Investigation of Cultivable Bacteria Isolated from Longstanding Retreatment-resistant Lesions of Teeth with Apical Periodontitis Fernanda G.C. Signoretti, DDS, MSc, PhD,* Brenda P.F.A. Gomes, DDS, MSc, PhD,* Francisco Montagner, DDS, MSc, PhD, † and Rogerio C. Jacinto, DDS, MSc, PhD* ‡ Abstract Introduction: The objective of this research was to inves- tigate the presence of viable bacteria in tissue samples from persistent apical lesions and to correlate the microbi- ological findings with the histopathological diagnosis of the lesion. Methods: Twenty persistent apical lesions associated with well-performed endodontic retreatment were collected. Tissue samples were processed through culture techniques including serial dilution, plating, aerobic and anaerobic incubation, and biochemical tests for microbial identification followed by histopathological diagnosis. Results: Cysts were more frequently diag- nosed (13/20). Strict anaerobic species predominated in both cysts (80.4% of the species detected) and granu- lomas (65% of the species detected). Viable gram- positive bacteria were frequently recovered from apical lesions (cysts = 70.6%, granulomas = 84.4%). Gemella morbillorum and Propionibacterium acnes were the most frequently recovered species from cysts and granulomas, respectively. At least 1 gram-positive bacte- rial species was present in almost every sample (cysts = 12/13, granulomas = 7/7). No significant correlation was found between histologic findings and bacterial species. Conclusions: In conclusion, although cysts were more frequent than granulomas in cases of failure of endodontic retreatment, bacteria were isolated from both types of lesions, with a predominance of gram- positive species, suggesting that these species can survive outside the root canal and might be related to the persistence of the pathological process even after accurate endodontic retreatment. (J Endod 2013;39:1240–1244) Key Words Apical periodontitis, apical surgery, endodontic failure, endodontic outcome, nonsurgical retreatment S urgical endodontic treatment is recommended for teeth with longstanding apical lesions that persist even after accurately executed conventional endodontic retreat- ment (1, 2). The main goal of apical surgery is to remove the etiologic agent, which is normally associated with extraradicular biofilm on longstanding apical lesions (3–6), periapical actinomycosis (7), foreign body reactions triggered by extruded endodontic materials (8), accumulation of endogenous cholesterol crystals in the apical tissues (9), or unresolved cystic lesions (10). Microorganisms that reach the environment beyond root canal foramen are recognized by the immune system, initiating a local inflammatory response through a series of events aiming to eliminate the infection and providing conditions to restore host balance (11). However, many pathogens might survive because of their ability to bypass, respond, or resist to the host defense mechanisms, colonizing external root surface and leading to biofilm formation (12). Microbial species associated with complex bacterial communities organized on a biofilm possess characteristics that differ from their planktonic forms such as increased metabolic diversity and efficiency; resistance to phagocytic cells, antimicro- bial agents, and environmental stresses; and enhanced pathogenicity (13, 14), which make the extraradicular biofilm a tough challenge to overcome by means of endodontic nonsurgical approach. Despite a vast discrepancy in the incidence of different types of periapical lesions, granulomas are the most common pathology (3,15–22). Previous studies reported the frequency of cysts ranging from 6%–55% (16–22), whereas the occurrence of periapical granulomas varies between 46% (19) and 94% (17). To better understand the microbial species associated with complex bacterial communities, the objective of this research was to identify the pres- ence of viable bacteria in persistent apical periodontitis and to correlate the microbio- logical findings with the histopathological diagnosis of the lesion. Materials and Methods Patient Selection Twenty patients selected for this research (between 18 and 65 years old; 6 men and 14 women) gave informed consent according to the Ethics Committee in Research of the Piracicaba Dental School, Universidade Estadual de Campinas, Campinas, Brazil, and were referred to the Endodontics