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ORIGINAL RESEARCH
Year : 2018  |  Volume : 10  |  Issue : 1  |  Page : 32-36

Application of fluorescent In situ hybridization for rapid detection of aggregatibacter actinomycetemcomitans in patients with chronic periodontitis


Maratha Mandal Central Research Laboratory, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India

Date of Web Publication6-Jul-2018

Correspondence Address:
Dr. Kishore G Bhat
Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, R.S.No. 47A/2, Bauxite Road, Belgaum - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jicdro.jicdro_28_17

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   Abstract 


Background: Aggregatibacter actinomycetemcomitans is a proven periodontal pathogen. In dentistry, there is a need to identify and quantitate the organisms from the diseased sites quickly and reliably. Since culture requires several days, molecular methods are being used frequently to detect A. actinomycetemcomitans. Among them, fluorescent in situ hybridization (FISH) is rapid, sensitive and quantitative. An attempt is made here to evaluate the applicability of this technique as a diagnostic tool in periodontology. Materials and Methods: A total of 77 healthy individuals and 77 patients with chronic periodontitis were enrolled for the study. Subgingival plaque was collected, fixed with paraformaldehyde and subjected to FISH. Oligonucleotide probe labelled with 6-carboxyfluorescein (FAM) was used for hybridization. After the procedure, the fluorescently stained A. actinomycetemcomitans were identified and counted from the smear and quantitated using a simple grading. Results: The data revealed that plaques from 84.5% of healthy individuals and 98.7% of chronic periodontitis showed the presence of A. actinomycetemcomitans. However, the number of these bacteria were very low in most positive samples from healthy subjects in contrast to patients with chronic periodontitis, who had higher number of organisms. Statistical analysis using Mann–Whitney test revealed a significant difference among the two groups with P ≤ 0.001 and Z = −5.833. Conclusions: The procedure used in the study is simple, rapid and can be easily adaptable. It also has a high sensitivity and has the ability to detect a single bacterial cell. The method can be directly applied to the clinical samples and can be used as a rapid diagnostic tool in periodontics.

Keywords: Chronic, hybridization, periodontitis, probe


How to cite this article:
Bhat KG, Chhatre A, Kumbar VM, Kugaji MS, Patil S. Application of fluorescent In situ hybridization for rapid detection of aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. J Int Clin Dent Res Organ 2018;10:32-6

How to cite this URL:
Bhat KG, Chhatre A, Kumbar VM, Kugaji MS, Patil S. Application of fluorescent In situ hybridization for rapid detection of aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. J Int Clin Dent Res Organ [serial online] 2018 [cited 2018 Dec 15];10:32-6. Available from: http://www.jicdro.org/text.asp?2018/10/1/32/236093




   Introduction Top


The oral microbial flora is highly complex and the application of molecular techniques in the recent years has revealed its enormous diversity.[1] The bacterial phylotypes present in the oral cavity is estimated to be around 2 × 104 and many of them remain uncultivable.[2] However, there are several key organisms implicated in the pathology of periodontal diseases as well as various extra oral/systemic illnesses such as atherosclerosis, Crohn's disease, obesity, diabetes and pregnancy complications.[3] One such organism is Aggregatibacter actinomycetemcomitans.[4]

Periodontitis is a chronic inflammatory disease associated with loss of connective tissue and alveolar bone around teeth and is one of the most common bacterial infections of man.[5]A. actinomycetemcomitans is known to be involved in the aetiology of aggressive periodontitis.[6] Several studies have shown that this bacterium is present in sites of chronic periodontitis and also in healthy individuals.[7]

This organism is facultative Gram-negative bacillus and prefers an atmosphere of 5%–10% CO2 for optimum growth. Even though it is relatively easy to culture, several days are needed before it produces growth on the medium and for further biochemical identification.[8] Hence, several molecular techniques with a high specificity and sensitivity are being used for rapid detection of these bacteria directly from clinical samples. These methods include polymerase chain reaction, loop mediated isothermal amplification, hybridization techniques such as fluorescent in situ hybridization (FISH), checkerboard hybridization and microarray and high throughput 16s rRNA cloning and sequencing.[1] Each of these methods have their own advantages and limitations and selection of proper technique is of utmost importance to achieve optimum outcome.

Majority of the molecular methods provide only qualitative results indicating the presence or absence of bacteria or semi quantitative results that are obtained through DNA or RNA amplification. Among them, the FISH technique provides information about the morphology, number and spatial distribution of various microbes in dental plaque.[9] Surprisingly, the utility of FISH as a diagnostic test for periodontal pathogens has not been explored much. Keeping this in mind, the present study was aimed at rapid detection and quantitation of A. actinomycetemcomitans in subgingival plaque of healthy individuals and patients with chronic periodontitis using FISH.


