Frequency of bacetrial content finding in persistant periapical lesions

Objectives To determine the percentage of persistant apical lesions positive for bacterial nucleic acids, to detect microorganisms difficult to cultivate in persistant apical lesions by PCR and relate them to endodontic failure, clinical symptoms and diabetes mellitus. Materials and methods The samples of persistent apical lesions were collected during apicoectomy. Bacterial ubiquitous primer 16S rRNA was used to detect 16S ribosomal RNA in 36 samples. A species–specific PCR was performed with primers targeted to the bacterial 16S rRNA genes of Prevotella Nigrescens, Pseudoramibacter alactolyticus, and Propionobacterium propionicum. Results Six samples (16.67%) were positive for bacterial ribosomal RNA. Pseudoramibacter alactolyticus was detected in three samples. Propionibacterium propionicum and Prevotella nigrescens were detected in one sample each. The prevalence of infection of such lesions with P. intermedia, P. propionicum and P. alactolyticus is low. Conslusion The study we conducted gave insufficient data about extraradicular infection and its connection with diabetes mellitus and clinical symptoms. Conclusions Apical lesions persisting after endodontic treatment could harbor microorganisms other than Actinomyces and Propionicum species.


Introduction
The persistence of apical lesions after endodontic treatment is associated with intraradicular infection, extraradicular infection, foreign body reaction, cyst formation and fibrous scar tissue healing (1,2).
A number of studies dealt with the detection and identification of microorganisams in the root canal of root-filled teeth (3,4,5,6,7), and the persistence of microorganisms in the apical part of the root canal was recognized as the major cause of endodontic treatment failures, even after lege artis performed endodontic procedures (1). This may occur due to inability of endodontic instruments and irrigants to reach all parts of the root canal system and effectively remove microorganisms. Root canal microflora between primary endodon-Uvod Perzistencija apikalnih lezija nakon endodontskog liječenja povezana je s intraradikularnom infekcijom, ekstraradikularnom infekcijom, reakcijom na strano tijelo, stvaranjem ciste i cijeljenjem vezivnim ožiljkom (1,2).
Intraradicular microorganisms can overcome periapical defense barrier and establish extraradicular infection, which may cause acute apical abscess. Microorganisms that prevail in extraradicular infection are anaerobic bacteria such as Actinomyces spp., Propionibacterium propionicum, Treponema spp., Porphyromonas endodontalis, Porphyromonas gingivalis, Treponema forsythia, Prevotella spp. and Fusobacterium nucleatum (10,11). In classic histology studies, micro-organisms were consistently present in the periapical tissue of cases with clinical signs and symptoms such as acute abscessi and draining sinus tracts, but asymptomatic persistant periapical lesions were generally not infected (1). There are, however, reports on extraradicular biofilms in asymptomatic periapical periodontitis and chronic apical abscesses with sinus tracts (12,13). There are, also, reports on extraradicular infection of asymptomatic periapical lesions persisting after proper endodontics using microbial culture and molecular methods, where strict aseptic sampling procedures were used (10,11).
A clear understanding of the etiology and pathogenesis of the microbes causing persistent endodontic lesion helps in deciding on the use of intracanal medicaments and irrigants in endodontic retreatment cases with periapical lesions, or whether apical surgery should be preferred. The aim of this study was to detect microorganisms difficult to cultivate in periapical lesions by PCR, and to relate them to endodontic failure, clinical symptoms, and diabetes mellitus.
The exclusion critera were: (i) teeth with periodontal pockets, (ii) treatment with antibiotics within the last three months, (iii) serious systemic diseases, (iv) pregnant or breast feeding female patients, (v) participation in other clinical studies within the last three months.

