| Abstract|| |
Background and Aims: Although Streptococcus mutans has been implicated as the major etiologic agent in the development of dental caries, however, this organism has not been found to be present in all children with caries. Thus it remains to be elucidated whether a single or specific consortium of bacteria is responsible for the caries process. The aim of this study is to evaluate the oral microflora of Indian children suffering from dental caries and to compare the same in children with no caries. Methods: The study was carried out on 67 out-patient 2-14 years old children who reported to the department of Paediatric Dentistry. Dental plaque samples from superficial and deep carious lesions and caries free surfaces in caries active children (n = 35) were collected using a sterile excavator in storage vials and subjected to various conventional and molecular microbial techniques. Caries free children (n = 32), who did not have any carious lesion served as controls. The data obtained was subjected to Pearson's Chi Square/Fischer's Exact tests to determine the statistical difference between the microflora of groups. Results: Main organisms isolated were: Streptococcal species mainly S anginosus, S salivarius, S gordonii: Lactobacilli spp; Coagulase negative Staphylococci, Staph epidermidis, Staph aureus. Other organisms such as Klebsiella spp, Acinetobacter, Enterococcus, E coli could also be found. There was a statistical difference in the frequency of isolation of non-mutans Streptococcal species (P = 0.008) and Lactobacilli species (P = 0.0001) in the two groups. Conclusions: The study suggests that caries activity in this population of children is associated with a diverse microbial flora without detectable S mutans. Main organisms associated with dental caries in this population are: Gram-positive cocci and bacilli mainly the non-mutans Streptococcus and Lactobacilli. Frequent consumption of sugar containing food promotes the presence and growth of cariogenic organisms.
Keywords: Caries active, caries free, dental caries, dental plaque, micro-flora, microbial analysis
|How to cite this article:|
Bansal K, Chaudhary R, Mathur VP, Tewari N. Comparison of oral micro-flora in caries active and caries free Indian children using culture techniques and PCR analysis. Indian J Dent Res 2020;31:420-5
|How to cite this URL:|
Bansal K, Chaudhary R, Mathur VP, Tewari N. Comparison of oral micro-flora in caries active and caries free Indian children using culture techniques and PCR analysis. Indian J Dent Res [serial online] 2020 [cited 2020 Sep 18];31:420-5. Available from: http://www.ijdr.in/text.asp?2020/31/3/420/291485
| Introduction|| |
Dental caries is dieto-bacterial, the most common disease of oral cavity during the childhood and a serious public health problem globally. It results from the interactions among susceptible host, cariogenic flora and cariogenic diets. Most of the researchers earlier have mainly focused around one or two micro-organisms in pure cultures through the perspective of Koch's postulates. Still, the cause of severe dental caries remains unpredictable. In majority of the investigations, acidogenic and acid-tolerant bacterial species,
Streptococcus mutans has been implicated as the major etiologic agent in the development of dental caries., However, it has been observed that S. mutans is detected in caries free populations as well and may not present in all children who have caries.
It has been documented that the initial acquisition of Mutans Streptococci occurs at the median age of 26 months during a discrete period which they referred to as “window of Infectivity'. However, some children present with severe caries before this window period raising a question about this hypothesis about window of infectivity. Thus, S mutans may not be the only cariogenic organism; other bacterial species may also have role in pathogenesis of caries.
Quantitative and qualitative analyses of plaque bio-films is extremely complicated as it has been reported that as many as 700 or more bacterial species are seen in oral cavity out of which 35% are not cultivable., The inhabitants of mouth are extremely diverse and mutualistic interactions are perpetual. Each individual harbours a unique microbiome that plays an important role in health and aetiology of disease within the body. Hence, the disease manifests and progresses differently among different individuals. Moreover, the inter-individual diversity in the oral bacterial flora is affected by factors such as age, diet and medical health. The environmental factors (e.g. nutrient availability and red-ox potentials) and host factors such as the availability of specific binding receptors also govern preferential bacterial colonization.
Since, no data is available on the microbiota of the caries active and caries free children in Indian population, the present study was planned with the objectives to determine and compare the cultivable micro-flora in caries active and caries free children. We used conventional cultivation techniques combined with Polymerase Chain Reaction (PCR) analysis of the cultivated colonies and automated microbial identification techniques using Matrix Assisted Laser desorption and Ionization time of flight mass spectrometry (MALDI-TOF).
| Methods|| |
Institutional ethical approval was obtained prior to the commencement of the study. Subjects from the age of 2-14 years were recruited from the department of Pedodontics and Preventive Dentistry in the Centre for Dental Education and Research between August 2016 to May 2018 whose parents consented to participate in study. Criteria to include the child in the study was that he/she should be free from any systemic illness and should not have taken systemic antibiotics in last three months.
