducted in order to determine an appropriate time to

produce confluent adherent biofilms. The biofilm was developed in a low oxygen

tension environment at 37ºC, and two coupons were sampled weekly over four weeks

for SEM analyses.

70

Materials and Methods

The flow cell accommodated steel coupons with a human dentine sample attached to each one. Once filled with media, E.

faecalis

ATCC51299 (Tetracycline resistant strain)

Figure 2. Flow cell showing coupons and dentine slices in situ

2.5 Scanning electron microscopy (SEM)

The dentine slices were stored overnight in fixative (4% paraformaldehyde/1.25%

glutaraldehyde in PBS + 4% sucrose). They were then washed in PBS + 4% sucrose

and dehydrated in ascending concentrations of ethanol as follows:

70% ethanol – 15 minutes

90% ethanol – 15minutes

95% ethanol – 15minutes

100% ethanol – 3 changes of 10 minutes each

Following critical point drying, the samples were coated with platinum and analysed

under a scanning electron microscope (Philips XL30 field emission scanning electron

71

microscope). The biofilm was assessed for confluence, bacterial density and tubular

penetration of the dentine substrate.

2.6 Test Agents Used

- 4% sodium hypochlorite (Asis Scientific, Adelaide)

- Ledermix (DENTSPLY, Australia)

- Odontopaste (Australian Dental Manufacturing)

- Calxyl – calcium hydroxide (OCO Präparate GmbH)

- Ledermix/calcium hydroxide

- Odontopaste/calcium hydroxide

- 0.2% chlorhexidine gel (Professional Dentist Supplies Pty. Ltd.)

Each test agent in paste or gel formulation was introduced individually into the flow

cell, and applied to cover each dentine surface, while the control samples were removed

from the flow cell and placed into sterile PBS for corresponding time periods. This was

to prevent any antimicrobial effects from dissolved test agents on the control

specimens. The dentine samples were exposed to each medicament for either 24 or 48

hours.

The sodium hypochlorite was in liquid form, and the testing regimen involved

removing samples from the flow cell and submerging them in 2mls of sodium

hypochlorite, while corresponding negative control samples were placed into sterile

72

PBS. The times of exposure were 1 minute, 10 minutes, 30 minutes and 60 minutes.

Each protocol was performed in duplicate.

2.7 Determining Antimicrobial Efficacy

2.7.1 Biofilm Harvesting

Following exposure to the test agent or the control solution, each coupon was washed

three times with sterile phosphate buffered saline (PBS) to remove residual test agent.

Neutralising solutions were not used, as these were not available for all the agents

tested and consistency in the protocol precluded its use for some but not all test agents.

Following washing, the biofilm attached to each coupon was detached by sonication

into 2 mls of sterile PBS. SEM analysis showed that <1% of cells remained on the

dentine surface following sonication (data not shown).

To investigate the possibility that viable bacteria may reside in the dentinal tubule and

were not removed by the surface sampling protocol, dentine samples were exposed to

NaOCl for 10 minutes, 30 minutes and 60 minutes. They were then crushed using a

sterile metal sleeve and cylinder, and suspended in 2 mls of sterile PBS. An aliquot

(100μl) was placed into sterile saline and viable counts determined. The viable counts

obtained through crushing the dentine samples could then be compared with viability

counts obtained from surface sampling after sonication.

73

2.7.2 Cellular viability and protein measurement

Cellular viability was determined using serial plating onto Todd Hewitt agar plates

(Oxoid Aust.). Viable counts were done in duplicate for each treatment procedure.

Plates were incubated aerobically at 37ºC for 48 hours, and the number of colony

forming units (CFU) per ml was counted.

Cellular protein levels were measured to quantitate the amount of biofilm harvested.

0.9mls of the biofilm suspension used for viability counts was combined with 0.1mls of

1M NaOH, and boiled for 30 minutes. 150uL of each sample was pipetted into a

microplate well, and combined with 150uL of the Coomassie Plus protein assay reagent

(Pierce biotechnology). The microtitre plate was mixed for 30 seconds and the

absorbance read at 595nm using a microplate reader (Bio-Tek Instruments, Winooski,

VT, USA). Protein concentrations were standardised against dilutions of bovine serum

albumin (BSA) which were assayed in parallel with the unknown samples.

Colony forming units were then divided by cellular protein levels so that treatment

protocols could be compared. Comparisons with the cellular viability in control

biofilms (no treatment) allowed calculations of the percentage kill of bacteria following

treatment with the test agents.

74

3. Results

3.1 Biofilm Growth

Preliminary experiments using scanning electron microscopy were conducted to

determine an appropriate time to produce confluent adherent biofilms.

Figure 3. Scanning electron microscopy images showing biofilm structure at different

stages of development. a) 1 week b) 2 weeks c) 3weeks and d) 4 weeks

Figure 3 shows the development of the biofilm over 4 weeks. At week one, the cells

appeared quite dispersed, with few dense aggregates. Interconnecting networks of

polymer strands can be observed. At two weeks, denser cellular aggregates become

apparent, but the coverage is not confluent. Three weeks post inoculation showed a

a b

c d

75

more confluent biofilm, with thick bacterial growth covering the dentine surface,

leaving small areas of loosely organised cells. At four weeks, cells are present in dense,

multi-layered, confluent aggregations, generally arranged in short chains. Uncolonised

sections of dentine were absent.

