Detection of pathogens from periodontal lesions

Correspondence to: Mario J Avila-Campos Laboratório de Anaeróbios Departamento de Microbiologia ICB/ USP Av. Prof. Lineu Prestes, 1374 05508-900 São Paulo, SP, Brasil E-mail: mariojac@usp.br Partially financied by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp Process n. 00/07785-3 and 00/ 07582-5). Based on thesis presented to Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 2002. Received on 16/6/2003. Reviewed on 26/11/2003. Approved on 5/2/2004. Detection of pathogens from periodontal lesions Detecção de patógenos de lesões periodontais


INTRODUCTION
It is known that a great number of different microorganisms exist in the oral cavity but only some species, particularly anaerobic bacteria, have been implicated in the etiology of periodontal disease.Both Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum are Gram-negative bacteria involved in the pathogenesis of human periodontitis but they can also be associated to other endogenous infections. 9 A. actinomycetemcomitans is an important periodontopathogen that is involved in the etiology of different forms of periodontal diseases, particularly in the localized juvenile and adult periodontitis, and also in several extra-oral infections such as endocarditis, pericarditis, pneumonias, septicemias, and abscesses. 3,10,17 nucleatum has also been considered an important periodontopathogen for the development of gingivitis and periodontitis and as the most common anaerobic species found in human and animal infections, particularly in the oral cavity. 2 The isolation and identification of periodontal pathogens characterize an important tool for increasing knowledge on periodontal microbiota as well as on the etiology and progression of periodontal infections.However, molecular methods have contributed to the detection of putative periodontopathogens in several oral or extra-oral infections.15 Even so, there have been some limitations of bacterial culture such as high cost and time-consuming procedures, besides the fact that it may fails to uncultivable organisms.Additionally, cell viability is necessary for isolation but can be partially lost during transport and in the sampling procedure.6 Several methods for the rapid detection of periodontal pathogens have been reported such as immunologic and immunoenzymatic assays, protein electrophoresis, and DNA-DNA hybridization.However, these methods show different limitations leading to false-positive results as well as crossreactivity.1,4 Polymerase chain reaction (PCR) is an excellent tool used to identify putative periodontopathogens directly from subgingival samples.Also, it is a fast and efficient method to detect, identify, and differentiate periodontal organisms due to its sensitivity and specificity but appropriate standardization is necessary. 5e aim of this study was detect the presence of A. actinomycetemcomitans and F. nucleatum from clinical subgingival samples of periodontal patients using two different methods.

METHODS
Fifty patients with adult periodontitis and 50 healthy subjects aged between 20 and 60 years old, of any sex or race, seen at an university of dentistry,in Brazil, were selected.Periodontal patients showed pockets deeper than 5 mm and bone loss, as diagnosed by radiographic examination.None of them was taking antibiotics during a three-month-period prior to sample collection.All subjects involved in this study were informed concerning the study's nature and procedures, and a consent form was obtained.The Ethics Commission of the Instituto de Ciências Biomédicas.University of São Paulo, approved this study.
Initially, the supragingival plaque was removed and subgingival samples were obtained using three sterilized paper points inserted in the deepest site of periodontal pocket or gingival crevice.After 60 seconds, the paper points were transferred into tubes containing 3.5 ml of the viability maintaining microbiostatic medium (VMGA III) transport medium. 7mples were mixed and diluted 10-fold in VMGA I solution, 7 and then plated on trypticase soy agarbacitracin-vancomycin (TSBV). 13Plates were incubated in anaerobiosis (90% N 2 + 10% CO 2 ) at 37 o C for four days.
To obtain a pure culture, characteristic colonies of both organisms were subcultured on blood agar.Then, they were presumptively identified by Gram-staining, and catalase, H 2 S, and indole production as well as by their susceptibility to sodium fluoride. 14Definitive identification was achieved using biochemical tests. 14ference strains of A. actinomycetemcomitans ATCC 29522, ATCC 33384, ATCC 43718, and KC 517 CDC (Centers for Disease Control and Prevention, GA, USA) and of F. nucleatum ATCC 10953 were used as controls.
Biotypes of A. actinomycetemcomitans isolates were detected by examining the ability to ferment dextrose, maltose, xylose, and mannitol. 12GA III media containing clinical samples were warmed at 37 o C for 10 minutes and then homogenized.Next 300 µl aliquots were washed three times with sterilized Milli-Q water.Pellets were resuspended in 300 µl water and then boiled for 10 minutes; supernatants were used as templates after centrifugation (14,000 x g, 10 minutes). 1so, bacterial DNA was extracted from 10 colonies of A. actinomycetemcomitans and five colonies of F. nucleatum.They were suspended in 500 µl sterile Milli-Q water, homogenized, and then boiled for 10 minutes.Then, after centrifugation, the supernatant (DNA) was used as template.
A termocycler Perkin Elmer, Gene Amp PCR System 2,400, was programmed to: 1 cycle at 94ºC (5 minutes); 30 cycles at 94ºC (30 seconds), annealing temperature for each specific primer pair (Table 1), and 72ºC (30 seconds); 1 cycle at 72ºC (5 minutes) until the final DNA extension.Specific primers used for identifying A. actinomycetemcomitans and F. nucleatum are listed in Table 1.
PCR products were detected by electrophoresis in 1% agarose gel in 1x TBE at 70 V for 2.5 hours.Gels were stained with an ethidium bromide solution (0.5 µg/ml), and then photographed on a UV transilluminator using the Electrophoresis Documentation and the Analysis System 120.A 1 kb DNA ladder was used as molecular mass standard.
To assess concordance between both detection methods (culture and PCR) as well as to determine their sensitivity and specificity, data were analyzed using a Kappa Index (K).