The relevant complete information of the enrolled children such as; caries status and 24-hour diet recall to know the frequency of sugar intake through diet was recorded. The children were classified on the basis of caries experience into caries active (CA) and caries free (CF) children. Dental caries was recorded when there was a definite cavity, soft floor on probing, decayed leathery dentin and/or undermined enamel.
Collection of dental plaque samples
The dental plaque sample from each caries active participant was collected from i) surface of cavitated lesions, ii) the caries free surfaces of teeth which exhibited moderate to heavy plaque accumulation using a sterile spoon excavator, ii) thirdly, carious dentin from deep carious lesions. Each subject's sample was collected from these sites and pooled together and placed in transport medium (Robertson Cooked medium in a vial). In the caries-free children, the plaque samples were collected from the labial/buccal and lingual/palatal surfaces of all teeth and pooled. The sampling was done approximately 2 hours after the consumption of last meals. The collected samples were transferred immediately to the Microbiology Laboratory in bacterial transport medium as well as in Eppendorf tubes for molecular procedures.
Microbial processing of plaque samples
In the laboratory, the dental plaque sample from each participant was subjected to the aerobic and anaerobic microbial culture and identification by conventional and automated culture techniques. For anaerobic bacterial culture and identification, the samples were inoculated into Robertson cooked medium (RCM) for 48 hours and kept at 37°C in anaerobic system. After this, plaque samples were sub-cultured from RCM onto Brain heart infusion blood agar (BHIBA), Bacteroides Bile Esculin (BBE) Agar for anaerobic microbial identification. For the identification of Lactobacillus further inoculation onto, de Mann Rogosa and Sharpe (MRS) agar was used.
For aerobic microbial identification; Blood agar, MacConkey and Mitis Salivarius bacitracin agar were used. Each aerobic culture and subcultures were performed after 24 hours of aerobic incubation at 37°C. The identifications of bacterial colonies were carried out by conventional biochemical method. The cultivable colonies were also identified using automated microbial identification techniques using Matrix Assisted Laser desorption and Ionization time of flight mass spectrometry (MALDI-TOF) using the bioMérieux VITEK MS system (IVD database version 2.0) (USA).
Molecular identification of lactobacillus species
Isolated colonies of lactobacillus were sub-cultured onto MRS agar. The log phase colonies were used for DNA extraction. The colonies were washed with sterile Phosphate Buffered Saline. Then the washed pellet was treated with 180 μl of Lysozyme solution, mixed thoroughly and incubated at 37°C for 30 min. DNA extraction was done using Qiagen Mini Kit following manufacturer's instructions.
Identification of lactobacilli isolates by PCR
Genus specific PCR assays were carried out using forward primer LbLMA-1 and reverse primer R-161 The PCR reaction was carried out in a total volume of 50 μl containing 20 pmol/μl forward and reverse primers each, 0.5 μl Taq DNA polymerase, 0.5 μl of DNA template and 2.5 μl PCR buffer containing Tris-HCl, KCl, MgCl2 and 0.5 μl of dNTP mix. The volume was made up by adding water. The PCR reaction was carried out in a thermal cycler (Applied Biosystems) by programming the cycling profile consisting of an initial denaturation step of 5 min at 95°C followed by an amplification for 35 cycles with denaturation for 30 seconds at 95°C, annealing for 30 seconds at 55°C, and final extension of 10 min at 72°C. The PCR amplified DNA fragments were resolved by 1% agarose gel Electrophoresis containing 0.5 mg/ml ethidium bromide followed by visualization under UV light in trans-illuminator.
Primary aim of the study was to identify the microbial species in caries active and caries free children. In addition, the percentage of children in both groups having specific microbial species were estimated. The degree of association between the type of organisms and the dietary sugar frequency has also been studied.
The data was compiled in excel sheets and analysed using Stata 12.1 (College Station, Texas, USA). The microbial species (presence) identified in each group are expressed as the number (percentage) of positive samples in both groups. The difference in the proportion of specific organism between two groups was compared using Pearson's Chi Square/Fischer's Exact test. The correlation between the diet frequency and the type of organism was assessed using Fischer's Exact test. The P value less than 0.05 was considered statistically significant.
| Results|| |
In all, 80 children were recruited for the collection of the plaque sample. However, 13 samples were rejected due to no growth or contamination. In the final analysis, the micro-flora of 67 samples have been enumerated using the culture techniques, PCR analysis of Lactobacilli species and MALDI-TOF technique.
