Download Isolation of root canal pathogens from primary endodontic infection

University J Dent Scie 2016; No. 2, Vol. 1
ISOLATION OF ROOT CANAL PATHOGENS
FROM PRIMARY ENDODONTIC INFECTION AND
RETREATMENT CASES –
A CLINICAL COMPARATIVE STUDY.
1
Vineet R.V., 2Moksha Nayak, 3Subbannayya Kotigadde, 4Beena Antony
Senior Lecturer, Department of Conservative Dentistry & Endodontics,
Sree Mookambika Institute of Dental Sciences, Kulasekharam,
2
Principal & Professor, Department of Conservative Dentistry & Endodontics,
KVG Dental College & Hospital, Sullia
3
Professor, Department of Microbiology, K.V.G Medical College & Hospital, Sullia
4
Professor, Department of Microbiology , Father Muller Medical College, Mangalore
1
ABSTRACT Aim: The purpose of this study was to isolate and compare the prevalence of common root
canal pathogens; Streptococcus mitis, Lactobacillus acidophilus and Enterococcus faecalis from primary
endodontic infections and retreatment cases.
Materials and method: 60 subjects scheduled for root canal treatment or retreatment were divided into two
groups comprising of 30 subjects with primary endodontic infections and 30 subjects with failed
endodontic treatment. Root canal samples were collected as per Moller's criteria using sterile paper point.
Advanced microbiological culture techniques were used to isolate Streptococcus mitis, Enterococcus
faecalis and Lactobacillus acidophilus from primary root canal infections and retreatment cases. Statistical
analysis was done using Student's T test and Pearson's Chi-Square test.
Results: A total of 50 cultivable isolates were recovered from 60 root canal samples. Among them 26
isolates were from primary infection and 24 isolates were from secondary infection. Streptococcus
mitis(53.3%) was significantly more associated with primary infection. On the other hand Enterococcus
faecalis (46.7%) was the most predominant microorganism in retreatment cases.
Conclusion: Higher prevalence of Streptococcus mitis in primary infection and Enterococcus faecalis in
secondary infection, suggest that specific type of root canal infection has an affinity to specific group of
microorganisms. Knowledge regarding this association may be beneficial in adopting strategies to combat
root canal infections for better prognosis.
INTRODUCTION :
Endodontic disease is the result of both the pathogenic effects
of the microbes and the response of the host. The goal of
clinicians is to disrupt and destroy the microbial ecosystem
associated with the disease process. Understanding of
microbial ecology of the pulp-dentine complex is important
because it provides a rational basis for disease prevention and
treatment.[1] Endodontic treatment involves treatment of
both primary and secondary infections in the root canal
system. Primary infections are caused by microorganisms
that colonize the necrotic pulp tissue. It can be regarded as the
initial or 'wild' infection and are the cause of primary apical
periodontitis.[2] Clinical studies have reported a success rate
of endodontic therapy ranging from 87% to 95%.[3]
Secondary infections are caused by microorganisms that were
not present in the primary infection, but were introduced in
the root canal at some time after professional intervention[4].
University
Journal of
Dental Sciences
Clinical Papers
and
Comparative
Studies
Keywords :
Isolation, Streptococcus
mitis, Lactobacillus
acidophilus,
Enterococcus faecalis,
primary infection,
retreatment cases.
Source of support : Nil
Conflict of interest: None
The composition of the microflora of root canals differs in
primary infection and retreatment cases of endodontic
origin.[5] Microorganisms from dental caries have proved to
be the most common causative factor of pulpal disease.
Studies have shown that Streptococcus sp., Lactobacillus sp.,
Enterococcus sp. are strongly associated with dental
caries.[6] A relatively wide range of bacteria have been
isolated from root canals using standard culture techniques.
