Athanassiadis, Basil

Walsh, Laurence J.

University of Queensland eSpace

102 Australasian Dental Practice May/June 2008
W ith a range of new methods and technolo-gies to support modern endodontictreatment, it is useful to take stock and
consider the prime goals of therapy and how these
are best achieved.
Current approaches to treatment are based on
instrumentation, irrigation and medication of the
root canal system, with the primary goal of elimi-
nating bacteria prior to placing a root canal filling.
Molecular methods have demonstrated that bac-
teria can be detected in the root canal system in
virtually all cases of root-filled teeth which have
persisting periapical radiolucencies. These bacteria
are found not only within the root canal itself, but
also in dentinal tubules, accessory canals, canal
ramifications, apical deltas, fins and transverse
anastomoses - areas which are difficult to access
using mechanical instrumentation and irrigation
when re-treatment is contemplated.
As we and others have described in recent litera-
ture reviews,1,2 disinfection of the root canal system
is not an easy objective to achieve. Where bacterial
biofilms are well established in the root canal, the
joint issues of contact time with antimicrobial
agents and penetration into biofilms come to the
fore. Even when using potent antimicrobial agents
and techniques, achieving reliable and complete
disinfection remains a challenge. The published lit-
erature indicates that current treatment protocols
with rotary NiTi instrumentation and copious
antibacterial irrigation will render some 50%-70%
of infected canals free of microorganisms by cul-
ture-based methods. This does not, however, mean
an absence of viable microorganisms.
The biofilm challenge
Biofilms provide a powerful barrier to the 
diffusion of medicaments, and it is essential that
materials and techniques used for endodontics are
tested not in plate or broth cultures but rather using
biofilm models - since in the latter the microorgan-
isms will be in a metabolically less active 
state, making them less prone to inactivation by 
antimicrobial agents.
Resistance of bacterial biofilms to antibiotics
can occur due to several factors.1 Firstly, the
polysaccharide matrix of the biofilm retards diffu-
sion of the antibiotic. Secondly, the chemical
milieu in the biofilm (low oxygen tension, low pH,
waste products, enzymes) can antagonize the
effects of the antibiotic. Thirdly, in response to
depletion of substrate and/or the accumulation of
waste products, bacteria in the biofilm enter a qui-
escent non-growing state, rendering them safe
from agents which target the metabolic activity of
bacterial replication. Finally, subpopulations of
bacteria in a biofilm may enter a phenotypic state
with altered gene expression which is akin to spore
formation. Because of these changes, bacteria in
biofilms may display a 1000-1500 times greater
resistance to antibiotics than when in the freely
dispersed planktonic form.
Resistant species
With incomplete disinfection of the root canal, 
the spectre of selection and dominance of resistant
species emerges. In dentistry, it is easy to 
overlook the fact that Enterococcus faecalis
possesses several characteristics akin to the 
The challenge of endodontic 
“superbugs” in clinical practice
By Laurence J. Walsh and Basil Athanassiadis
“disinfection 
of the root canal
system is not an
easy objective 
to achieve. Even
using potent
antimicrobial
agents and 
techniques,
achieving reliable
and complete
disinfection
remains a 
challenge...”
infection | CONTROL
“superbugs” associated with hospital-
based nosocomial infections. Of all the
organisms which have been found in root
canals, E. faecalis has the most well
developed (and well deserved) reputation
as a difficult target for antibiotic and
microbial therapy.
In relation to antibiotics, the family of
enteroccoci have both intrinsic resistance