On the basis of these findings, the growth period for subsequent biofilm development

was maintained at 4 weeks.

3.2 Dentine surface prior to inoculation

To confirm the effectiveness of the disinfection protocol used to prepare the dentine

sections, sample dentine surfaces were analysed under SEM (Philips XL30 field

emission scanning electron microscope) and are shown in Figure 4. The surfaces

appeared free of debris and microbes, and dentinal tubules appeared patent.

Figure 4 SEM images of dentine surfaces following disinfection a) 8000× magnification

b) 3500× magnification

a b

76

3.3 Effective removal of bacterial biofilm

SEM was used to observe the efficiency of removing the biofilm from the dentinal surface (Figure 5). Following 5 minutes of sonication only a small number of cells remain on the tooth surface.

Figure 5. Dentine surface following the removal of a 4 week old biofilm using

sonication.

3.4 Sodium Hypochlorite

The first experiment investigated the effect of 4% NaOCl on an E. faecalis biofilm

using exposure times of 30 minutes, 60 minutes and 6 hours. Viable bacteria could not

be cultured at any of these times compared to the control samples. An additional

experiment was then conducted to further examine the relationship between the time of

exposure and the level of bacterial killing. These experiments confirmed that total

bacterial elimination occurred at 30 and 60 minutes (Fig. 6). However, it was also

77

discovered that a 95% drop in bacterial viability occurred at 1 minute, and this

increased to 99.99% at 10 minutes of exposure.

*Viable cells expressed as colony forming units per microgram of protein

Figure 6. Effect of 4% sodium hypochlorite on E. faecalis biofilm viability. E. faecalis

biofilm established over a 4 week period was exposed to 4% sodium hypochlorite for

either 1 minute, 10 minutes, 30 minutes, 60 minutes or 360 minutes. The bacterial

viability, measured as colony forming units per microgram of protein, was then

determined for each time exposure. The data presented represents the averages of

duplicate samples from 3 flow cells.

SEM was also used to determine the effect of NaOCl treatment on the dentinal surface.

After exposure times of 10 minutes (Figure 7) and 30 minutes (Figure 8), dentine

surfaces appeared free of microbes and debris. The dentinal tubules appeared patent.

1

10

100

1000

10000

100000

1000000

0 1 1 control

10 10 control

30 30 control

60 60 control

360 360 control

*cfu

/ug

P

rote

in

Time (minutes)

Effect of 4% NaOCl on E. faecalis biofilm viability

78

Figure 7. Dentine surface following a 10 minute exposure to 4% sodium hypochlorite

(800× magnification)

Figure 8. Dentine surface following a 60 minute exposure to 4% sodium hypochlorite

(1000× magnification)

79

3.5 Crushed Samples

E. faecalis biofilm established over a 4 week period was exposed to 4% sodium

hypochlorite for 10 minutes, 30 minutes and 60 minutes. The bacterial viability,

measured as colony forming units per microgram of protein was then determined for

each time exposure, using both surface sonication and tooth crushing sampling methods.

The tooth crushing method detected viable cells (86) after 30 minutes of exposure to

NaOCl, whereas surface sampling did not detect viable cells (Fig. 9). Neither method

demonstrated bacterial viability after 60 minutes of exposure to NaOCl.

*Viable cells expressed as colony forming units per microgram of Protein

Figure 9. Detection of viable E. faecalis cells in crushed and non-crushed dentine slices

following NaOCl exposure for 10, 30 and 60 minutes. Data based on 3 samples.

0

1

2

3

4

5

6

7

10 30 60

*cfu

/ug

Pro

tein

Time (minutes)

Bacterial viability: crushed vs surface sampling

Crushed Samples

Surface Sampling

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3.6 Ledermix and Odontopaste

Ledermix and Odontopaste had no apparent effect on the cellular viability of a 4 week

E. faecalis biofilm even after 48 hours of exposure to each medicament (Fig. 10 and

12). SEM analysis of the biofilm following exposure to Ledermix (Fig. 11) and

Odontopaste (Fig. 13), showed an apparently undisturbed, thick biofilm covering the

entirety of the dentine surface.

*Viable cells expressed as colony forming units per microgram of Protein

Figure 10. Effect of Ledermix paste on E. faecalis biofilm viability. E. faecalis biofilm

established over a 4 week period was exposed to Ledermix paste for either 24 or 48

hours. The bacterial viability, measured as colony forming units per microgram of

protein was then determined for each time exposure. The data presented represents the

averages of duplicate samples taken from 2 flow cells.

1

10

100

1000

10000

100000

1000000

10000000

0 24 48 48 control

*cfu

/ug

P

rote

in

Time (hours)

Effect of Ledermix Paste on E. faecalis biofilm viabililty

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Figure 11. Dentine surface following 48 hours of exposure to Ledermix paste

*Viable cells expressed as colony forming units per microgram of Protein

Figure 12. Effect of Odontopaste on E. faecalis biofilm viability. E. faecalis biofilm

established over a 4 week period was exposed to Odontopaste for either 24 or 48 hours.