Specialization Clinic of the Piracicaba Dental School, State University of Campinas, Sao Paulo, Brazil, for endodontic surgery (confirmed by the presence of a persistent radiolucent area in the periapical region From the *Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School, State University of Campinas, Piracicaba, S~ ao Paulo, Brazil; † Endodontics Division, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; and ‡ Endodontics Division, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil. Supported by the Brazilian Agencies FAPESP (2011/09047-4, 2011/50510-0), CNPQ (302575/2009), and CAPES (BEX: 3679/10-1). Address requests for reprints to Dr Rogerio C. Jacinto, Endodontics Division, Department of Semiology and Clinics, Federal University of Pelotas, Rua Gonc ¸ alves Chaves, 457, Pelotas, RS, Brazil, CEP: 96015-560. E-mail address: [email protected]0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.06.018 Clinical Research 1240 Signoretti et al. JOE — Volume 39, Number 10, October 2013
Transcript
Clinical Research
Investigation of Cultivable Bacteria Isolated fromLongstanding Retreatment-resistant Lesions of Teethwith Apical PeriodontitisFernanda G.C. Signoretti, DDS, MSc, PhD,* Brenda P.F.A. Gomes, DDS, MSc, PhD,*Francisco Montagner, DDS, MSc, PhD,† and Rog�erio C. Jacinto, DDS, MSc, PhD*‡
Abstract
Introduction: The objective of this researchwas to inves-tigate the presence of viable bacteria in tissue samplesfrom persistent apical lesions and to correlate themicrobi-ological findings with the histopathological diagnosis ofthe lesion. Methods: Twenty persistent apical lesionsassociated with well-performed endodontic retreatmentwere collected. Tissue samples were processed throughculture techniques including serial dilution, plating,aerobic and anaerobic incubation, and biochemical testsfor microbial identification followed by histopathologicaldiagnosis. Results: Cysts were more frequently diag-nosed (13/20). Strict anaerobic species predominated inboth cysts (80.4% of the species detected) and granu-lomas (65% of the species detected). Viable gram-positive bacteria were frequently recovered from apicallesions (cysts = 70.6%, granulomas = 84.4%).Gemellamorbillorum and Propionibacterium acnes werethe most frequently recovered species from cysts andgranulomas, respectively. At least 1 gram-positive bacte-rial species was present in almost every sample (cysts =12/13, granulomas = 7/7). No significant correlationwas found between histologic findings and bacterialspecies. Conclusions: In conclusion, although cystswere more frequent than granulomas in cases of failureof endodontic retreatment, bacteria were isolated fromboth types of lesions, with a predominance of gram-positive species, suggesting that these species cansurvive outside the root canal and might be related tothe persistence of the pathological process even afteraccurate endodontic retreatment. (J Endod2013;39:1240–1244)
From the *Department of Restorative Dentistry, Endodontic D†Endodontics Division, Federal University of Rio Grande do Sul, PortoRio Grande do Sul, Brazil.
Supported by the Brazilian Agencies FAPESP (2011/09047-4, 20Address requests for reprints to Dr Rog�erio C. Jacinto, Endodon
Chaves, 457, Pelotas, RS, Brazil, CEP: 96015-560. E-mail address: r0099-2399/$ - see front matter
Copyright ª 2013 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.06.018
1240 Signoretti et al.
Surgical endodontic treatment is recommended for teeth with longstanding apicallesions that persist even after accurately executed conventional endodontic retreat-
ment (1, 2). The main goal of apical surgery is to remove the etiologic agent, which isnormally associated with extraradicular biofilm on longstanding apical lesions (3–6),periapical actinomycosis (7), foreign body reactions triggered by extruded endodonticmaterials (8), accumulation of endogenous cholesterol crystals in the apical tissues(9), or unresolved cystic lesions (10).
Microorganisms that reach the environment beyond root canal foramen arerecognized by the immune system, initiating a local inflammatory response througha series of events aiming to eliminate the infection and providing conditions to restorehost balance (11). However, many pathogens might survive because of their ability tobypass, respond, or resist to the host defense mechanisms, colonizing external rootsurface and leading to biofilm formation (12).