(where the gene for intrinsic resistance
resides on the chromosome) and acquired
resistance (from mutations in existing
DNA or the acquisition of new DNA).
They are inherently more resistant to
antimicrobial drugs than any other clini-
cally important Gram-positive bacteria
encountered in dentistry or medicine.
Enterococci are intrinsically resistant to
many common antimicrobial agents,
including cephalosporins, clindamycin,
penicillinase-resistant penicillins, and
vancomycin, and they have acquired 
resistance to many other classes of 
antimicrobials, including tetracyclines,
erythromycin, chloramphenicol, ciprofloxacin
and vancomycin. Because of this heredity,
E. faecalis has intrinsic resistance to 
clindamycin, cephalosporins and amino-
glycosides. There is strong evidence that
most clinical strains of E. faecalis isolated
from the oral cavity are resistant to both
clindamycin and to tetracyclines3-8 - pro-
viding a good reason not to use these in
root canal medicaments (Table 1).
These resistance patterns explain why
clinical microbiological studies using cur-
rent products show persisting bacterial
growth after the use of current inter-visit
medicaments. In a recent (2006) study,
samples were taken before and after two-
visit endodontic treatment from 88 canals
with apical periodontitis. All of the canals
but one had cultivable growth before 
treatment. After dressing with Ledermix,
Septomixine, or Calasept (calcium
hydroxide), the percentages of canals with
positive growth on culture were 48% 
(13 of 27), 31% (8 of 26), and 31% (11 of
35), respectively. In the Ledermix group, 
38 strains of bacteria were recovered, with
25 from the Septomixine group, and 25
from the Calasept group.9 A key finding in
this study was that Gram-positive faculta-
tive anaerobic cocci - such as Enterococci
- were the survivors, whereas the Gram-
negative obligate anaerobic rods were
easily inactivated.
The great unknown?
Adding to this is the issue of non-cultivable
flora present in root canals and other sites
within the oral cavity - of the more than 700
different species which can be detected by
molecular methods, 50% are non-cul-
tivable, and thus our understanding of
endodontic infections is not absolute. In all
likelihood, there are other species, yet to be
cultivated, which may share characteristics
of E. faecalis (Table 2).
Studies using bacterial cultivation
methods have shown that infected necrotic
pulps, and pulpless, infected teeth (i.e. teeth
without any previous endodontic treatment,
have a polymicrobial flora with 4-7 species
present, mostly strict anaerobes, with
approximately equal proportions of gram-
negative and gram-positive organisms.12,13
In contrast, previously root-filled teeth with
apical periodontitis have 1-2 cultivable
species, and these are dominated by facul-
tative anaerobic Gram-positive bacteria
such as E. faecalis. 14-16 Using molecular
methods, which recover the non-cultivable
species, gives a rather different picture,
with 1-5 species present in well treated
cases with sound coronal restorations, and
2-11 species in teeth with defective coronal
restorations.16,17 Based on the published lit-
erature, a reasonable estimate would place
the number of bacterial species in infected
root canals between 10 and 30 - a much
greater challenge to disinfection than previ-
ously thought. In fact, the argument could
be made that endodontic procedures, if
done poorly, select for the more robust and
resilient organisms (such as E. faecalis),
because the more susceptible Gram-nega-
tive anaerobes are relatively easily
eliminated. Data from culture and molec-
ular studies indicate that E. faecalis may be
present in from 29% to 70% of root-filled
teeth with periradicular lesions.18,19
May/June 2008 Australasian Dental Practice 103
infection | CONTROL
Table 1. Antibiotic Resistance in Enterococci
Antibiotic family Type of resistance
ß-Lactams (penicillins, cephalosporins) Intrinsic resistance. Can also acquire
