Distribution of Aggregatibacter actinomycetemcomitans in deep caries lesions

Summary Introduction Deep caries is a reversible process where caries lesion has affected bigger part of dentin and only thin layer of softened dentin that separates lesion from the pulp is remained. The objective of this study was to identify and determine serotypes of Aggregatibacter actinomycetemcomitans in teeth with deep caries lesions at the beginning of their treatment. Material and methods Clinical research included 29 patients of both genders, aged 16 to 40 and 45 permanent teeth with diagnosed deep caries lesions based on medical history, clinical and radiographic examination. After cavity preparation and removal of softened dentin, microbiological swab was taken from the bottom of the cavity. Swabs were disposed in special sterile micro tubes and stored at the temperature of −80°C until serotyping was done (determination of serotypes of A. actinomycetemcomitans bacterium). Results In one of the 3 samples two serotypes of A. actinomycetemcomitans (b and c) were identified which is relatively rare finding, while in the second and third sample serotypes (a) and serotype (b) was identified, respectively. Conclusion In the three samples the 3 serotypes were found (a, b and c) and one of the samples was carrying even two different serotypes, which is a rare phenomenon. For more serious epidemiological study of A. Actinomycetemcomitans serotypes at the population level incomparably larger starting material is necessary, at least few hundred of samples.


INTRODUCTION
Dental caries is a chronic complex bacterial infection that results in milligram loss of minerals from infected tooth. Some authors have defined caries as a disease of hard dental tissues (enamel, dentin and cementum) with characteristic processes of demineralization and remineralisation. Deep caries can be classified as clinically visible lesion in dentin characterized by close topographic relation to the pulp, followed by weakening of the sidewalls due to the progression of caries both in width and depth [1,2].
One of the factors affecting the occurrence of caries is dental plaque that represents adhered deposits of bacteria and their products and it exists on every surface of teeth. Dental plaque contains pyogenic bacteria such as Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actynomycetemcomitans [3]. According to the new classification based on phylogenetic similarity, A. actynomycetemcomitans was in 2006 grouped together with Haemophilus aphrophilus and H. paraphrophilus in the new Aggregatibacter genus. A. actynomycetemcomitans was the first time described in 1912 by Klinger as coccobacillary bacteria isolated together with Actinomyces from actinomycotic lesions of man and it was therefore originally grouped into the Actinomyces genus.
A. actynomycetemcomitans is a non-motile, slowgrowing, capnophilic Gram-negative coccobacilus. It grows slowly at 37°C, both aerobically and anaerobically. Five stereotypic groups of A. actinomycetemcomitans were classified based on surface polysaccharide, where serotypes a, b and c are the most prevalent in the oral cavity. A particular clone of serotype b shows enhanced leukotoxic activity. It is predominantly associated with the cases of localized aggressive periodontitis, while the serotype c is usually found in healthy subjects [4][5][6][7]. Takahashi et al. demonstrated in an animal model that SPA serotype b has an accentuated ability to stimulate interleukin-1 release from macrophages. The latest data based on molecular genetic analysis indicated significant divergent evolutions of genomes of bacteria serotype a in relation to serotypes b/c, and differences in genomes implied accentuated phenotypic differences [8]. Pajukanta et al. showed that the response of A. Actynomycetemcomitans to antimicrobials can vary depending on the serotype [9].
It is assumed that patients are usually infected with one serotype that is usually maintained over time, i.e. indicates stability [10]. However, more recent research based on molecular genetic testing and not on serological tests, indicated that serotype changes are possible over time. In the study of van der Reijden et al. performed on the population of Indonesian island Java, after 8 years (time interval of the study) there was a change noticed in the prevalence of serotypes at the population level [11]. Among the people who were tested at the beginning and at the end of the study, after 8 years 58% of them had the same bacterial serotypes, and 42% of them had other serotypes. They also reported rare cases of multiple serotype infections, around 10% while during the 8 years of research multiple infections increased from 12% to 17% [12].
Another characteristic of A. actynomycetemcomitans is that distribution of serotypes significantly fluctuates depending on geographic region of analysed population as well as periodontal status of teeth. Thus, for example, in the USA, in patients with localized juvenile periodontitis the serotype b is more common than serotypes a and c. The situation is similar in Finland population where serotype b dominates among the patients with periodontal disease, whereas serotype c is more commonly found among patients with no periodontal disease. In Japanese population, serotypes a, c and e were the most common [13].
The aim of this study was to determine and identify serotypes of Aggregatibacter actynomycetemcomitans in teeth with deep caries lesions at the beginning of deep caries treatment.