Tissue samples
The samples of persistent apical lesions were collected during apicoectomy procedures. The patients washed their mouth with 0.2% chlorhexidine gluconate solution for 30 seconds prior to surgery. The Nowak-Peter incision was applied and full-thickness mucoperiosteal flap was reflected. The operative field was subsequently washed with sterile saline and the apex was approached using sterile carbide bur. The entire lesion was enucleated and apicectomy was performed using Lindeman carbide bur. Periapical tissue samples were transferred to lysis buffer from the extraction kit QiaAmp † DNA Mini Kit (Qiagen, GmbH, Germany) according to the manufacturer instruction, and subsequently stored in liquid nitrogen,and then in criotubes at -80°C prior to DNA extraction.

DNA extraction
DNA extraction was performed using commercial kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The samples were taken out of the criotubes, warmed to room temperature (20-25°C), and cut to smaller pieces using sterile scissors. Less than 25 mg of each sample was placed into 1,5 ml tube (Eppendorf Safe-Lock Tubes, Eppendorf, Garmany) with previously prepared emzymatic solution (20 mg/ml lysosime; 29mM TrisHCl, 2 mM ED-TA; 1,2% tritione) for the degradation of gram positive bacteria wall. Subsequently, the samples were incubated for 1 h at 37°C.

DNA amplification
At the beginning, the bacterial ubiquitous 16S rRNA primer was used to detect the 16S ribosomal RNA in all 36 samples. Subsequently, a PCR mixture with oligonucleotide primers specific for rDNAs was used.
The quality of extractracted DNA was checked spectrophotometrically (NanoDrop 1000 Spectrophotometer, Thermo Fisher Scientific, Wilmington, USA). A species-specific PCR was performed with primers targeted to the bacterial 16S rRNA genes of Prevotella Nigrescens All PCR essays were performed in a 50 μL reaction mixture containing 1 μM of each specific primer, 2 mM Mg-Cl2, 0.2 mM dNTP, the 0.25 μL AmpliTaq Gold DNA polymerase, and 10 μL extracted DNA (all reagents from Applied Biosystems, Lewinsville. USA).
Amplification was carried out in a thermal cycler (Applied Biosystems 2720 Thermal Cycler, Singapore) with an initial denaturation at 95°C for 5 min followed by 36 cycles of denaturation at 95°C for 30 s, primer annealing step at 55°C for 1 min, an extension step at 72°C for 1 min (apart from 2 min for Prevotella nigrescens), and a final step at 72°C for 2 min (except 10 min for Prevotella nigrescens).
PCR products were examined by 2% agarose gel electrophoresis performed at 5 V/cm. Agarose gel was made by melting agarose in Tris-acetate EDTA buffer in the microwave owen until boiling. After that, it cooled down to the temperature of approximately 50°C, and ethidium bromide of 0.5 μg/ml concentration was added. The gel was then solidified at room temperature and placed in 1X TAE buffer. Amplification products were mixed with a buffer with bromphenole-blue (BlueJuice, invitrogen, California, USA) in a ratio 6 to 1. The gel was photographed using Kodak DC 290 Zoom Digital Camera (Kodak corp, Rochester, New York, United States) under UV light (λ=302 nm). For determinig the length of bacterial DNA strand, a commercial standard with DNA strands ranging in length from 67 to 1114 bp was used (Roche, Indianapolis, USA).
One sample was positive for Pseudoramibacter alactolyticus and Propionibacterium propionicum, one sample was positive for Prevotella nigrescens and Pseudoramibacter alactolyticus, while one sample was positive only for Pseudoramibacter alactolyticus.
There was no statisticaly significant correlation between either of three microorganisms and diabetes mellitus ( Table  2) or symptms (Table 3). However, considering a low number of infected tissue samples, it is hard to consider the results of this particular statistical analysis as highly relevant. Due to this fact, this study did not give sufficient data about extraradicular infection and its connection with diabetes mellitus and symptoms.