Beta lactamase production.
Tetracyclines Acquired
Lincosamides (clindamycin) Low level intrinsic resistance.
Can acquire high level resistance.
Macrolides (erythromycin, etc) Acquired
Aminoglycosides (gentamycin, etc) Low level intrinsic resistance.
Can acquire high level resistance.
Glycopeptides (vancomycin, etc) Acquired
Based on Refs 3,7,8,10 and 11
Table 2. Survival factors for E. faecalis and 
related non-cultivable Gram positive facultative anaerobes
 Natural resistance to many antibiotics
 Low susceptibility to many biocides
 Can live and persist in the poor nutrient environment of endodontically-treated teeth
 Can survive in a quiescent non-cultivable phase with low metabolic activity for
extended periods of time
 Can tolerate a broad range of pH values (from 4-11)
 Forms dense biofilms
 Can invade dentinal tubules up to 300 microns
 Can tolerate calcium hydroxide
Taking this on board and looking at the
range of agents currently used as inter-
appointment medicaments, a range of
problems can be identified (Tables 3 and
4). Despite recent trends to use other
antibiotics in these medicaments, from
first principles, in a polymicrobial infec-
tion the decision to use a single antibiotic
can be questioned. It is more likely a com-
bination of agents would be needed to
address the diverse flora encountered. If a
combination of antibiotics were used, this
would decrease the likelihood of resistant
strains developing. The typical antibiotic-
related long term problems of resistance
and sensitization would remain, however.
Clues from the past
Given these problems, it may be timely to
revisit other strategies for dealing with 
persistent bacteria which do not rely only
upon inactivating a specific biochemical
pathway (as do antibiotics), but instead
taking a wide approach by using biocides.
Because biocides have a broader spectrum
of activity, working on multiple target sites
within microorganisms, bacterial resistance
to biocides does not develop, although it
should be recognized that some bacteria are
naturally resistant to certain biocides
because of their cell wall structure.
Biocide activity is affected by several
factors - notably their concentration, period
of contact, pH, and temperature. The pres-
ence of organic matter is a major issue,
since this can interfere with the antimicro-
bial effects exerted by some biocides - with
chlorhexidine being a good example. Con-
versely, the activity of biocides can be
enhanced by the use of chemical agents
such as EDTA which increase the perme-
ability of bacterial cell membranes.
Whilst there are many biocides which
could be used, chlorhexidine (CHX)
would rate well amongst other candidates
in the field, despite some of its issues.
Slow release forms of CHX and mixtures
of CHX with other agents (such as calcium
hydroxide) appear promising20-22 in terms
of penetration into dentinal tubules and
antibacterial effects against E. faecalis.
Back to the future
Manipulating the physical, ionic, and
metabolic factors which modulate the prop-
erties of the biofilm may provides a new
approach to dealing with endodontic
pathogens - as an adjunct to effective antimi-
crobial medicaments. These approaches are
now being explored for dental caries.23,24
Biofilm properties may be manipulated by
influencing cell to cell signaling within the
biofilm. Blocking this “quorum sensing”
would reduce the ability of the biofilm to tol-
erate stresses such as reductions in nutrients
or assault by external chemical agents (such
as biocides). Slowing the biofilm accumula-
tion rate may be possible using agents which
affect quorum sensing. “Magic bullet” and
“smart bomb” therapies are of ongoing
interest. Using photosensitization via
endogenous (protoporphyrin) or exogenous
(sensitizer) molecules in biofilms with light
as the vector may overcome the penetration
problem,25-27 and remains an important area
for further investigation.