   Materials and Methods Top


The present study was carried out in the Central Research Laboratory of Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka. This investigation was part of a funded project from Rajiv Gandhi University of Health Sciences, Bengaluru on “A study of microbial diversity of subgingival plaque: Comparison of FISH, denaturing gradient gel electrophoresis and dot blot hybridization.” This study included 77 healthy subjects (Group I) and 77 patients with chronic periodontitis (Group II) belonging to both the sexes and between the age group of 18 and 60 years. A written informed consent was obtained from each participant before enrolling in the study. Approval from Ethics committee of the Institution was taken before commencement of the project.

The participants were enrolled only if they fulfilled certain selected definitions. The inclusion criteria for the healthy group were; no signs of gingival inflammation, absence of bleeding on probing, probing depth of ≤3 mm, and no clinical attachment loss. The criteria for inclusion of chronic periodontitis include patients with presence of >20 natural teeth, signs of gingival inflammation, clinical attachment loss of >3 mm in at least 4 teeth and bleeding on probing. The exclusion criteria for both the groups were subjects who had undergone periodontal therapy and/or received antibiotics within 3 months prior to sampling, pregnant and lactating women, tobacco users, presence of diabetes and other systemic illnesses.

From each participant, after selection and isolation of the tooth, the supraginigival plaque was stripped and the subgingival plaque was collected by curetting and transferred to vial containing Tris-ethylenediaminetetraacetic acid buffer. Upon receipt in the laboratory, portion of the plaque was transferred to a vial containing ice-cold paraformaldehyde (4%), fixed for 4 h, washed three times with ice-cold phosphate buffered saline (PBS) and then resuspended in equal proportions of PBS and ethanol (96%) and stored at −20°C till use.[10]

The probe used for targeting A. actinomycetemcomitans was species specific, was complementary to 16s rRNA of the organism and had the sequence 5'-TCC ATA AGA CAG ATT C-3'.[11] These probes were labelled with fluorescent dye 6-carboxyfluorescein (FAM) on the 5' end (procured from Bioserve, USA). The procedure was optimized by testing the probe against standard strain of A. actinomycetemcomitans (ATCC 43718) [Figure 1]a. The specificity of the probe was confirmed by testing against known strains of Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia, Treponema denticola, Actinomyces viscosus and Streptococcus mutans.
Figure 1: (a) standard strain of A. actinomycetemcomitans in FISH analysis (belongs to this study), (b) occasional A. actinomycetemcomitans in healthy individuals (belongs to this study)

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For preparation of slides, 10 μl of the fixed sample was applied to gelatin coated slide, allowed to dry and was dehydrated by exposing the material to serial concentrations of ethanol. The slides were then dried at 46°C for 10 min and FISH was performed. Briefly, fresh hybridization buffer was prepared with 30% formamide concentration, and oligonucleotide probe was diluted in this buffer to achieve a concentration of 20 picomoles in 100 μl of buffer and applied to the plaque on the slide. It was kept in a moist petri dish and incubated at 48°C for 2 h. The hybridization buffer was then decanted and slides were washed in wash buffer at 48°C for 10 min, the slides were then washed with ice cold distilled water, quickly air dried and mounted with cover slip using DPX mountant. They were wrapped in aluminium foil and stored at −20°C till studied under the microscope.[10],[11]

The demarcated area of the plaque material was studied using fluorescent microscope with ×40 and ×100 objectives. A. actinomycetemcomitans appear as brightly fluorescent coccobacilli/bacilli. They were counted and graded by using a simple formula devised by us: No bacteria seen – 0/negative, 1–5 bacilli/smear – occasional/0.5, 6–10/smear – 1+, 10–100/smear – 2+, >100 bacteria/smear – 3+, clumps of bacteria seen – 4+.

Comparison of the data between the two groups was performed by applying Mann–Whitney test and the results were expressed as z and P values.


   Results Top


Analysis of the data showed that A. actinomycetemcomitans could not be detected in 15.5% of healthy individuals where as only 1.3% of patients with chronic periodontitis were negative for the organism. This difference was statistically significant (P ≤ 0.001, Z = −5.833).

The positive samples were further graded as 0.5+, 1+, 2+, 3+ and 4+ based on the number of bacteria in each smear as mentioned in the methodology. It could be seen that among healthy individuals, even though 84.5% of plaque samples had A. actinomycetemcomitans, their number was low in most of the samples. In 36.4% (28) samples, only occasional bacilli could be detected and in 40.2% (31) samples, the bacterial load was <10 [Figure 1]b and [Graph 1].

In patients with chronic periodontitis, 98.7% of plaque samples were positive for A. actinomycetemcomitans. Among them, only 10.8% (8) patients had occasional bacilli, 52% (40) had a bacterial count of up to 10/smear and a significant proportion of patients had higher bacterial numbers (37%) [Figure 2]a, [Figure 2]b and [Graph 1].
Figure 2: (a) A. actinomycetemcomitans in patients with chronic periodontitis. (Belongs to this study), (b) cluster of A. actinomycetemcomitans in patients with chronic periodontitis (belongs to this study)

Click here to view



In plaque samples from patients with chronic periodontitis, it was observed that whenever the bacterial load was higher, the organisms appeared to be in clusters rather than being discreet.