The bacterial viability, measured as colony forming units per microgram of protein was

then determined for each time exposure. The data presented represents the averages of

duplicate samples taken from a single flow cell.

1

10

100

1000

10000

100000

1000000

0 24 48 48 control

*cfu

/ug

Pro

tein

Time (hours)

Effect of Odontopaste on E. faecalis biofilm viability

82

Figure 13. Dentine surface following 48 hours of exposure to Odontopaste paste

To assess the susceptibility of a different strain of E. faecalis to Ledermix paste, a

tetracycline sensitive strain, ATCC 29212, was used to repeat the experiment. Despite

the use of this strain, no reductions in viability were detected over the 48 hour

experiment (Fig. 14). This was consistent with results from the agar diffusion test,

which showed that Ledermix was unable to achieve zones of inhibition for either of the

E. faecalis strains.

83

*Viable cells expressed as colony forming units per microgram of Protein

Figure 14. Effect of Ledermix paste on tetracycline sensitive E. faecalis biofilm. E.

faecalis biofilm established over a 4 week period was exposed to Ledermix paste for

either 24 or 48 hours. The bacterial viability, measured as colony forming units per

microgram of protein was then determined for each time exposure. The data presented

represents the averages of duplicate samples taken from a single flow cell.

3.7 Calcium Hydroxide and 50:50 combinations of Calcium

Hydroxide with either Ledermix or Odontopaste

Exposure to calcium hydroxide for 24 and 48 hours reduced the viability of E. faecalis

by > 99.9% (Fig. 15). SEM images of the dentine surface following 48 hour exposure to

calcium hydroxide show a disturbed biofilm,but with residual cells clearly present (Fig.

16). The high magnification image shows E.faecalis cells residing within the dentinal

tubules.

1

10

100

1000

10000

100000

1000000

0 24 48 48 control

*cfu

/ug

Pro

tein

Time (hours)

Effect of Ledermix on Tetracycline sensitive E. faecalis biofilm viability

84

*Viable cells expressed as colony forming units per microgram of Protein

Figure 15. Effect of calcium hydroxide on E. faecalis biofilm viability. E. faecalis

biofilm established over a 4 week period was exposed to calcium hydroxide for either

24 or 48 hours. The bacterial viability, measured as colony forming units per

microgram of protein was then determined for each time exposure. The data presented

represents the averages of duplicate samples taken from a single flow cell.

Figure 16. Dentine surface following 48 hours of exposure to calcium hydroxide

a) at 2000×magnification, b) at 10000×magnification

1

10

100

1000

10000

100000

1000000

0 24 48 48 control

*cfu

/ug

Pro

tein

Time (hrs)

Effect of Ca(OH)2 on E. faecalis biofilm viability

a b

85

Compared to the controls, calcium hydroxide combined with Ledermix and calcium

hydroxide combined with Odontopaste, were able to reduce viability of E. faecalis by >

99.9%. This was similar to the microbial reductions achieved with the sole use of

calcium hydroxide (Figure 17). SEM images of the dentine surface following 48 hour

exposures to the Ledermix/calcium hydroxide combination (Fig.18) and the

Odontopaste /calcium hydroxide combination (Fig.19), showed a disturbed biofilm, but

with residual cells clearly visible.

*Viable cells expressed as colony forming units per microgram of Protein

Figure 17. Effect of calcium hydroxide, calcium hydroxide combined with Ledermix,

and calcium hydroxide combined with Odontopaste on E. faecalis biofilm viability. E.

faecalis biofilm established over a 4 week period was exposed to calcium hydroxide for

either 24 or 48 hours. The bacterial viability, measured as colony forming units per

microgram of protein was then determined for each time exposure. The data presented

represents the averages of duplicate samples taken from 3 flow cells.

1

10

100

1000

10000

100000

1000000

0 24 48 48 control

*cfu

/ug

Pro

tein

Time (hours)

Effect of endodontic medicaments on E. faecalis biofilm viability

Calcium Hydroxide

50:50 Ledermix and Calcium Hydroxide

50:50 Odontopaste and Calcium Hydroxide

86

Figure 18. Dentine surface following 48 hours of exposure to a 50:50 Ledermix and

calcium hydroxide (10 000× magnification)

Figure 19. Dentine surface following 48 hours of exposure to 50:50 Odontopaste and

calcium hydroxide (10 000× magnification)

87

3.8 Chlorhexidine gel

Compared to the controls, 0.2% chlorhexidine gel reduced bacterial numbers by 97%

after 24 and 48 hours of exposure (Fig. 20). SEM images of the dentine surface

following a 48 hour exposure time showed a disturbed biofilm, but with residual cells

clearly visible (Fig. 21).

*Viable cells expressed as colony forming units per microgram of protein

Figure 20. Effect of 0.2% chlorhexidine gel on E. faecalis biofilm viability. E. faecalis

biofilm established over a 4 week period was exposed to calcium hydroxide for either

24 or 48 hours. The bacterial viability, measured as colony forming units per

microgram of protein was then determined for each time exposure. The data presented

represents the averages of duplicate samples taken from a single flow cell.