Microbial species associated with complex bacterial communities organized ona biofilm possess characteristics that differ from their planktonic forms such asincreased metabolic diversity and efficiency; resistance to phagocytic cells, antimicro-bial agents, and environmental stresses; and enhanced pathogenicity (13, 14), whichmake the extraradicular biofilm a tough challenge to overcome by means ofendodontic nonsurgical approach. Despite a vast discrepancy in the incidence ofdifferent types of periapical lesions, granulomas are the most common pathology(3,15–22). Previous studies reported the frequency of cysts ranging from 6%–55%(16–22), whereas the occurrence of periapical granulomas varies between 46%(19) and 94% (17). To better understand the microbial species associated withcomplex bacterial communities, the objective of this research was to identify the pres-ence of viable bacteria in persistent apical periodontitis and to correlate the microbio-logical findings with the histopathological diagnosis of the lesion.
Materials and MethodsPatient Selection
Twenty patients selected for this research (between 18 and 65 years old; 6 menand 14 women) gave informed consent according to the Ethics Committee in Researchof the Piracicaba Dental School, Universidade Estadual de Campinas, Campinas, Brazil,and were referred to the Endodontics Specialization Clinic of the Piracicaba DentalSchool, State University of Campinas, Sao Paulo, Brazil, for endodontic surgery(confirmed by the presence of a persistent radiolucent area in the periapical region
ivision, Piracicaba Dental School, State University of Campinas, Piracicaba, S~ao Paulo, Brazil;Alegre, Rio Grande do Sul, Brazil; and ‡Endodontics Division, Federal University of Pelotas, Pelotas,
11/50510-0), CNPQ (302575/2009), and CAPES (BEX: 3679/10-1).tics Division, Department of Semiology and Clinics, Federal University of Pelotas, Rua [email protected]
even after a 1-year follow-up of endodontic retreatment). The followingclinical/radiographic features were found and recorded: the presenceof swelling, pain on palpation, tenderness to percussion, size of theradiographic lesion $5 mm and <5 mm, and time after retreatmentof 12–24 months and >24 months. Patients with teeth featuring sinustracts (in order to exclude chronic apical abscess), periodontalprobing depths >4 mm, or root fracture and patients treated with anti-biotics over the last 3 months were excluded from this study.
Radiographic FeaturesTeeth with satisfactory canal filling (ie, uniform radiodensity and
adaptation of the filling to the root canal walls, root canal filling endingshort of the apex by 2 mm or less, no extruded gutta-percha beyond theapex, and the presence of a periapical lesion related to endodonticallyretreated tooth) were selected (Fig. 1A). Patients requiring retreatmentbecause of missing canals, separated endodontic instruments, rootcanal deviations, perforations or calcified root canals in which theapex was inaccessible, and inadequate endodontic filling (under- oroverfilling) were excluded. All sampled teeth presented appropriatepermanent coronal restorations with no secondary carious lesions,marginal defects, fractures, or material loss.
Sample CollectionThe protocol used for sample collection was based on the work of
Subramanian and Mickel (23). A 1-minute-long rinse with 0.2% chlo-rhexidine gluconate and swabbing of the surgical area with 2% chlo-rhexidine gel (Endogel; Essencial Farma, Itapetininga, SP, Brazil)were performed to avoid external contamination. After local anesthesia,a full-thickness mucoperiosteal flap was reflected using intrasulcularincision, and the root end was accessed with a surgical bur (KG Soren-sen, S~ao Paulo, SP, Brazil) cooled with sterile saline. After the reflectionof the flap, periosteal tissue samples were collected from areas adjacentto the surgical site using curettes and absorbent paper cones to test forbacterial contamination. A portion of the periradicular tissue removedby curettage (Fig. 1B) was stored in an Eppendorf tube containing 1.0mL transport medium Viability Medium G€oteborg Agar III (24), whichwas immediately transported to the laboratory for culture analysis, andthe remaining tissue was immersed in 10% neutral buffered formalinfor further histopathological examination. All patients were treated by
Figure 1. (A) A radiographic image of failed endodontic retreatment with adequa
JOE — Volume 39, Number 10, October 2013
the same surgeon (FGCS) under high magnification with a dental oper-ating microscope (DF Vasconcellos SA, S~ao Paulo, Brazil).
