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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 2  |  Page : 79-86

Surveillance of microorganisms associated with dental caries patients attending at a Tertiary Care Indian Teaching Hospital


1 Medical Research Laboratory, IMS and SUM Hospital, Siksha O Anusandhan Deemed to be University, Bhubaneswar, Odisha, India
2 Department of Oral Pathology, Institute of Dental Science, Siksha O Anusandhan Deemed to be University, Bhubaneswar, Odisha, India
3 Medical Research Laboratory, IMS and SUM Hospital, Siksha O Anusandhan Deemed to be University; Division of Microbiology, ICMR - National Institute of Occupational Health, Ahmedabad, Gujarat, India

Date of Submission15-Dec-2018
Date of Acceptance16-May-2019
Date of Web Publication19-Jun-2019

Correspondence Address:
Mahesh Chandra Sahu
Division of Microbiology, ICMR - National Institute of Occupational Health, Meghani Nagar, Ahmedabad - 380 016
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/am.am_95_18

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  Abstract 


Backgrounds: Dental caries patients are developed day by day in all age groups and predominantly in children. It is caused due to microorganisms mainly. Here, we find the organisms associated with dental caries of patients attending at Institute of Dental Science, Bhubaneswar, India. Materials and Methods: This prospective study was carried out from February to May 2018 at Institute of Dental Science, Bhubaneswar, India. A total number of 135 samples were collected from dental caries patients and processed in different agar media for identification of organisms, and the percentage of antibiotic susceptibility was evaluated. Results: Out of 135 patient samples, 112 organisms were isolated and identified with both culture and biochemical tests. Among all, 9 Enterococcus sp. (6.66%), 27 coagulase-negative Staphylococcus aureus sp. (20%), 6 Bacillus sp. (4.44%), and 30 Streptococcus sp. (22.22%) were found as Gram-positive cocci; 3 Acinetobacter sp. (2.22%), 6 Pseudomonas sp. (4.44%), 21 Shigella sp. (15.55%), and 6 Citrobacter freundii sp. (4.44%) were identified as Gram-negative bacteria; and 6 Candida glabrata (4.44%), 3 Candida albicans (2.22%), and 3 Candida tropicalis (2.22%) were identified as budding yeast colonies. In antibiotic resistance patterns, it was revealed that penicillin is the most resistance (92%) and gentamycin is the most sensitive antibiotic (79.2%) for bacteria associated with dental caries. Conclusion: Dental caries caused due to microbial infection is cured with antibiotics. Out of all antibiotics, gentamicin is more sensitive and preferable for dental caries with this study. Hence, a clinician can prescribe gentamicin beside amoxicillin and cefixime for early treatments.

Keywords: Antibiotics, dental caries, drug resistance, gentamicin, Streptococcus mutans


How to cite this article:
Lenka S, Debta P, Sahu MC. Surveillance of microorganisms associated with dental caries patients attending at a Tertiary Care Indian Teaching Hospital. Apollo Med 2019;16:79-86

How to cite this URL:
Lenka S, Debta P, Sahu MC. Surveillance of microorganisms associated with dental caries patients attending at a Tertiary Care Indian Teaching Hospital. Apollo Med [serial online] 2019 [cited 2019 Jul 20];16:79-86. Available from: http://www.apollomedicine.org/text.asp?2019/16/2/79/260693




  Introduction Top


Among all chronic infectious diseases, dental caries is one of the most common affecting to human population and it is predominative to pediatric age groups.[1],[2] It can occur in very young children, shortly after the eruption of teeth, and may be severe. For many children, early childhood caries is a source of pain and impaired quality of life, and for some, it results in serious infection, hospitalization, and even fatality.[3] In this young age, cohort treatment must often be completed under general anesthesia, accounting for a disproportionate fraction of total dental expenditures.[4] The microbial etiology of the onset of caries, since preventive interventions such as probiotics or vaccines, will be most effective if they interrupt the process before irreversible damage is done to teeth.