1

10

100

1000

10000

100000

0 24 48 48 control

*cfu

/ u

g P

rote

in

Time (hrs)

Effect of 0.2% Chlorhexidine gel on E. faecalis biofilm viability

88

Figure 21. Dentine surface following 48 hours of exposure to 0.2% chlorhexidine gel

(10 000× magnification)

89

4. Discussion

4.1 Antibiotic-containing medicaments

The results of this study indicate that antibiotic-containing medicaments alone have

little quantitative effect on the viability of E. faecalis. Exposure to either Ledermix

paste or Odontopaste for up to 48 hours did not register any observable decreases in

microbial numbers. SEM analyses found a complete inability of these medicaments to

remove biofilm. This was consistent with recent findings by Norrington et al., who

qualitatively examined mixed biofilms exposed to a variety of antibiotics (amoxicillin,

clindamycin, metronidazole, doxycycline and azithromycin) for 3 and 8 days. Scanning

electron microscope images showed that the antibiotics had no observable effect on the

biofilms (Norrington et al. 2008).

A major reason for the lack of effectiveness of either Ledermix paste or Odontopaste is

the intrinsic resistance of E. faecalis to several commonly used antibiotics, and perhaps

more importantly, its ability to acquire resistance to all currently available antibiotics,

either by mutation or by horizontal gene transfer (Sood et al. 2008). A significant

proportion of enterococcal isolates demonstrate acquired resistance to tetracyclines

(Dahlén et al. 2000; Geijersstam et al. 2006) and intrinsic resistance to clindamycin

(Singh et al. 2002). Geijersstam noted that 57 of 59 endodontic isolates of E. faecalis

displayed the classic LSA phenotype which confers intrinsic resistance to streptogramin

A and lincosamides such as clindamycin (Geijersstam et al. 2006). Similarly, Dahlén et

al. found 25 out of the 26 E. feacalis species isolated from root canals to be resistant to

clindamycin (Dahlén et al. 2000). In terms of tetracycline resistance, Dahlén et al.

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found 4 out of 29 enterococcal strains to be highly resistant to tetracycline, while

Geijersstam et al. identified 17 tetracycline resistant isolates out of 59 (Geijersstam et

al. 2006). The close proximity of bacterial cells within the biofilm readily allows the

exchange of genetic information, including the transfer of antibiotic resistance. Two

strains of E. faecalis with reportedly different levels of tetracycline sensitivity were

used in this experiment to test the efficacy of Ledermix paste. No significant reductions

in bacterial viability could be noted for either strain and this was supported by the lack

of susceptibility to Ledermix paste by the two strains using the plate diffusion method.

This may well be an indication that the level of tetracycline in Ledermix paste is too

low to affect antibiosis. We were unable to source a strain of E. faecalis susceptible to

clindamycin, an indication of how widespread resistance to this antibiotic is among

enterococcal species.

In addition to the intrinsic or acquired antibiotic resistance of E. faecalis, the

organisation of cells in a biofilm structure could be a further factor contributing to the

lack of antimicrobial efficacy of Ledermix paste and Odontopaste. Cells growing in

biofilms have been shown to be up to 1000 times more resistant to antimicrobial agents

than their planktonically growing counterparts (Ceri et al. 1999). This was highlighted

by Rossi-Fedele and Roberts, who identified the MIC of plate-grown E. faecalis to

tetracycline to be 32 μg ml-1. In the root canal model, where the microbe had

presumably established a biofilm, cells survived irrigation with a tetracycline

concentration of 30 mg ml -1, almost 1000 times more than the concentration that had

been effective in the plate culture (Rossi-Fedele and Roberts 2007).

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Concern has been raised about the capacity of demeclocycline HCl in Ledermix to

diffuse to the peripheral parts of dentine in sufficient concentrations to inactivate

bacteria (Abbott et al, 1990). In this set of experiments, however, even the superficial

surface of the dentine remained laden with E. faecalis biofilm seemingly unaffected by

the antimicrobial effects of the antibiotic. This was also seen with the more recently

introduced Odontopaste. Laboratory and clinical research is urgently needed to assess

the properties of this new product. Its clinical benefit is likely to be derived from the

potential anti-resorptive and sedative properties of the steroid component, rather than

the antibacterial effects of the antibiotic, but this is yet to be supported by evidence, as

are the manufacturers’ claims that it causes no dentinal discolouration (ADM).

4.2 Calcium Hydroxide

Our results showed that calcium hydroxide was able to achieve reductions in microbial

viability of 99.9% at 24 and 48 hours of exposure. This finding was at variance with

reports showing that the antimicrobial effectiveness of calcium hydroxide is abolished

in the presence of dentine (Haapasalo et al 2000; Portenier et al 2001). This

inconsistency in findings is most likely a reflection of methodological differences, with

the present study involving sections of dentine rather than the vast active surface area of

dentine in powder form. Consistent with other reports, calcium hydroxide was found to

be unable to achieve total elimination of E. faecalis cells. The persistence of a small

proportion of bacteria was confirmed through scanning electron microscopy. Many of

the persisting cells were seen to be present in the protective environment of the dentinal

tubules. Similar findings were noted when calcium hydroxide was combined with either

Ledermix or Odontopaste. The combining of antibiotic based medicaments with

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calcium hydroxide did not appear to elicit any synergistic action, nor did it diminish the

effectiveness of calcium hydroxide when used alone. It is therefore unlikely that adding

antibiotic based medicaments will improve disinfection within the root canal. Whether

the high alkalinity of the calcium hydroxide influences the properties and diffusability

of the steroid component of Ledermix or Odontopaste, and the therapeutic effect

achieved through these combinations has yet to be adequately investigated.