104 Australasian Dental Practice May/June 2008
infection | CONTROL
Table 4. Factors which reduce the 
effectiveness of topical antimicrobial agents
 Resistance
 Poor diffusion
 Inadequate exposure (dose and time)
 Poor topical delivery system (bio-availability)
 Reservoirs of re-infection such as foreign bodies
 Agent is not active (e.g. because of pH)
 Inactivation by host or bacterial factors 
Table 3. Issues with contemporary endodontic medicaments
Calcium hydroxide pastes Effects are limited by buffering of alkaline pH by dentine
proteins, particularly in the apical third of the canal.
Final pH and hydroxyl ion release vary according to the
vehicle used in the paste. Limited if any penetration into
dentine tubules. Only limited inactivation of E. faecalis.
Ledermix (Lederle), Based on 3.2% Demeclocycline hydrochloride. This
Endopaste (ADM) tetracycline has a bacteriostatic rather than bactericidal
action. Effects are short-lived because of diffusion. 
Demineralizes and complexes with dentine, resulting in
staining, a process accelerated by light. Limited 
inactivation of E. faecalis because of natural resistance 
to tetracyclines.
Odontopaste (ADM) Based on 5% Clindamycin. Does not have the dentine
staining issue of tetracyclines. Limited inactivation of E.
faecalis because of natural (intrinsic) low-level resistance
to lincosamides, which is increased by acquired resis-
tance in clinical conditions.
Septomixine Forte Contains neomycin, polymyxin B sulphate, and
(Septodont) tyrothricin. Issues with limited spectra of activity, since
neomycin is bactericidal against Gram-negative bacilli
but is ineffective against Bacteriodes spp, while
polymyxin B is ineffective against Gram-positive bac-
teria. Little or no inactivation of E. faecalis.
Chlorhexidine pastes Inactivated by residues of sodium hypochlorite, forming
para-chloro-aniline as a by-product. Binds to the surface
and has substantivity, but does not penetrate into tubules.
Inactivated by high organic loads. Partly inactivated by
serum, dentine proteins and hydroxyapatite. Moderately
effective against E. faecalis but only as an irrigant.
References
1. Athanassiadis B, Abbott PV, Walsh LJ.  The use of
calcium hydroxide, antibiotics and biocides as antimi-
crobial medicaments in endodontics. Aust Dent J.
2007; 52(1 Suppl):S64-S82.
2. Orstavik D. Root canal disinfection: a review of
concepts and recent developments. Aust Endod J
2003; 29:70-4.
3. Murray BE. The life and times of Enterococcus.
Clin Microbiol Rev 1990; 3: 46-65.
4. Clewell DB, Courvalin P, Dunny GM, Murray BE,
Rice L. The Enterococci. Pathogenesis, molecular
biology and antibiotic resistance. ASM Press 2002.
5. Dina J, Malbruny B, Leclercq R. Nonsense muta-
tions in the lsa-like gene in Enterococcus faecalis
susceptible to lincosamides and streptogramins A.
Antimicrob Agent Chemo 2003; 47:2307-9.
6. Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G,
Di Rosa R, Baldassarri L. Survey for virulence determi-
nants among Enterococcus faecalis isolated from
different sources. J Med Microbiol 2004; 53:13-20.
7. Sedgley C, Lennan DB, Clewell DB. Prevalence,
phenotype, and genotype of oral enterococci. Oral
Microbiol Immunol 2004; 19:95-101.
8. Sedgley CM, Molander A, Flannagan SE, Nagel AC,
Appelbe OK, Clewell DB, Dahlén G. Virulence, phenotype
and genotype characteristics of endodontic Enterococcus
spp. Oral Microbiol Immunol 2005; 20:10-9.
9. Chu FC, Leung WK, Tsang PC, Chow TW, 
Samaranayake LP. Identification of cultivable
microorganisms from root canals with apical peri-
odontitis following two-visit endodontic treatment
with antibiotics/steroid or calcium hydroxide dress-
ings. J Endod. 2006; 32(1):17-23. 
10. Woodford N. Biological counterstrike: antibiotic
resistance mechanisms of Gram-positive cocci. Clin
Microbiol Infect Dis 2005; 11(Suppl 3):2-21.
11. De Vera ME, Simmons RL. Antibiotic-resistant
enterococci and the changing face of surgical infec-