The causes of sex differences in dental caries experience are not fully understood, although possible explanations include earlier tooth eruption (and thus longer exposure to cariogenic processes) in females, as well as sex differences in dietary and oral hygiene behaviors, utilization of oral health care, hormones/physiology, and characteristics of saliva.[5],[6] The differential actions of genes in men and women also have been proposed.[7] Genetic variants related to tooth eruption, dietary preferences, physiology, saliva, or other unknown caries risk factors may have different effects in men versus women; such genetic effects are called gene-by-sex interactions.

Dental caries occurs as the result of a shift in the composition of a biofilm community specific to the human tooth surface. Frequent carbohydrate intake can disrupt the ecology of this community by the selection of acidogenic and acid-tolerant species, and these acidogenic communities are responsible for caries development.[8] Streptococcus mutans appears to be the most common acid producer in caries initiation.[9] Intake of dietary sugars is the most important risk factor for dental caries.[10] Low levels of dental caries, especially when observed in the permanent dentition, are of concern because dental caries is a lifelong progressive and cumulative disease that tracks to adulthood, even with exposure to fluoride through water or mouth care products.[11],[12]

Nowadays, multiple drug resistance has been developed in human pathogenic microorganisms due to the haphazard utilization of commercial antibacterial drugs frequently used in the treatment of infectious diseases.[13] Resistance of numerous bacterial pathogens to many antibiotics continues to increase worldwide. Frequencies, pattern, and distributions of resistant bacteria diverge extensively with geographic regions and often reflect the usage patterns of antibiotics.[14] Factors that increase in resource-poor and resource-rich nation include total antibiotic consumption as well as underuse through lack of access, inadequate dosing, poor adherence, and substandard antimicrobial treatment.[13] Moreover, inappropriate antibiotic prescribing and its use have been identified as major factors in the appearance of antibiotic resistance. Nowadays, a shift from narrow-spectrum antibiotic prescriptions which included penicillin to broad-spectrum aminopenicillins which include amoxicillin by dental professionals has been reported and the increase of bacterial isolates resistant to the former antibiotics is blamed for such a shift in prescription practices.[15] Fluoroquinolones are quinolone antimicrobials which are active against many β-lactam-resistant bacteria. Amoxicillin/clavulanic acid, a combination of a β-lactam antibiotic (amoxicillin trihydrate) and a β-lactamase inhibitor (potassium clavulanate), has broad antimicrobial spectrum and effective against amoxicillin-resistant bacteria that produce β-lactamase.[16] Such antimicrobial agent may prove valuable for managing dental infections.

People infected with drug-resistant organisms are more likely to have longer and more expensive hospital stays and may have a higher mortality rate. The antibacterial susceptibility patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients. Therefore, the present study was carried out to determine drug resistance trends among isolates of dental caries which will help health-care authorities for further planning, implementation, and evaluation in public dental health service by building the effective management program for it. We evaluate the organisms associated with dental caries and performed the antibiotic susceptibility patterns and find the suitable antibiotics for early treatment of dental caries.


  Materials and Methods Top


Collection of sample

This prospective study was carried out from February 2018 to May 2018. All the dental samples were collected from the patients of the Institute of Dental Science Hospital, Bhubaneswar, Odisha. Dental sample collection from the patients was done very cautiously using aseptic technique. The dental sample was collected from the patients using sterilized swab sticks.

Sample processing

Collected samples were kept in refrigerator and were processed within 6 h of collection. The dental swabs were first inoculated on blood agar plates and MacConkey (MAC) agar plate. The inoculated plates were incubated at 37°C for 24 h and for 48 h in case of negative results. Colony characterization on media for samples having positive growths was studied and noted. The microbial isolates were again cultured on nutrient agar plates to obtain a pure culture for further biochemical tests and were subcultured periodically.

Biochemical testing for identification of microbial isolates

A single colony from pure cultures was used for further biochemical testing. First, a Gram's stain was done to identify Gram-positive and negative bacteria. Gram-positive bacterial isolates were subjected to catalase and coagulase testing. Gram-negative isolates were subjected to lactose fermenting and were processed with oxidase and methyl red (MR) tests, whereas lactose-fermented bacteria were subjected to indole, MR, Voges–Proskauer (VP), and citrate test, triple sugar iron (TSI) test, and urease test. The results were noted and analyzed for identification. Budding yeast colony (BYC) isolates were grown on Candida differential agar (CDA), and the resulting coloration of isolates was noted for identification.