MATERIAL AND METHODS
The clinical study was conducted on 45 permanent teeth of patients, aged 16 to 40. Different morphology groups of permanent teeth with deep caries lesions were included in the study. Deep lesions considered dental caries followed by sensitivity to thermal stimuli, affecting more than ¾ of the tooth crowns with lots of softened dentin. After cavity preparations and removal of softened dentin, the swab was taken from the bottom of the cavity. Taken swabs were disposed in special sterile micro tubes and stored at the temperature of -80 o C until serotyping was performed (determination of serotypes of A. actinomycetemcomitans).
The samples were tested at the Institute for Human Genetics, Faculty of Dentistry, University of Belgrade using the multiplex PCR method that enables simultaneous amplification of various gene sequences using multiple pairs of primers. Familiar sequences of primers were used for PCR reactions. Serotyping of A. actynomycetemcomi-tans was also based on the multiplex PCR reaction that included the use of five pairs of primers specific for a, b, c, d and e serotypes of this microorganism, as well as highly specific amplification conditions that are appropriate for all oligonucleotide primers.
The length of products of gene amplification for certain serotypes, with upper pairs of primers were as follows: serotype a: 428 bp, serotype b: 258 bp, serotype c: 559 bp, serotype d: 690 bp and serotype e: 211 bp. Reactions were conducted in the total volume of 25 microliters.
PCR conditions are given in the Table 1.

RESULTS
Oligonucleotide primers specific for the group of genes involved in the biosynthesis of bacterial serotype-specific polysaccharide antigens were designed to be able to identify five main serotypes of A. actynomycetemcomitans (a, b, c, d and e) by using the multiplex PCR. In laboratory conditions, multiplex PCR optimization has proven to be technically demanding and that is why it was possible to serotypically define only a small percentage of samples. A serotype was conditionally established after a number of repeated attempts in only 3 samples. The interesting fact is that two serotypes (b and c) were found in one of the 3 samples, which is relatively rare finding. Figure 1 shows that gel was given after one attempt of serotyping where only one out of 10 samples showed the corresponding strips (10b sample). In the samples # 18 and # 23 arrows represent strips that do not correspond to any known serotype and which could be the PCR artefacts. One of repeated serotyping successfully identified serotypes in 2 more samples: 7c (serotype a) and 6 (serotype c). During repeated multiplex PCR reaction, a nonspecific strip that was present in the first experiment was now lost in the sample 23 ( Figure 2).