However, only 50% of the bacteria in the oral cavity are
cultivable.[7] Use of advanced anaerobic microbiological
techniques and culturing in selective media have been proven
to be beneficial in this regard. A selective medium contains
antimicrobial agents that are capable of suppressing all other
microorganisms other than one for which the medium is
designed. Selective culturing constitutes an efficient
technique to detect very low proportions of specific
microorganism from a sample.[8]
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India
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University J Dent Scie 2016; No. 2, Vol. 1
To date there are no comparative data regarding the
association of Streptococcus mitis, Lactobacillus acidophilus
and Enterococcus faecalis in primary endodontic infection
and in retreatment cases using selective media. In addition,the
microbial flora that colonize the root canals vary with the
geographic location of the patients studied.[9] Literature on
microorganisms isolated from root canals in Indian
population is sparse. Hence, it is prudent to study the gram
positive microorganisms especially Streptococcus mitis,
Lactobacillus acidophilus, Enterococcus faecalis from
primary endodontic infections and retreatment cases using
advanced culturing technique among Indian population.
MATERIALS AND METHOD :
Study population: Root canal samples were collected from 60
subjects who had been referred for root canal treatment or
retreatment to the Department of Conservative dentistry and
Endodontics, K.V.G. Dental College & Hospital, Sullia. The
Ethical Committee of K.V.G Dental College &
Hospital,Sullia approved the study protocol. The subjects
were informed of the study protocol and written consent was
obtained before the sampling procedure was performed.
60 subjects selected for the study were divided into two
groups.
Group 1: 30 subjects with primary endodontic infections.
Group 2: 30 subjects with failed endodontic treatment
requiring retreatment.
cavity. It was then transferred to a vial containing 1ml of
liquid transport medium and assessed for bacterial growth. If
growth occurred the patient sample was disqualified from the
study.
All instruments used for access cavity preparation were
sterile. In case of a treated canal, canal filling material was
removed with the use of Gates Glidden drills and endodontic
files without the use of any chemicals. Canal was rinsed with
sterile saline to remove the remnants of filling material and
debris, and to moisten the canal before sampling. Working
length of the canal was determined radiographically using a
20 K-file 0.5mm short of the radiographic working length.
Sampling was performed by placing a sterile paper point in
the canal to its full length for 60seconds. In case of
multirooted teeth, a single root canal was sampled in order to
confine the microbial evaluation to a single ecologic
environment. The criteria used to choose the canal for
sampling were the presence of exudation, or in its absence, the
largest canal or the canal associated with periapical
radiolucency. Before sampling the selected canal of
multirooted teeth, the entrance of the other canals was closed
with sterile cotton pellets. Following removal from the canal,
the paper point was immediately placed in a transport medium
containing 3ml of sterile reduced transport fluid which was
further transferred to Robertson cooked meat medium and
glucose broth.
Both males and females aged 20-70 years; immunocompetent
subjects were included in the study. In group 1 patients with
deep carious lesion involving pulp or with a diagnosis of
pulpal necrosis were included. In group 2 patients requiring
retreatment of endodontically treated teeth with a diagnosis of
apical periodontitis, root filled teeth with radiographic
evidence of periradicular disease and the termini of the root
canal fillings atleast 2mm short of the radiographic apex were
included in the study. Subjects with any systemic diseases,
pregnancy and lactation, use of any antibiotics during past 3
months, immunocompromised patient, teeth that cannot be
isolated with rubber dam, calcified canals and teeth having
periodontal pockets greater than 4mm were excluded from the
study.
Root canal sampling: Aseptic techniques were used
throughout the root canal sampling procedure as proposed by
Moller. The tooth was isolated with rubber dam. All coronal
restorations and carious lesions was completely removed. The
tooth and surrounding field was rinsed using 30% hydrogen
peroxide and then with 2.5% sodium hypochlorite solution for
30seconds. The solution was inactivated with sterile 5%
sodium thiosulfate. To assess the efficacy of the disinfection
procedure,a sterile cotton pellet was used to swab the access
Figure 1: Root canal sampling technique
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India
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University J Dent Scie 2016; No. 2, Vol. 1
Bacterial culture method: The root canal samples obtained
were then immediately submitted to the Department of
microbiology, K.V.G Medical College and Hospital, Sullia
for culturing. The root canal samples were shaken in a vortex
mixer for 60 seconds. The culture inoculation, incubation and
identification procedures were done under the supervision of
a trained microbiologist. After vortexing, 50ìl of sample was
plated onto selective culture media. Mitis-Salivarius
agar(Himedia) was used for Streptococcus mitis, Mac
Conkey agar(Himedia) was used for Enterococcus faecalis
and Rogosa SL(Himedia) agar was used for Lactobacillus
acidophilus. For culturing Streptococcus mitis a candle jar
was used and incubated at 370C for 5 days. Aerobic culturing
technique was used for Enterococcus faecalis and incubated
at 370C for 2 days. Advanced anaerobic culturing technique
using anaerobic glass jar and gaspak (Himedia) was used for
Lactobacillus acidophilus and incubated at 370C for 7 days.