   Discussion Top


A. actinomycetemcomitans is considered to be one of the keystone periodontal pathogens. It is known to be definitively involved in the aetiology of aggressive periodontitis in young adults. It possesses several virulence factors including leukotoxin, cytolethal distending toxin, lipopolysaccharide and fimbriae among others (1). Even though this organism is a facultative anaerobe, it is often overshadowed by other faster growing bacteria from the dental plaque samples, compelling the use of one of the several selective media for its recovery.[12] Since the isolation and identification of A. actinomycetemcomitans takes several days by culture, many investigators tend to rely on culture independent molecular methods for their study. These molecular microbiology techniques can be used as rapid screening tools and serve as important diagnostic approaches in clinical dentistry. However, majority of these techniques are either qualitative indicating the presence or absence of organisms or yield semi-quantitative results.[9] Among these various techniques, whole cell hybridization with fluorescently labelled 16s rRNA oligonucleotide probes has become popular. It allows detection of specific bacteria at the single cell level and aid determination of their abundance in complex microbial communities such as dental plaque.[13]

The FISH technique provides information about the morphology, number and spatial distribution of various microorganisms in dental plaque. In addition, this technique provides direct quantitative results without need for prior culture. The method is rapid, sensitive and can be very specific.

In dentistry, FISH has been mainly used as a research tool to study the interrelationship between different oral bacteria in in vitro created biofilm [14],[15],[16],[17] and to study the invasive capability of these bacteria including A. actinomycetemcomitans on buccal epithelial cells.[18],[19] Surprisingly, there are very few studies that have utilized this technique as a diagnostic tool to detect periodontal pathogens.[20]

The main intent of the present study was to assess the applicability of FISH as a diagnostic tool in detecting A. actinomycetemcomitans from dental plaque specimens from patients with chronic periodontitis and healthy individuals. We did extensive literature survey and prepared a protocol that was simple, rapid and could be easily adapted to any clinical laboratory. We performed initial trials with different probe concentrations, hybridization temperatures and incubation timings for optimization of the protocol. The specificity of the assay was confirmed by testing the probe against known strains of several oral bacteria.

The results of the present study clearly showed that A. actinomycetemcomitans was present in plaque samples of many healthy individuals (84.5%), but their numbers were very low in most of these subjects. Since the samples showing very occasional bacteria were also considered as positive could be the reason for such a high prevalence. The same could be true even for patients with chronic periodontitis (with 98.7% positivity). However, the samples showing occasional bacteria were not many and most samples had significant number of bacteria. Since the major thrust of the study was on FISH technique, we have not attempted to correlate the results with clinical signs and indices of chronic periodontitis.

Although A. actinomycetemcomitans is linked to the aetiology of aggressive periodontitis, the organism is also found in other forms of periodontal diseases and variable number of healthy individuals. A number of studies have shown that they occur at diseased sites in high numbers in both aggressive and in chronic periodontitis.[21],[22],[23] They are also known to be present in the oral cavity of healthy individuals and the prevalence is known to vary from 0% to 78%(7). It has been reported that the main difference between the clinical groups may not be the prevalence but rather the load of pathogens found in positive samples.[24]


   Conclusion Top


One of the main requirements in periodontal microbiology is to quantify organisms quickly and reliably. This technique which requires just 3 h for completion after fixation of the samples can be used as a diagnostic tool for rapid detection and quantitation of A. actinomycetemcomitans from clinical samples. Since this method has the ability to detect even a single microbial cell against a mixed background, it can be considered very sensitive and useful for follow up posttherapy.

The only limitations of the technique are need for fluorescent microscope which is pretty expensive and availability of expertise to separate the fluorescent stained bacteria from the nonspecific background material. Attempts are being made to improve the specificity of the method still further by either employing probes of a larger length and use of peptide nucleic acids as probes.[25] We feel that the method described here is simple, rapid, and easily adaptable and has great potential for accurate identification of A. actinomycetemcomitans directly from plaque samples.

Acknowledgment

We express our sincere gratitude to the advanced research committee, Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka for funding this project.

Financial support and sponsorship

The study was supported by Advance Research Grants by Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka, India.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Al-Ahmad A, Follo M, Selzer AC, Hellwig E, Hannig M, Hannig C, et al. Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization. J Med Microbiol 2009;58:1359-66.  Back to cited text no. 17
    
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Rudney JD, Chen R, Sedgewick GJ. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are components of a polymicrobial intracellular flora within human buccal cells. J Dent Res 2005;84:59-63.  Back to cited text no. 18
    
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Tomita S, Komiya-Ito A, Imamura K, Kita D, Ota K, Takayama S, et al. Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in Japanese patients with generalized chronic and aggressive periodontitis. Microb Pathog 2013;61-62:11-5.  Back to cited text no. 23
    
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