Microbial CultureMicroorganism isolation and characterization were performed as
previously described by Gomes et al (25). Briefly, aliquots of 50 mLwere retrieved from the vortexed VMGA tubes of each sample in ananaerobic environment (Anaerobic Chamber; Don Whitley Scientific,Bradford, UK) at 37�C, inoculated, and incubated as follows: plates con-taining brain-heart infusion agar (Himedia, Mumbai, India) + 5%sheep blood were incubated in O2 (aerobically) at 37
�C for 2 daysfor the detection of facultative anaerobes and plates containing prere-duced fastidious anaerobe agar (Lab M, Bury, UK) + 5% sheep blood+ 600 mL menadione (Vitamin K3, 2-methyl-1,4-naphthoquinone[Sigma-M5625; Sigma-Aldrich, St Louis, MO]) + 600mL hemin (heminbovine minimum 80% [Sigma-H5533]) were incubated at 37�C in ananaerobic atmosphere with 10% H2, 10% CO2, and 80% N2 up to 14days to detect fastidious microorganisms.
Phenotypic characterization was performed under a stereomicro-scope (3� magnification) (Lambda Let 2; Atto Instruments Co, HongKong, China) according to the macroscopic features, and each differentcolony was subcultured onto both a FAA prereduced plate and a brain-heart infusion blood agar plate to test for their gaseous requirement.Pure cultures were subjected to Gram stain (Newprov; Laboratory Prod-ucts, Pinhais, PR, Brazil) and tested for catalase production. The oxidasetest was performed in gram-negative and catalase-positive strains.
Appropriate biochemical testing kits (bioM�erieux SA, Marcyl‘Etoile, France) specific for each strain according to their previouslydetermined features was selected as follows: Rapid ID 32A (bioM�erieuxSA) for gram-negative and gram-positive anaerobes; API Staph (bio-M�erieux SA) for staphylococci and micrococci (gram positive and cata-lase positive); API Strep (bioM�erieux SA) for streptococci (grampositive and catalase negative); API 20 E (bioM�erieux SA) for Entero-bacteriaceae (enteric gram-negative bacilli, catalase positive, andoxidase negative); API NH (bioM�erieux SA) for Eikenella, Haemophi-lus, Neisseria, and Actinobacillus (gram-negative, oxidase-positivefacultative anaerobes, cocci or bacilli); and API C AUX (bioM�erieuxSA) for yeasts.
te root canal filling. (B) Periradicular tissue curettage.
Investigation of Cultivable Bacteria 1241
Clinical Research
Tissue ProcessingSamples stored in formalin were washed in running water for
5 minutes and processed for embedding in paraffin. Five-micrometer-thick sections were cut from the paraffin blocks, stainedwith hematoxylin-eosin, and examined under light microscopy for diag-nostic confirmation of a cyst or granuloma. The following features wereobserved: the presence and distribution of acute (polymorphonuclearleukocytes) and chronic inflammatory cells, granulomatous tissue,lining epithelium, and fibrous connective tissue capsules. Five of theseparaffin blocks were submitted to Brown-Brenn staining to verify thepresence of bacteria in the lesions (Fig. 2A and B).
Statistical AnalysisThe data collected for each case (clinical features and bacteria iso-
lated) were entered into a spreadsheet and statistically analyzed usingSPSS for Windows (SPSS Inc, Chicago, IL). The Pearson chi-squaretest or the 1-sided Fisher exact test, as appropriate, was chosen totest the null hypothesis that there was no relationship between bacteriaspecies such as endodontic clinical/radiographic signs/symptoms andthe presence of a specific group of bacteria in the lesion samples.P < .05 was considered statistically significant.