After incubation, each plate was biochemically analysed for
growth and identification of bacteria using the colony
morphology and gram staining.
Figure 2. Microbial culture technique
STATISTICAL ANALYSIS :
The data collected were typed onto a spreadsheet and
statistically analysed using SPSS 17.0 (SPSS Inc., Chicago,
IL, USA). The results were statistically evaluated using
Student unpaired T test and Pearson chi-square test.
RESULTS :
The study population age ranged from 20 to 70 years. Primary
infection(mean age = 36 years) and secondary infection(mean
age = 32 years) groups included equal proportion of men and
women. Unpaired 't' test showed no significant difference
between the groups in terms of age and gender distribution.
In the present study, microorganisms were isolated from
73.3%(44/60) of selected teeth microbiologically sampled
from primary and secondary infection by culture
method.There were 26.7% of teeth(16/60) with negative
culture on the plates. Primary infection yielded a total of 26
isolates. Streptococcus mitis was the most predominant
microorganism isolated from 53.3% of primary root canal
infection followed by Enterococcus faecalis (20%) and
Lactobacillus acidophilus (13.3%).Chi-square test showed
statistically significant association between Streptococcus
mitis and primary infection (p= 0.018) as shown in Table 1. In
secondary infection a total of 24 isolates were recovered.
Enterococcus faecalis (46.7%) was the most frequently
isolated microorganism followed by Streptococcus mitis
(20%) and Lactobacillus acidophilus (13.3%).
TABLE 1: Prevalence of Streptococcus mitis, Lactobacillus
acidophilus and Enterococcus faecalis in patients with
primary and secondary infections
DISCUSSION :
Our findings indicate that amongst the isolates, Streptococcus
mitis(53.3%); a facultative anaerobic gram-positive species
were predominant in infected root canals, which is
corroborating with the studies of Sundqvist et al. and
Molander et al. in which they also found facultative
anaerobic and gram-positive species were predominant in
primary infected root canals.[10] In contrast Baumgartner et
al. and Gomes et al. have revealed the composition of necrotic
pulp presents a polymicrobial flora characterized by a wide
variety of combinations of bacteria, averaging 4-7 species per
canal, predominantly anaerobic, with approximately equal
proportions of Gram-negative and-positive bacteria.[11]
Most of the previous studies have used non-selective culture
methods. However,the present study did not evaluate gram
negative microorganisms and it is noteworthy that the most
prevalent species in primary infections may vary from study
to study, which can be explained by several factors such as the
sensitivity and specificity of the identification method,
sampling technique, geographic location, and accuracy or
divergence in clinical diagnosis.[12]
In the present study Streptococcus mitis was significantly
associated with primary root canal infections. Nisengard et al.
has specified that the use of Mitis Salivarius agar helps in
cultivation of only Streptococcus mitis suppressing the
growth of other microorganisms.[13] Our study is in
accordance with the study of Ercan et al. who also obtained
Streptococcus mitis as one of the prevalent microorganism in
primary root canal infections.[11] Moreover, Studies by
Chavez et al. have revealed that Streptococci are present in
high prevalence in infection of endodontic origin, including
both chronic and acute periradicular diseases.[14]
Enterococcus faecalis was isolated from 46.7% of retreatment
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University J Dent Scie 2016; No. 2, Vol. 1
cases in the present investigation, this is in line with the
findings of Molander et al.(47%) , Peciuliene et al.(38%) ,
Pinheiro et al. (55%) in which E. faecalis was the species
most frequently isolated from retreatment cases.[1] This
indicates the importance of this resistant microorganism in
root canal treatment failure. The results of the present study
showed that Enterococcus faecalis was isolated from 20% of
primary root canal infection. Stuart et al. by using culture
method have concluded that Enterococci constitute a small
percentage of the microbial species isolated from root canals
of teeth with necrotic dental pulps.[15] Mac Conkey agar is a
specific media for definitive isolation of Enterococcus
faecalis. However, PCR studies by Gomes et al. demonstrated
E. faecalis to be as frequent in teeth with necrotic pulp as in
teeth with failing endodontic treatment.[16] E. faecalis was
present in 18% of the cases of primary endodontic infections
as detected by Rocas et al. using nested PCR.[17]
This raises the suspicion that the involvement of E. faecalis
with failed cases can be a result of persistent infections. Based
on this assumption, E. faecalis would take advantage of
elimination of inhibitory members of the endodontic
microbiota, or of its ability to resist intracanal procedures of
disinfection.[18] Although the occurrence of E. faecalis in
root-filled teeth associated with periradicular lesions can be
presumed to be a result of persistent infections, the possibility
of participation of this microorganism in secondary infections
should not be disregarded. For the latter to occur, the
microorganism should gain entry into the root canal during
treatment, between appointments, or even after conclusion of
the treatment. The origin of E. faecalis infecting root-filled
teeth is worth elucidation for future research.