The antimicrobial activity of calcium hydroxide is due to the release and diffusion of

hydroxyl ions, which produce a highly alkaline environment incompatible with

microbial survival. For calcium hydroxide to act effectively as an intracanal dressing,

the hydroxyl ions must be able to diffuse through dentine and pulpal tissue remnants.

Studies have demonstrated that hydroxyl ions derived from a calcium hydroxide

medication do diffuse through root dentine, but the pH values decrease as a function of

distance from the main canal (Nerwich et al. 1993). One of the limitations of our study

was the relatively short time of exposure of E. faecalis to the calcium hydroxide

containing medicaments. Forty-eight hours may not have been sufficient to allow

hydroxyl ions to reach the deeper layers of root dentine. Other reports, however, have

demonstrated the inability of calcium hydroxide to eliminate E. faecalis from dentinal

tubules, even superficially, after incubation periods of 10 days (Haapasalo and Ørstavik

1987), and 7 days (Heling et al. 1992). It is, therefore, unlikely that total bacterial

elimination would have been achieved had the exposure time been extended.

Abdullah et al. found calcium hydroxide to be equally effective against bacteria in a

planktonic and biofilm presentation. Although this medicament was not able to achieve

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100% elimination of E. faecalis cells, a greater than 4 minute exposure resulted in

99.9% bacterial reduction (Abdullah et al. 2005), a finding replicated by the present

study. Much of the recent research has been critical of calcium hydroxide, yet in the

context of a microbial biofilm (Abdullah et al. 2005) and in clinical studies (Byström et

al. 1985; Sjögren et al. 1991; Shuping et al. 2000), it has been shown to be highly

efficacious. A possible explanation for its efficacy against microbial biofilms is that

calcium hydroxide is capable of dissolving organic tissue, and is therefore less inhibited

by dense cellular aggregations and exopolysaccharide matrix. Tissue dissolution is a

property that is neglected in investigations of antimicrobial efficacy, probably because

it is redundant in study designs incorporating agar diffusion or bacterial suspension

methods. In clinical endodontics, however, the capacity of a medicament to physically

dissolve necrotic pulpal tissue or microbial colonies is critical to achieving disinfection.

Despite its ability to drastically reduce bacterial numbers, calcium hydroxide failed to

achieve sterilisation of the dentine surface. One explanation for this is the well

recognised capacity of E. faecalis to resist the antibacterial effect of calcium hydroxide

(Haapasalo and Ørstavik 1987; Safavi et al. 1990; Siqueira and de Uzeda 1996).

Byström et al. showed that the pH needs to reach levels of 11.5 or higher to exert a

bactericidal effect on E. faecalis (Byström et al. 1985). Investigations of the

mechanisms involved in the resistance of E. faecalis to calcium hydroxide identified

that its survival at high pH was related to the effective function of its proton pump.

When cells were exposed to calcium hydroxide at pH 11.1 for 30 minutes, there was a

20-fold reduction in cell survival in the presence of the proton pump inhibitor CCCP

(Evans et al. 2002). Futhermore, the buffering effects of dentine may not allow a

sufficiently high pH to be achieved in the dentinal tubules (Haapasalo et al. 2000). The

94

biofilm forming ability of E. faecalis constitutes another important survival strategy

(Distel et al. 2002), while its proficiency at invading dentinal tubules affords the

organism physical protection from chemomechanical root canal preparation and

intracanal dressings (Love 2001).

4.3 Chlorhexidine gel

Chlorhexidine gel was able to eliminate 97% of bacteria at 24 and 48 hours of exposure,

making it less effective than calcium hydroxide (99.9%), calcium hydroxide combined

with Ledermix (99.9%) and calcium hydroxide combined with Odontopaste (99.9%).

Chlorhexidine was also considerably less effective than sodium hypochlorite (100%), a

consistent finding in biofilm studies. According to Dunavant et al., 6% sodium

hypochlorite achieved >99.99% elimination of E. faeacalis cells after a 5 minute

exposure, while 2% chlorhexidine achieved only a 60.5% bacterial reduction (Dunavant

et al. 2006). In comparison to these findings, chlorhexidine performed considerably

better in our experiment. This is most probably due to the longer exposure time, and the

greater initial volume of biofilm to which the persisting bacterial load is related to in the

calculations of percentage kill. Quantitatively, 3% bacterial survival may represent a

very significant number if the initial bioburden was extensive. SEM images showed

residual bacteria and exopolymeric material persisting on the dentine surface. This is

consistent with findings by Clegg et al., who noted a virtually intact biofilm after

exposure to chlorhexidine, and suspected the antimicrobial of fixation (Clegg et al.