Discussion
The role of microorganisms in primary endodontic infections was early established (16). Also, residual intraradicular infection was recognized as the major cause of persistant apical periodontitis (1). Apart from intraradicular infection, the absence of post-treatment healing can be caused by: aloplastic material in periapex (e.g. extruded root canal filling materials), cholesterol crystals accumulations, and true cysts formation. Also, periapical radiolucency can be caused by scar tissue healing and extraradicular infection that is according to Nair (1) generally in the form of periapical actinomycosis. Nevertheless, there are studies that have shown the presence of biofilms on the outer-extraradicular side of the root using scanning electron microscope, and studies reporting that Actinomyces species are not the only infective agents found in unresolved periapical lesions (13). Wang et al. (13) investigated extraradicular bacterial flora in persistent apical periodontitis, and although the prevalences of Actinomyces sp. and Propionibacterium were the highest, bacterial species were multiple and included Prevotella sp., Streptococcus, Porphyromonas endodontalis, and Burkholderia. In cases with vital and necrotic pulps there were no microorganisms on extraradicular surfaces, which suggested that extraradicular biofilm is associated with persistent infection after failed endodontics (13).
We chose to evaluate the tissue samples of closed periapical lesions for the presence of 3 microorganisms that were reported to be isolated from root canals with necrotic pulp and/ or from the root canals of the teeth with failed endodontic procedure. Prevotella Nigrescens was chosen as a representative of bacterial species that are highly prevalent in root canals with necrotic pulp, and frequently isolated in symptomat-
Mi smo odabrali ispitati uzorke tkiva zatvorenih periapikalnih lezija na prisutnost triju mikroorganizama izoliranih iz korijenskih kanala s nekrotičnom pulpom i/ili korijenskih kanala neuspješno endodontski liječenih zuba. Prevotella nigrescens odabrana je kao reprezentativna bakterija visoko prevalentnih bakterija u korijenskih kanalima s nekrotičnom ic endodontic infections (17). Pseudoramibacter alactolyticus and Propionobacterium propionicum were detected in root canals of patients with failed endodontic treatment (4). Exclusion criteria were applied to the patients that had been taking antibiotic therapy during the previous three months, due to a possible decrease in the number of anaerobic bacteria caused by antibiotic therapy. In this research, the intention was to study microbiological aspects of periapical tranparencies persisting after endodontic treatment that are "closed" i.e. not communicating with periodontium or oral cavity through caries, not adequately condensed root canal fillings, unfilled root canals, and leaking coronar restoration. Also, root fracture, sinus tract, communication with maxillary sinus, and tooth mobility indicate communication of periapical lesion with oral ecosystem, and similar cases were also exculded from our research before or during operative procedure.
It must be pointed out that we took great care about the samples in order to prevent contamination with oral bacteria. For this purpose, the rules of aseptic surgical sampling were applied. Marginal incision was performed to reflect mucoperiostal flap and reach periapical region, because it was reported that the risk of contamination was not significantly higher when compared to submarginal incision, and significantly smaller then with Partsch incision (18). Mucosa was rinsed with 0.2% chlorhexidin-gluconate, and microbiological samples were cultivated, and although with marginal incision, 100% of the samples from bone were positive, as compared to only 20% with submarginal incision, the ratio of periapical samples positive for microorganisms did not significantly differ between the two groups, and the microorganisms cultivated from mucosa, bone and periapical tissues of the same patient differed. The authors concluded that mucoperiostal flap reflection was not responsible for positive findings. Still, even if care is taken to avoid contamination from the mouth, it is still possible that the lesion gets infected by the intraradicular bacteria during sampling, and the positive finding of microorganisms in such samples can be reporeted as extraradicular infection (1). Apart from the mentioned contamination of the samples, molecular techniques such as PCR in detecting microorganisms in the samples of periapical tissue are questionable as well. Is not possible to differentiate between viable and non-viable microorganisms nor is it possible to distinguish microbes and their structural elements in phagocytes from extracellular microorganisms using the PCR method (1, 4). Therefore, although molecular methods are quite sophisticated, they do not solve the primary issue of avoiding contamination of periapical lesion during sampling.