DISCUSSION
A. actynomycetemcomitans, ie its serotype c, is a part of normal flora of the oral cavity in healthy patients. This bacterium can also be pathogen because it has significant virulence factors (one of them is adhesion) that enable colonisation of bacteria and intensify its destructive potential in oral diseases [14]. Serotyping of bacteria is a suitable typization method for epidemiological studies. Primarily it is easy to perform and more sensitive compared to other methods that require additional equipment and are more costly [15]. Dental caries is multibacterial disease but serotyping is the only method to determine certain serotypes of bacteria that play role in aetiology. Number of studies has shown that certain bacteria detected in dental plaque are closely associated with the occurrence of caries while large caries lesions often communicate with subgingival biofilm bacteria. Streptococcus mutans and Aggregatibacter actynomycetemcomitans are oral pathogenic bacteria associated with caries and periodontal disease. Psoter et al. (2011) aimed to determine colonization of these two microorganisms in dental plaque of adolescents from rural areas of Haiti by using two different methods of polymerase chain reactions (PCR): Standard PCR and Quantitative real-time PCR (qPCR). This study included 152 plaque samples from 104 patients aged 12 to 19 years. Total genomic DNA of these bacteria was isolated from the samples while dental caries or periodontal changes were found in all subjects during clinical examination. The results showed moderate to high prevalence of S. mutans and A. actinomicetemcomitans in all samples [16][17][18][19][20].
A large number of teeth affected with deep caries in both, upper and lower jaw (regardless the morphological group of teeth) could primarily be explained with socioeconomic and health conditions, not only in war but also in the post-war period in our region. Certainly the most important factor is the level of health care that does not meet basic health needs of the population. Also, difficult social situation and struggle for pure existence often put dental health as second priority. More frequent occurrence of deep caries on the upper and lower molars could be explained by the fact that occlusal surfaces of these teeth is susceptible to caries, due to their morphology and the existence of fissures and cusps. It is also well-known fact that nutrition, use of fluorides and oral hygiene have dominant influence on the occurrence of caries.
During clinical examination in this study, poor oral hygiene was noticed among the respondents. Sofrata et al. (2008) tested the antibacterial effect of Miswak sticks for oral hygiene against bacteria involved in the occurrence of periodontal disease and caries including A. actynomycetemcomitans. The Miswak sticks were standardized by size and weight (0.07 and 0.14 g) and they were tested against S. mutans, Lactobacillus acidophilus, A. actynomycetemcomitans, Porphiromonas gingivalis and Haemophilus influenzae. The inhibitory effect of the pieces of Miswak sticks embedded in the agar plate was most pronounced on P. gingivalis, A. actynomycetemcomitans, H. influenzae and less on S. mutans and L. acidophilus [21].
The literature indicates that it is impossible to determine the serotype in 3 to 8% of samples of A. Actynomycetemcomitans [22]. Unfortunately, this percentage was significantly higher in our study. We tried to overcome technical problems in many ways by changing the number of experimental parameters but we got unsatisfactory results. Multiplex PCR was performed with different amounts of starting material and different duration of particular steps of reaction. Also, the number of cycles was modified (25,30,35) as well as MgCl 2 concentrations and hybridization temperature. However, even after all the effort and repeated attempts the success of serotyping was moderate. We were able to determine A. Actynomycetemcomitans serotypes in only 21% of the samples.
Numerous studies have come to conclusion that A. actynomycetemcomitans is most frequently associated with periodontal diseases [23]. The study of Cortelli et al. (2005) showed the presence of A. actynomycetemcomitans in 41.6% of the subjects with chronic periodontal disease and 72% of subjects with acute periodontal disease [24]. Tinoco et al. also found A. actynomycetemcomitans in 80% of young patients with periodontal disease and suggested that the presence of this bacterium in the oral cavity may serve as an indicator of risk for future tests of acute periodontal disease [25].
Based on research of deep caries lesions Simon-Soro and Mira found diverse ecosystem made of a large number of bacteria affecting the spreading of caries lesions. The results showed that S. mutans was present in a small percentage and that other bacteria including A. actynomycetemcomitans affect spreading of caries lesions [26].

CONCLUSION
Given the small initial number of teeth, relatively small percentage of samples positive for A. actynomycetemcomitans and finally, poor achievement of the multiplex reaction of serotyping, it is impossible to talk about the prevalence of certain serotypes in our population. In the 3 samples three serotypes of A. actinomycetemcomitans (a, b and c) were identified and two different serotypes were   identified in one of the samples that is a rare phenomenon. For more serious epidemiological study of serotypes of A. Actinomycetemcomitans at the population level and their relation to the formation of dental caries an incomparably larger starting material is necessary, at least a few hundred of samples.