2006).

95

Chlorhexidine has consistently been found less effective than sodium hypochlorite in

biofilm studies (Abdullah et al. 2005; Dunavant et al. 2006; Williamson et al. 2009).

This is in contrast to in vitro experiments using broth dilution tests on suspended cells

or in models where bacterial cells are used to inoculate an extracted root. Such study

models generally find the antibacterial effectiveness of chlorhexidine and sodium

hypochlorite to be similar (Gomes et al. 2001; Vianna et al. 2004; Oncag et al. 2003).

In vivo studies have generated less favourable results when evaluating the antimicrobial

efficacy of chlorhexidine. Many have found that the microbial reduction achieved

through mechanical instrumentation and the use of an antimicrobial irrigant could not

be improved upon with the use of an additional chlorhexidine medicament, and in some

cases the number of recoverable microorganisms actually increased after a

chlorhexidine dressing was placed (Manzur et al. 2007; Paquette et al. 2007). Vianna et

al. compared the degree of microbial reduction after chemo-mechanical preparation

using either sodium hypochlorite or chlorhexidine in vivo. Significantly greater

bacterial reduction was achieved through the use of NaOCl compared to chlorhexidine

(99.9% vs 96.6%) (Vianna et al. 2006).

The activity of chlorhexidine is pH dependent, and is greatly reduced in the presence of

organic matter (McDonnell and Russel, 1999). Its inability to dissolve organic tissues is

therefore a major shortcoming, and responsible for the reduced antimicrobial

effectiveness of chlorhexidine when challenged by an increased bioburden. Sodium

hypochlorite, on the other hand, acts as a solvent in the presence of organic tissue,

releasing chlorine which, combined with the protein amino group, forms chloramines

(Baumgartner and Cuenin 1992). This tissue dissolving ability is one of the features that

makes sodium hypochlorite such an effective endodontic irrigant.

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4.4 Sodium Hypochlorite

In this study, 4% sodium hypochlorite was the only agent capable of totally eliminating

the E. faecalis biofilm. Its antimicrobial efficacy was time dependant, with a 95% drop

in bacterial viability noted at 1 minute, 99.9% at 10 minutes, and no detectable bacteria

cultured at 30 or 60 minutes. Crushing the dentine revealed viable cells after 30 minutes

of sodium hypochlorite exposure, even though surface sonication failed to produce any

colony forming units from the same dentine slice. This highlights the limitation of

surface sampling methods in assessing disinfection, such as the paper point sampling

used in clinical endodontics. A negative culture result is merely an indication that

microbial numbers are below the point of detection, rather than a guarantee of sterility.

Scanning electron microscopy supported the quantitative findings, with small numbers

of E. faecalis cells and polymeric matrix substance visible after 1 minute. Longer

exposure times produced dentine walls free of microbial cells or debris, with patent

dentinal tubules. Variable degrees of surface erosion were noted, and this appeared to

be a function of time. Clinically this indicates that sodium hypochlorite is capable of

both eliminating infecting microbes and physically removing them, but its action is

time-dependant. Continuous replenishing with fresh solution and ultrasonic activation

are mechanisms that may speed up the antimicrobial and tissue dissolving action of

sodium hypochlorite (Sjögren and Sundqvist 1987; Spoleti et al. 2003), although this is

yet to be shown against a bacterial biofilm. Consequently, the time required to exert its

effect in the root canal may be reduced, limiting the structural damage to dentine.

97

4.5 Limitations and future research direction

The length of time to establish the experimental biofilm, the limited number of samples

per flow cell and the need to include control and SEM samples in each cell limited the

number of experiments that could be conducted within the study period. As such, the

sample sizes are relatively small and preclude statistical analyses of the results. A flow

cell capable of accommodating a greater number of specimens would be more efficient

at achieving such statistical power. Attempts to use reverse transcriptase PCR (RT-

PCR) to survey cellular viability in samples obtained with sodium hypochlorite were

unsuccessful, and further work to streamline the experimental protocol relating to PCR

is being undertaken. This would be particularly useful in investigating whether E.

faecalis cells adopt the VBNC state as a function of antimicrobial agent exposure.

Live/dead stain and quantitative (real time) PCR would also have contributed positively

to this research. Further limitations of this study are that a bacterial monospecies was

used to establish the biofilm. Microbiological studies using culturing techniques have

frequently isolated an E. faecalis monoinfection in failing root canals (Sundqvist et al.

1998; Pinheiro et al. 2003), although this is more likely a reflection of the limitations of

the sampling and culturing technique than the true state of infected post-treatment root

canals.

The biofilm in our study received a continuous and plentiful nutrient supply in the form

of Todd Hewitt Broth which flowed over the biofilm surface at a constant rate. It has

been documented that certain biofilm characteristics such as density of growth and

depth of dentine tubule penetration are influenced by the level of nutrition and oxygen

concentration to which they are exposed (George et al. 2005). The growth environment

98

in the enclosed cell provided the cells with very low oxygen concentration, but

repeating the experiment with a reduced nutrient supply may have altered some aspects

of the biofilm structure and potentially influenced its susceptibility to the test agents.