ResultsThe following clinical features were observed in the cases
analyzed: swelling (17/20), pain on palpation (18/20), and tendernessto percussion (20/20) (Table 1). In the 20 cases with persistent symp-tomatic apical periodontitis investigated, only 1 did not present culti-vable bacteria. Periosteal tissue collected from surrounding areas tothe surgical site showed no microbial growth, indicating the absenceof external contamination during the sampling procedures. Bacterialcolonies were visualized in the apical tissues of 5 samples that weresubmitted to Brown-Brenn stain as shown in Figure 2A and B. Cystswere more frequently diagnosed (13/20). Strict anaerobic species pre-dominated in both cysts (80.4% of the species detected) and granu-lomas (65% of the species detected). Viable gram-positive bacteriawere frequently recovered from apical lesions (cysts = 70.6%, granu-lomas = 84.4%). Gemella morbillorum and Propionibacteriumacnes were the most frequently recovered species from cysts and gran-ulomas, respectively. At least 1 gram-positive bacterial species waspresent in almost every sample (cysts = 12/13, granulomas = 7/7).No significant correlation was found between histologic findings andbacterial species.
A maximum of 7 different species were found per sample, andgranulomas accounted for more different species per sample than cysts.
Figure 2. (A) Bacterial colonies are visualized in the periradicular tissue indicaAnother section in which bacterial colonies are visualized (Brown-Brenn stain, or
1242 Signoretti et al.
Positive associations were found between the following: Actinomycesnaeslundii and Propionibacterium propionicum (P = .01), Bacter-oides ureolyticus and Eubacterium limosum (P = .032), and B. ure-olyticus and Prevotella oralis (P = .004). The most commonpathogens isolated in cysts were G. morbillorum (61.5%), B. ureoly-ticus and E. limosum (30.8%), and Anaerococcus prevotii (23.1%),whereas a predominance of P. acnes (57.1%), Gemella haemolysans,Micrococcus spp, and A. naeslundii (42.9%) (Table 2) was observedin granulomas.
Soft and bright yellow sulfur granules were observed in only 1 case(S15). After being cultured, the following microorganisms wereisolated: A. naeslundii, E. limosum, Parvimonas micra, and P. pro-pionicum.
DiscussionThe present study investigated the presence of viable bacteria in
apical lesions associated with failure of endodontic retreatment; 65%were cysts, and 35% were granulomas. There is no consensus in theliterature regarding the incidence of different types of chronic apicallesions (16–18, 20); this can be attributed to sampling procedures,processing and interpretation of the histologic material, and clinicalfeatures associated with samples. Most of these studies analyzedspecimens obtained from wide sources of routine histopathologicaldatabases (18). It must be pointed out that in the present research tissuecollection was performed through curettage, not including the apicalportion of the affected tooth. Therefore, it was unfeasible to know theirstructural relation. Because of this, the periapical lesions were catego-rized as granulomas or cysts without any further subclassification.
The high incidence of cysts detected in this study differs with previousreports (11,17–20, 23); this might be a result of the restrictive patientselection criteria used compared with other studies. For instance,patients presenting with sinus tracts were excluded because sinus tractsconstitute a communication between periapical lesions and the oralcavity, modifying the periapical environment and changing the localmicrobial profile. Additionally, all samples analyzed had previouslyreceived root canal retreatment over a 1-year follow-up, showinga periapical radiolucent area and/or other signs that suggested failure.
Except for 1 case in which no microbial growth was found, theprevalence of 1 to 7 microorganisms was detected both in cysts or gran-ulomas, supporting the polymicrobial feature of persistent apicallesions (6, 7, 24). Gram-positive bacteria accounted for 70.6% of theisolates in cysts and 84.4% in granulomas. Both cysts and granulomaspresented with remarkably similar microbiological profiles witha predominance of viable gram-positive bacteria, highlighting thata cyst is a direct evolution of a granuloma lesion.
ted by the arrows (Brown-Brenn stain, original magnification: 400�). (B)iginal magnification: 1000�).