The participants' characteristics such as age, caries status and daily sugar intake frequency is given in the [Table 1]. Caries active children were further classified into high and low caries active children on the basis of caries score, those having score equal to or below 3 were regarded as low caries active while with more than 3 carious lesions in the oral cavity were classified as high caries active children. Caries-free children were not found to have sugar exposure level to more than 2 times per day. Most of caries-free children reported with chief complaints of either misaligned teeth or crown fracture due to traumatic dental injury. Various organisms isolated and identified from various techniques were compiled as the number and percentage of plaque samples showing various specific microbial species [Table 2].
|Table 2: Percentage of plaque samples with microbial species in Caries Active (n=35) and Caries Free (n=32) Children|
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We also found association between the degree of sugar exposures and the isolation frequency of caries associated microbial species - Streptococci and Lactobacilli species (P < 0.05) [Table 3]. The caries free children had relatively more gram-negative bacilli in the dental plaques and can be attributed to lower availability of fermentable carbohydrates in their diet.
|Table 3: Association of daily frequency of sugar exposures and number (%) of Plaque samples with specific microbial species|
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| Discussion|| |
This is the first time that the micro-flora of the dental plaque of Indian children has been assessed using both conventional cultivation techniques and molecular techniques; 16s rRNA gene sequencing technique was utilized for identification of Lactobacillus species. Bacterial identification directly from colonies using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has developed as a revolutionary tool perfectly adapted to the clinical microbiology laboratory. This method is fast, relatively cheap, does not create much waste, and most importantly, the results are reliable and can be supported with a high level of confidence.
A lot of heterogeneity in the micro-flora was observed in plaque samples in both, CA and CF children. The reasons for this diversity are that sample from each subject were collected from various tooth sites in the oral cavity such as superficial cavitated lesions, deep dentin lesions and caries free surfaces in CA children and pooled for analysis and in CF group from various sound teeth surfaces. The sample from each site was not adequate enough to be cultivated on different culture media as the subjects did not refrain from their normal brushing regime. It has been known that different oral habitats that exist in the human mouth are colonized by distinct microbial communities., Thus, site specific variations in micro-flora could have resulted in the diversity in the microbial population observed in the present study. In RNA-based studies as well, the composition of active bacteria has been revealed to be distinct in initial, enamel lesions from that in more advanced dentin cavities.
Gram positive cocci and bacilli isolated more frequently than Gram negative bacilli. Gram negative cocci (Veillonella parvula) were seen in only one sample in the caries free group. Most predominant bacterial species isolated from both CA and CF children were Streptococcus, Lactobacilli, Staphylococcal species. Relatively less number of samples from caries active children showed growth of Gram-negative bacilli like Klebsiella, Enterococcus, E faecalis, E coli, Enterobacter, Acinetobacter as compared to the samples from the caries free children. However, most commonly implicated organism in caries aetiology, S mutans could not be detected in all caries active children in our study.
About a century ago, in his pioneering work, Clarke in 1924 observed that caries sometimes develops in the absence of detectable S. mutans. This suggested that other species, or combinations of species, could produce similar amounts of acid to S. mutans and hence cause disease. Corby et al.
reported that some subjects with severe caries did not have detectable levels of S. mutans, whereas few caries-free children had detectable levels of S. mutans. It has been documented in several studies that the level of Mutans Streptococci is not necessarily high in biofilms associated with caries, especially the microflora associated with non-cavitated stages of lesion formation., Instead, non-mutans acidogenic and aciduric bacteria, including non-mutans streptococci and Actinomyceshave been proposed to be closely involved with the initiation of caries. Other investigators have also observed that non-mutans aciduric bacteria other than non-mutans streptococci and Actinomyces from dental biofilms covering white-spot lesions. Further, it has been documented using second generation sequencing and metagenomic techniques that S. mutans accounts only for 0.1% of the bacterial community in dental plaque and 0.7–1.6% in carious lesions., From all the established deep dentin cavities, Lactobacilli could be cultivated which confirms its role in the advanced caries process. The findings of the study corroborate with that of Munson et al. who noted that Gram-positive species dominated the sample, and lactobacilli were the most prevalent of all species present.