Lactobacillus acidophilus was isolated from 13.3% of
primary infection and retreatment cases. There was no
statistical significant association between Lactobacillus
acidophilus isolated from primary and secondary infection.
This result is similar to Chavez et al. who found that
Lactobacillus acidophilus are normal inhabitants of plaque
and carious lesions but are not frequently identified in root
canal infections.[19] Ercan et al. also isolated Lactobacillus
from only 7.1% of infected root canals.[11] The fastidious and
anaerobic nature of Lactobacillus sp. makes cultivation of this
microorganism very difficult.[19] Further a brief exposure to
air during the sampling procedure might be sufficient to kill
anaerobic microorganisms.[20]
In our study 26.7% of evalauated sample had no cultivable
microorganism. Successful cultivation relies on viable
microorganisms that live and grow on culture plates. The
finding that 26.7% of root canals had no cultivable bacteria
was not entirely unexpected; earlier studies were also unable
to isolate bacteria from 55.6%(Sundqvist et al.), 26.6%
(Molander et al.) and 17.5% (Peciuliene et al 2001) of
teeth.[21] The present study evaluated only three
microorganism, other microorganisms may also be associated
with root canal infection. However, failure to detect bacteria
does not prove their absence. Although the sampling
techniques and laboratory procedures used in this study have
been shown to be highly effective, it is indeed a technique
sensitive method.[22] It is possible that some microorganisms
present could have been lost, especially if the number of
microorganisms present in the root canal was very low or if
they were present in areas such as anatomical branches and
apical areas obliterated by previous treatment. Furthermore,
some microorganisms could have been removed together
with the previous root filling and debris.[23]
Follow-up studies on the root canal treatment report success
of retreatment of teeth with apical periodontitis is lower, with
an overall success rate of 66%.3 Molander et al. suggested
that this poorer prognosis in root canal retreatments may be
associated with difficulties in the elimination of the particular
microflora in cases of root canal treatment failure.[24]
Facultative anaerobic and Gram-positive bacteria, such as
Enterococcus, Streptococcus are more resistant to
instrumentation and to antiseptic agents, and therefore can be
expected to persist more frequently in the root canal after
inadequate root canal preparation and obturation.[19]
Persisting microorganisms or their products can maintain an
infectious process and cause treatment failure.[10] Hence,
thorough knowledge of this microflora could guide new
strategies to combat infection, leading to a better prognosis
for root canal retreatments.
CONCLUSION :
Within the parameters of this study it can be concluded that
Streptococcus mitis was most predominant in primary
endodontic infection whereas Enterococcus faecalis was
most frequently isolated from retreatment cases. This
suggests that there is an affinity for specific microorganisms
to be associated with specific type of endodontic infection.
Accurate knowledge of the occurrence of the major putative
endodontic pathogens and their implication in pathogenesis
of periradicular diseases has the potential to afford subsidies
for development of antimicrobial strategies effective in
treating both primary and secondary infection.
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CORRESPONDENCE AUTHOR:
Dr. Vineet R. V.
Department of Conservative Dentistry & Endodontics,
Sree Mookambika Institute of Dental Sciences,
Kulasekharam, Kanyakumari District, Tamil Nadu, India.
Email: [email protected]
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India
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