tions. Arch Surg 1996; 131:338-42.
12. Byström A, Sundqvist G. Bacteriologic evaluation of
the efficacy of mechanical root canal instrumentation in
endodontic therapy. Scand J Dent Res 1981; 89:321-8.
13. Gomes BPFA, Pinheiro CR, Gadê-Neto EL, Sousa
ELR, Ferraz CCR, Zaia AA, Teixeira FB, Souza-Filho
FJ. Microbiological examination of infected dental
root canals. Oral Microbio Immunol 2004; 19:71-6.  
14. Molander A, Reit C, Dahlén G, Kvist T. Microbial
status of root filled teeth with apical periodontitis. Int
Endod J 1998; 31:1-7.
15. Sundqvist G, Figdor D, Persson S, Sjögren U. Micro-
biologic analysis of teeth with failed endodontic treatment
and the outcome of conservative re-treatment. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 1998; 85:86-93.
16. Siqueira JF Jr, Rôças IN. Molecular methods to
explore endodontic infections: Part 2- redefining the
endodontic microbiota. J Endod 2005; 31:488-498.
17. Hommez GMG, Verhelst R, Claeys G, Vanee-
choutte M, De Moor RJG. Investigation of the effect of
the coronal restoration quality on the composition of
the root canal microflora in teeth with apical peri-
odontitis by means of T-RFLP analysis. Int Endod J
2004; 37:819-27.
18. Kaufman B, Spånberg L, Barry J, Fouad A. Entero-
coccus spp. in endodonticaly treated teeth with and
without periradicular lesions. J Endod 2005; 31:851-856.
19.Siqueira JF Jr, Rôças IN. Polymerase chain reac-
tion based analysis of microorganisms associated with
failed endodontic treatment. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 2004; 97:85-94.
20. Lin S, Zukerman O, Weiss EI, Mazor Y, Fuss Z.
Antibacterial efficacy of a new chlorhexidine slow
release device to disinfect dentinal tubules. J Endod
2003; 29:416-8.
21. Almyroudi A, Mackenzie D, McHugh S, Saunders
WP. The effectiveness of various disinfectants used as
endodontic intracanal medications: an in vitro study. J
Endod 2002; 28:163-7.
22. Schafer E, Bossmann K. Antimicrobial efficacy of
chlorhexidine and two calcium hydroxide formulations
against Enterococcus faecalis. J Endod 2005; 31:53-6.
23. Walsh LJ, Tsang AKL. Chairside testing for cario-
genic bacteria: current concepts and clinical strategies.
Internat Dent. 2008: in press.
24. Beighton D. The complex oral microflora of high-risk
individuals and groups and its role in the caries process.
Commun Dent Oral Epidemiol 2005; 33:248-255.
25. Walsh LJ. Germ warfare in the dental surgery. Aus-
tralas Dent Pract 2003;14: 22.
26. Soukos NS, Ximenez-Fyvie LA, Hamblin MR,
Socransky SS, Hasan T. Targeted antimicrobial pho-
tochemotherapy. Antimicrob Agents Chemother 1998;
42: 2595-2601.
27. Soukos NS, Mulholland SE, Socransky SS,
Doukas AG. Photodestruction of human dental plaque
bacteria: enhancement of the photodynamic effect by
photomechanical waves in an oral biofilm model.
Lasers Surg Med 2003; 33: 161-168.
About the authors
Professor Laurence J. Walsh is the tech-
nology editor of Australasian Dental
Practice magazine. He is also a noted
commentator on and user of new technolo-
gies and is the Head of The University of
Queensland School of Dentistry.
Basil Athanassiadis is a general dental
practitioner in suburban Brisbane who
has had a long standing interest in 
dental materials and endodontics. He
recently completed a research masters
degree on the antibacterial properties of
endodontic medicaments which was
supervised by Professors Paul Abbott and
Laurie Walsh.
106 Australasian Dental Practice May/June 2008
infection | CONTROL
CPD POINTS AVAILABLE
Continuing Education credits are 
available on this article for subscribers
by answering the questionnaire at
www.dentalpractice.com.au


The challenge of endodontic "superbugs" in clinical practice

http://espace.library.uq.edu.au/view/UQ:159917/Endo_superbugs.pdf

The challenge of endodontic "superbugs" in clinical practice

Abstract

Similar works

Full text

Available Versions

University of Queensland eSpace