The dentine sections maximised the area of contact between the bacteria and the

antimicrobials. An experimental model incorporating root canals of standardised sizes

may have offered a greater antimicrobial challenge.

Saline controls throughout this study showed a measurable reduction in viable cells.

This reduction can be attributed to the removal of the biofilm from its nutrient source,

leading to the death of some of the cell populations. After 48 hours in nutrient-poor

PBS this outcome was not unexpected. The osmolarity of the saline may have

contributed to additional cell damage.

One of the potential concerns of using sonication to harvest the biofilm following

exposure to test agents is that some cells may be physically damaged through this

process. However, this is a protocol that has been used previously in biofilm research

(Chai et al. 2007), without reports of cellular damage. Furthermore, high magnification

SEM images in this study confirm the presence of intact cells post-sonication,

indicating that any damaging influence of sonication must be minor.

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5. Conclusions

When used in isolation, antibiotic containing medicaments had no appreciable effect

on the viability of E. faecalis. Using a tetracycline sensitive strain did not improve the

antimicrobial effectiveness of Ledermix paste. Calcium hydroxide in isolation, and

when combined with Ledermix or Odontopaste, achieved a 99.9% reduction in E.

faecalis viability, compared to 97% achieved by chlorhexidine gel. The antimicrobial

effectiveness of sodium hypochlorite increased exponentially with increased exposure

time. Total bacterial elimination was predictably achieved at 30 minutes of exposure.

A comparison of sampling methods showed that crushing the dentine sample was able

to identify bacterial viability in specimens deemed sterile through surface sampling.

100

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113

7. Appendix

Ledermix

E. faecalis biofilm was exposed to Ledermix paste for 24 and 48 hours. The number of

colony forming units was calculated, and divided by the protein levels in order to

determine viability. The percentage kill could then be calculated. See Table 1,2 and 3.

Time of

exposure (hrs)

Viable count

(cfu/ml)

Protein (µg/ml) Viability

(cfu/mg protein)

Percentage kill

(%)

0 6.39 × 107 52.0 1.23 × 106

0 8.99 × 107 52.2 1.72 × 106

24 3.73 × 107 48.7 7.66 × 105 48

24 3.87 × 107 51.4 7.53 × 105 49

48 3.21 × 107 28.3 1.13 × 106 23

48 1.54 × 107 23.9 6.44 × 105 56

48 (control) 8.9 × 106 3.8 2.34 × 106 0

48 (control) 2.86 × 107 32.8 8.72 × 105 41

Table 1. Ledermix paste and tetracycline resistant strain ATCC 51299

114

Time of

exposure (hrs)

Viable count

(cfu/ml)

Protein (µg/ml) Viability

(cfu/mg

protein)

Percentage kill

(%)

0 4.25 × 106 31.5 1.35 × 105

24 1.5 × 107 26.6 5.6 ×105 0

24 1.5 × 107 17.3 8.7 ×105 0

48 5.0 × 106 12.32 4.1 ×105 0

48 1.34 × 106 13.33 1.0 ×105 26

Table 2. Ledermix paste and tetracycline resistant strain ATCC 51299

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

0 4.19 × 106 12.7 3.3 × 105

0 3.28 × 106 17.7 1.9 × 105

24 4.41 × 106 34.1 1.3 × 105 50

24 7.09 × 106 56.9 1.2 × 105 54

48 5.52 × 106 40.9 1.3 × 105 50

48 5.76 × 106 33.8 1.7 × 105 35

48 control 1.91 × 106 31.9 6.0 × 104 77

48 control 1.74 × 106 17.8 9.8 × 104 62

Table 3. Ledermix paste and tetracycline sensitive strain ATCC 29212.

115

4% Sodium Hypochlorite

E. faecalis biofilm was exposed to 4% NaOCl for 30 minutes, 60 minutes and 360

minutes. The number of colony forming units was calculated, and divided by the

protein levels in order to determine viability. The percentage kill could then be

calculated. See Table 4, 5, and 6 .

Time of

exposure

(minutes)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage

kill (%)

0 4.5 × 106 23.2 1.9 × 105

0 4.2 × 106 8.1 5.2 × 105

30 0 17.2 0 100

30 0 29.0 0 100

60 0 22.7 0 100

60 0 21.7 0 100

60 control 1.35 ×106 14.3 9.4 ×104 74

360 0 69.3 0 100

360 control 4.5 ×106 15.38 2.9 ×105 18

Table 4. Sodium hypochlorite

116

Time of

exposure (min)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

0 8.25 × 106 99.8 8.3 × 104

0 1.24 × 107 74.0 1.7 × 105

30 0 74.8 0 100

30 0 30.4 0 100

30 control 3.38 × 107 181.6 1.9 × 105 0

60 0 64.2 0 100

60 0 32.7 0 100

60 control 1.52 × 107 104.3 1.5 × 105 0

Table 5. Sodium hypochlorite

Time of

exposure (min)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

0 9.1 × 105 26.7 3.4 × 104

0 2.32 × 106 77.7 3.0 × 104

1 5.3 × 104 21.6 2.5 × 103 92

1 5.2 × 104 70.4 7.4 × 102 98

1 control 6.7 × 106 80.0 8.4 × 104 0

10 9 × 10 13.4 6.7 99.9

10 0 30.4 0 100

10 control 3.6 × 106 37.5 9.6 × 104 0

Table 6. Sodium hypochlorite

117

Crushed Samples 10, 30, 60 min

E. faecalis biofilm was exposed to 4% Sodium Hypochlorite for 10 minutes, 30

minutes, and 60 minutes. The number of colony forming units was calculated using

both surface sonication and tooth crushing sampling methods. This was divided by the

protein levels in order to determine viability. Percentage kill could then be calculated.