Staphylococcal species could be found in both caries active as well as in caries free children however only in a smaller number of subjects. It has been reported that Staphylococcal species can be isolated from the mouths of children and adults, both in health and disease. Another organism, Klebsiella species were also isolated in our population, almost in equal frequency from both groups and are known transient colonizers of the normal flora of the oral cavity. Ayars et al.
suggested that reduced salivary flow and the concomitant reduction of intraoral pH may predispose patients to bacterial colonization with K. pneumoniae.
Gram-negative rods were present in more than 1/4th samples, more significantly in caries free subjects. Conditions such as inadequate hygiene habits, salivation reduction and natural chewing movements favour the colonization and proliferation of Enterobacteriaceae in the oral cavity. Investigations have demonstrated that such species are normally residents of intestinal tract, but can be isolated from the oral cavity of healthy subjects.
In addition to these species, there are several other micro-organisms which were reported to be associated with dental caries like Bifidobacterium dentium and Scardovia wiggsiae and were not detected in the current study. This could be attributed to the difference in severity of disease at the time of sampling, difference in study populations, methodologies particularly the culture-based and DNA-based methods.
Prior to the advent of molecular techniques for bacterial identification, the role of S mutans in the causation of dental caries has been shown by a large number of investigations, A central role for the Mutans Streptococci in the initiation of dental caries on enamel and root surfaces has been reported by a systematic literature review. However, molecular studies have confirmed the diversity of micro-flora associated with health and disease. The possible assumption is that S mutans may be the prominent etiologic organism for dental caries owing to evidences that these are i) frequently isolated from cavitated caries lesions, ii) induce caries formation in animals fed a sucrose-rich diet, iii) are highly acidogenic and aciduric,, iv) able to produce surface antigens I/II and water-insoluble glucan, which promote bacterial adhesion to the tooth surface and to other bacteria. However, other organisms and combinations of organisms too have similar acidogenic potential to cause cavitation. Since S mutans could not be cultivated in this population, there is a likelihood that combination of other bacteria in the biofilm (Lactobacilli and Non-mutans Streptococci) are synergistically and mutualistically causing acidogenic conditions to cause dental caries in the present population.
In caries-free children, low sugar exposures in the oral biofilm does not preferentially select the growth of acidogenic species thereby creating the environment for the proliferation of gram-negative bacilli. The homeostasis in the oral cavity gets disturbed owing to excessive acid production by the bacterial metabolism after the consumption of easily fermentable carbohydrates. This leads to lowered pH conditions and enamel and dentin dissolution. It has been suggested that upon frequent carbohydrate consumption, oral bio-film undergoes complicated shift in the multiple groups of bacteria as low pH of bio-film selects more acidogenic and acid tolerant species and the process of demineralization continues thereby leading to deepening of the cavitation. Base formation from other species of the biofilm may partly dampen the demineralization processes. Therefore, it has been suggested that it is the proportions and numbers of acid-base-producing bacteria that are the core of dental caries activity.
The limitations of the study were that there was a significant difference between the mean age of caries active and caries free children. This was because caries-free children reported to hospital with the complaint of mal-aligned teeth and traumatic dental injuries which is usually after the age of 8-10 years, whereas children as young as 2 years had reported with dental caries as chief complaint. Another limitation is that PCR analysis was not performed directly from the plaque samples as the quantity of DNA is very low. This is because most of the subjects displayed fair oral hygiene at the time of reporting to the dentist, and the plaque sample collected on that day were not sufficient to isolate the required quantity of DNA to put the direct PCR for individual microorganisms including S mutans. It is quite possible that S mutans might be present in these samples but in very less quantities to be able to be cultivated.
| Conclusions|| |
This study suggests that a combination of species could produce similar amount of cariogenic activity as S mutans.
Main organisms associated with dental caries in this population are mainly the non-mutans Streptococcus and Lactobacilli. More frequent consumption of sugary snacks was associated with more caries associated organisms and vice-versa. Thus, we may assume that physiologic characteristics of the micro-flora rather than its composition seems to be the main determinant of the caries initiation. However, more studies are required using various molecular identification techniques and metabolome analysis to identify and differentiate the cultivable and not-yet cultivated micro-flora of Indian children; both healthy and having dental caries.
The authors acknowledge the help of Dr Deepak V Bamola, Scientist at Dept of Microbiology at AIIMS, New Delhi in the microbial analysis of the dental plaque samples.
Financial support and sponsorship
This Research work was conducted with the help of grant obtained from Intra-mural Research Grant Scheme from the All India Institute of Medical Sciences, New Delhi.
Conflicts of interest
There are no conflicts of interest.
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Dr. Kalpana Bansal
Department of Pedodontics and Preventive Dentistry, Centre for Dental Education and Research, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]