The use of universal bactreial primer in our research revealed bacterial infection in 16.67% of the samples. Our results also suggest that there was a low incidence of periapical infection with the three bacterial species examined. In our study Prevotella nigrescens and Pripionibacterium propionicum were detected in one sample each. Low prevalence of extraradicular infection with these two species was expected and is in agreement with the reported low prevalence of extraradicular biofilms (found in 6%) in the teeth with either untreated or treated root canals (14). In the cited study, pulpom i često izolirana u simptomatskim endodontskim infekcijama (17). Pseudoramibacter alactolyticus i Propionobacterium propionicum detektirani su u korijenskim kanalima pacijenata nakon neuspješnog endodontskog liječenja (4).
In our research, Pseudoramibacter alactolyticus and Propionibacterium propionicum were detected in 3 samples and 1 sample, respectively. In the studies dealing with endodontic failures, these species were detected in significant percentages 11%-50% (4, 23) and were, apart from E. fecalis, the most prevalent species detected in the root canals of the teeth with persistant periapical lesion (4). In these reports, the samples were taken from root canals. Hence, these results cannot be compared to ours. As already mentioned, we detected Pseudoramibacter alactolyticus in 3 tissue samples of persistant apical lesions. Although the percentage of the infected samples is low it, such a result has been expected and it is in accordance with the findings of Siqueira and Rôças (4). They found that E. faecalis, P. alactolyticus and P. propionicum are the most prevalent species in the root canal samples of the teeth with failed endodontics, and that all of the examined samples harbored at least 1 of the gram-positive bacterial species: E. faecalis, P. alactolyticus, or P. propionicum. It must be pointed out that that we analysed periapical tissues rather than root canal samples. Propionibacterium propionicum is a facultative anaerobe formerly known as Arachnia propionica. This microorganism shares similar invasive characteristics as actinomyces (3). Genera Actinomyces and Propionibacterium can cause a chronic, granulomatous, infectious disease called actinomycosis. These bacteria are Grampositive filaments that end in hyphae and they are non-acid, non-motile, and form intertwining filamentous colonies called "sulphur granules" (1). The endodontic infections of actinomyces are caused by Actinomyces israelii and Propionibacterium propionicum, commensals of the oral cavity. These microorganisms can establish cohesive extraradicular colonies resistant to the immune system. They are consistently isolated from the periapical lesions which did not heal after an adequate endodontic treatment (1).
The role of Enterococcus faecalis as a key-stone pathogen in posttreatment disease was questioned (24,25) and the species belonging to other genera including Prevotella, Propionibacterium, and Pseudoramybacter have also been detected in root canals of treated teeth (7,26,27). Rôças and Siqueira (27) found that Propionibacterium (52%) and Pseudoramibacter alactolyticus (14%) were more prevalent in root canal samples of the teeth with posttreatment apical periodontitis undergoing retreatment than Enterococcus faecalis (12%). Our findings are in line with recent studies that have questioned the status of E. faecalis as the main pathogen in posttreatment apical periodontitis (24,25). Moreover, although ceratin bacteria can be recognized as key-stone pathogens, it is the synergistic activity of the whole bacterial community that interferes with host immune defence and causes tissue destruction (14,13). Biofilm is a form of microbial community attached to a solid surface in a nutrient-containing fluid, where the microbial cells embedded in an extracellular matrix, interact with each other. The microorganisms living in a biofilm are able to self-organize, resist environmental changes, act synergistically and respond to the changes in the environment as a community (12,13,14). Bearing in mind the tendency of microorganisms to form intraradicular and extraradicular biofilms, the future metagenomic studies should be oriented on the pathologic potential of bacterial biofilms rather than on a single microorganism, despite relatively few bacterial species involved in persistant periapical periodontitis. Study designs using cultivation methods and PCR cannot reveal interaction potentials between different microbial species since it is hard to co-cultivate many species, and PCR does not differentiate between viable and non viable microorganisms.

Conclusions
We can conclude that lesions of apical periodontitis associated with failed endodontic treatment could harbor microorganisms other than Actinomyces and Propionicum species. The prevalence of infection of closed lesions of persistant apical periodontitis with P. intermedia, P. propionicum and P. alactolyticus is low. We have insufficient data for clearly determining extraradicular infection and its connection with diabetes mellitus and clinical symptoms.