See Table 7

Time of

exposure (min)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

10 471 73.6 6.4 99.9

30 86 107.9 0.8 99.9

60 0 104.2 0 100

Table 7. Crushed vs Surface sampling

118

Odontopaste

E. faecalis biofilm was exposed to Odontopaste for 24 and 48 hours. The number of

colony forming units was calculated, and divided by the protein levels in order to

determine viability. The percentage kill could then be calculated. See Table 8

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg protein)

Percentage

kill (%)

0 6.7 × 106 69.8 9.6 ×104

0 6.3 × 106 44.5 1.4 ×105

24 1.7 × 106 80.8 2.1 ×104 82.2

24 3.9 × 106 62.5 6.2 ×104 47.4

48 2.1 × 106 61.4 3.4 ×104 71.1

48 1.8 × 106 60.7 3.0 ×104 74.5

48 control 2.6 × 106 58.9 4.4 ×104 62.7

48 control 8.7 ×105 55.5 1.6 ×104 86.4

Table 8. Odontopaste

119

Calcium Hydroxide

E. faecalis biofilm was exposed to calcium hydroxide for 24 and 48 hours. The number

of colony forming units was calculated, and divided by the protein levels in order to

determine viability. The percentage kill could then be calculated. See Table 9.

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg protein)

Percentage

kill (%)

0 6.2 × 106 16.4 3.8 ×105

0 4.6 × 106 8.3 5.5 ×105

24 112 44.4 2.5 99.9

24 95 40.2 2.4 99.9

48 347 44.0 7.9 99.9

48 60 54.6 1.1 99.9

48 control 4.9 × 106 48.3 1.0 ×105 78.5

48 control 2.2 × 106 29.3 7.5 ×104 83.9

Table 9. Calcium Hydroxide

120

50:50 Ledermix and Calcium Hydroxide

E. faecalis biofilm was exposed to a 50:50 mixture of Ledermix and Calcium

Hydroxide for 24 and 48 hours. The number of colony forming units was calculated,

and divided by the protein levels in order to determine viability. The percentage kill

could then be calculated. See Table 10

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg protein)

Percentage

kill (%)

0 9.1 × 106 51.3 1.8 × 105

0 1.5 × 107 49.8 3.0 × 105

24 93 65.8 1.4 99.9

24 145 86.7 1.7 99.9

48 120 50.3 2.4 99.9

48 240 73.6 3.3 99.9

48 control 9.6 × 105 46.2 2.1 × 104 91.3

48 control 3.5 × 106 69.0 5.1 × 104 78.8

Table 10. 50:50 Ledermix and Calcium Hydroxide

121

50:50 Odontopaste and Calcium Hydroxide

E. faecalis biofilm was exposed to a 50:50 mixture of Odontopaste and Calcium

Hydroxide for 24 and 48 hours. The number of colony forming units was calculated,

and divided by the protein levels in order to determine viability. The percentage kill

could then be calculated. See Table 11

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

0 5.7 × 106 29.7 1.9 × 105

0 3.1 × 106 60 5.2 × 104

24 180 71.3 2.5 99.9

24 180 178.3 1.0 99.9

48 4.15 × 103 54.7 76 99.9

48 3.87 × 103 117.4 33 99.9

48 control 1.76 × 106 122.5 1.4 × 104 88.4

48 control 1.02 × 106 73.1 1.4 × 104 88.4

Table 11. 50:50 Odontopaste and Calcium Hydroxide

122

Chlorhexidine gel 0.2%

E. faecalis biofilm was exposed to 0.2% chlorhexidine gel for 24 and 48 hours. The

number of colony forming units was calculated, and divided by the protein levels in

order to determine viability. The percentage kill could then be calculated. See Table 12.

Time of

exposure

(hours)

Viable count

(cfu/ml)

Protein

(µg/ml)

Viability

(cfu/mg

protein)

Percentage kill

(%)

0 1.05 × 106 22.9 4.6 × 104

0 2.9 × 106 38.7 7.5 × 104

24 6.1 × 104 50.7 1.2 × 103 98

24 9.5 × 104 52.5 1.8 × 103 97

48 1.23 × 105 51.6 2.4 × 103 96

48 7.25 × 104 79.4 9.1 × 102 98

48 control 5.9 × 105 38.5 1.5 × 104 75

48 control 7.4 × 105 65.6 1.1 × 104 82

Table 12. Chlorhexidine gel