RESULTS
To detect A. actinomycetemcomitans and F. nucleatum, 50 subgingival samples from adult periodontitis patients and 50 from healthy subjects were analyzed.In a trypticase soy agar bacitracin-vancomycin (TSBV), nine (18%) clinical samples from peri- A. actinomycetemcomitans isolates were grouped in five biotypes (II, VI, VIII, IX and X).The most prevalent was biotype II.
Table 2 shows the sensitivity and specificity of the primer sets used for detecting A. actinomycetemcomitans and F. nucleatum from clinical samples.Primers AA were highly sensitive (100%) to detect A. actinomycetemcomitans from both subject groups.Furthermore, primers ASH and FU were also 100% sensitive to detect this organism in healthy subject samples.Primer FN5047, used to detect F. nucleatum, was more sensitive to detect this organism in patients or healthy samples than primer 5059S.On the other hand, primer ASH and 5059S were more specific to detect A. actinomycetemcomitans and F. nucleatum, respectively, in patients and healthy subject samples (Table 2).
All amplified products were compared and the primer pairs produced amplicons of predicted size (data not shown).Table 3 shows a comparison of the bacterial detection between culture method and each primer used in PCR.It can also be observed that the detection values were as significant to detect F. nucleatum in periodontal patients using primer FN5047 (p<0.002) as to detect A. actinomycetemcomitans in healthy subjects using primer ASH (p<0.000).
Statistical analysis showed that primers AA and FU had higher sensitivity, and primers ASH had higher specificity for detecting A. actinomycetemcomitans.However, primer FN 5047 showed higher sensitivity and primer 5059S showed higher specificity for detecting F. nucleatum (Table 3).

DISCUSSION
A. actinomycetemcomitans and F. nucleatum are important organisms of both human and animal indigenous microbiota, and they have been involved in several oral cavity infections.It is well known that improvements in diagnostic methods are useful in the prevention and treatment of periodontal disease and contribute for increasing knowledge on subgingival microbiota.Bacterial cultures are used for allowing to recovering cultivable organisms, although being a time-and-labor-consuming method.Several molecular tools are often used to identify periodontopathogens but PCR is considered to be an easy and fast detection method even in clinical samples. 11 this study, two methods of detecting A. actinomycetemcomitans and to F. nucleatum were compared.The primary isolation of both organisms studied was performed on selective TSBV medium. 13It is important to mention that although TSBV agar is used as the first choice to isolate A. actinomycetemcomitans, a high recovery rate of F. nucleatum was also observed.
Studies have detected A. actinomycetemcomitans using a PCR method but at different rates in populations with (19%) and without (70%) periodontal disease.These studies have also shown PCR sensitivity and the specificity in comparison to traditional bacterial culture.In the present study, primers AA and FU had higher sensitivity for detecting A. actinomycetemcomitans but primers ASH had higher specificity (Table 3).On the other hand, when compared to culture for detecting A. actinomycetemcomitans, primers AA and FU showed similarly sensitivity (100%), which confirms their specificity.DNA from A. actinomycetemcomitans and F. nucleatum strains were amplified by specific methods and produced predicted bands.It is important to note that both the annealing temperature and magnesium concentration are critical factors in PCR detection. 1F. nucleatum detection by PCR (primer 5059S) was similar to culture methods; however, when primer FN 5047 was used, PCR showed higher sensitivity than culture.The study results suggest that PCR is a sensitive and highly specific technique to detect A. actinomycetemcomitans and F. nucleatum in the subgingival plaque.
In conclusion, the present study demonstrated the usefulness of specific primers based on the PCR detection of periodontal organisms in subgingival samples.The use of molecular tools in the bacterial detection will provide faster and safer diagnostics of periodontal diseases and PCR reaction may be helpful in detecting putative periodontopathogens from subgingival samples.A single method could not be ideal, and using both traditional and molecular methods is recommended in the bacterial detection.

Table 1 -
Specific primers used in the detection of A. actinomycetemcomitans and F. nucleatum.with 17 isolates recovered, and ten (20%) were positive to F. nucleatum with 19 isolates.Two A. actinomycetemcomitans were isolated from one (2%) healthy subject and 18 F. nucleatum isolates were recovered from 12 (24%) healthy subjects.

Table 2 -
Sensitivity and specificity of specific primer pairs used to detect A. actinomycetemcomitans and F. nucleatum from clinical samples in VMGA III.

Table 3 -
Detection of A. actinomycetemcomitans and F. nucleatum strains from periodontal patients and healthy subjects.