Sampling of microorganisms in the apical area during surgery ischallenging because of possible contamination during these proce-dures. In the present study, all periosteal tissue samples collectedsubjacent to the surgical site showed no bacterial contamination, indi-cating that the surgical site was sterile. Another issue would be contam-ination during the excision of the lesion, even in the presence ofa negative control from adjacent areas. Although this possibility cannotbe completely discarded, the clinical procedures were performed bya highly qualified surgeon who collected the samples under magnifi-cation and was extremely careful to avoid contamination. Moreover,histologic examination with Brown-Brenn stain in some of the samplesrevealed the presence of bacteria inside the lesions. Therefore, it canbe assumed that the bacteria recovered in this study were present in thelesions before surgery.
The majority of gram-positive strains belonged to the followingspecies: G. morbillorum, P. acnes, A. naeslundii, A. prevotii, andE. limosum. Gomes et al (26) showed that G. morbillorum mightbe an important pathogen in endodontic infections because of itshigher percentage of recovery in both primary and secondary/persis-tent infections in 82% and 72% of the samples, respectively. Sunde et al(27) investigated the periapical microbiota of teeth with refractoryapical periodontitis, showing a wide variety of microorganisms, partic-ularly gram-positive species. It has been reported that structuralcomponents of the robust bacterial cell wall of gram-positives protectthem from noxious environmental factors. In addition, the majority ofthese microorganisms express fast-adaptive properties when exposedto extreme conditions, thus making them potential causal elements inposttreatment endodontic disease (28). Other previous reports havealso associated gram-positive species with persistent infections byinvestigating periapical lesions (2, 6, 29).
However, the literature is inconclusive regarding whether theseextraradicular infections are self-sufficient or dependent on intraradic-ular infections. Ricucci and Siqueira (4) investigated the prevalence ofintra- and extraradicular biofilm in both treated and untreated teeth,indicating their high frequency of being located in canal complexitiesout of reach of instruments and antimicrobial irrigants and assumingthat intraradicular biofilms may be responsible for the persistence ofthe extraradicular infection. In the present study, all teeth selectedhad been submitted to thoroughly nonsurgical retreatment with theaid of a surgical microscope, avoiding and overpassing operativecomplications and providing accurate root canal disinfection, at least1 year previously to the surgical approach. Adhered biofilm was notinvestigated, so extreme care was taken to avoid collecting materialfrom the external surface of the root apex during sampling procedures.Therefore, our results suggest that a viable polymicrobial community,with an adequate number of cultivable cells and interacting species,was present inside the lesion.
Clinically, such a situation is relevant, playing a role not only inthe persistence of infection but also in disease exacerbation (30). Inaddition, the probability of a systemic disorder occurring from oralfoci of infection has been suggested to be particularly prevalent insusceptible individuals with a lowered immune response, such asimmunocompromised elderly patients or patients with damaged heartvalves (31, 32).
In recent years, molecular biological methods such as randomcloning and 16S rRNA sequence analysis have been introduced inorder to profile the microbiota (33, 34). However, live and deadbacterial cells in the microbiota could not be differentiated by usingonly molecular biological methods. Using culture techniques, thepresent study showed the presence of viable bacteria in cysts orgranulomas in cases associated with failure of retreatment,suggesting that these species could be related to the maintenance of
Investigation of Cultivable Bacteria 1243
TABLE 2. Frequency of Bacteria Found in Periapical Lesions Dividedaccording to the Histopathological Diagnosis
the disease. However, the pathogenicity of live bacterial cells in theendodontic lesions remains to be determined.
In conclusion, although cysts were more frequent than granu-lomas in cases of failure of endodontic retreatment, bacteria were iso-lated from both types of lesions, with a predominance of gram-positivespecies, suggesting that these species can survive outside the root canaland might be related to the persistence of the pathological process evenafter accurate endodontic retreatment.
AcknowledgmentsThe authors deny any conflicts of interest related to this study.
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