For Gram-positive bacteria

After catalase test, if H2O2 solution gives air bubbles, then the result is positive and coagulase test is done. If coagulase test is positive, then the organism identified is Staphylococcus aureus, and if coagulase test gives negative, then the organism identified is coagulase-negative Staphylococcus aureus (CONS).

  • When catalase gave a negative result, i.e., no gas bubble found, then bile esculin agar (BEA) test is done
  • By BEA test, Enterococcus is identified from positive result and Streptococcus is identified by negative result.


For Gram-negative bacteria

For Gram-negative bacteria (GNB) and non-lactose fermenting on MAC plate, oxidase test was done. On positive result, the organism identified as Pseudomonas aeruginosa and on negative result of oxidase test, MR test, and VP test was done, i.e., MR, TSI, and urease tests. It was done serially, started from MR. On positive MR testing, TSI was done. On negative result, organism identified that Acinetobacter. ON lactose fermenting (LF) bacteria Indole, methyle red (MR), Voges–Proskauer (VP), Citrate, Triple Sugar Iron Agar (TSI) and urase tests were carried out.

  • In TSI test, on H2S production on tube, then urease test was done
  • On positive result of urease test organism identified Proteus sp. and on negative Urease  Salmonella More Details sp. was identified
  • On no H2S production in TSI, then the organism identified was Shigella sp. [Table 1].
Table 1: Identification of bacteria

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For fungus

BYC isolates were cultured on Sabouraud dextrose agar (SDA) plates, incubated at 37°C for 24 h, and then outside of incubator for better identification. Again, it was cultured on CDA plates by taking a single colony from SDA for final identification. Light green color on CDA, Candida albicans is identified, on cream white color colonies Candida glabrata, on purple color, Candida krusei and on blue-purple color Candida tropicalis were identified [Table 2].
Table 2: Identification of Candida species

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Antibiotic susceptibility testing

Isolates were tested for antimicrobial susceptibility testing using standard Kirby–Bauer's disc diffusion method. Standard inoculums were made by taking 1–2 colonies and were dipped into liquid nutrient broth and incubated on shaker for 3 h. Then, the liquid bacterial culture was spread platted on Muller-Hinton agar (MHA) plates using sterile spreader. Antimicrobial impregnated disks were placed onto the medium using an automated disk dispenser. MHA plates were then incubated at 37°C for 24 h. After 24 h, the clear inhibition zones were measured and interpreted by clinical and laboratory standards to check the resistance pattern of each microbial isolates [Figure 1]a and [Figure 1]b.
Figure 1: (a) Antibiotic susceptibility test of Gram-positive cocci on Muller-Hinton agar. (b) Antibiotic susceptibility test of Gram-negative bacteria on Muller-Hinton agar. (c) Antifungal screening of Candida species on Sabouraud dextrose agar

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Fungal isolates were tested for antifungal susceptibility testing using Standard Kirby–Bauer's disc diffusion method. Standard inoculums were made by taking 1–2 colonies and were dipped into liquid Sabouraud dextrose broth and put into shaker for 3 h. After 3 h the liquid fungal culture was spread platted on SDA plates or potato dextrose agar (PDA) plates using sterile spreader. Antifungal impregnated disks were placed onto the medium using an automated disk dispenser. SDA/PDA plates were then incubated at room temperature for 48 h. After 48 h, the clear inhibition zones were measured and interpreted by clinical and laboratory standards to check the resistance pattern of each microbial isolate [Figure 1]c.

Statistical analysis

All the data generated from the study were analyzed with SPSS Version 20, (IBM Corp., Armonk, NY, USA) software for statistical significance of the study. P ≤ 0.05 was considered for significance.


  Results Top


A total number of 112 colonies were identified with 135 dental caries samples. Among them, 20 samples (14.81%) did not reveal any growth and 3 samples (2.22%) revealed three or more colonies which were taken as contamination and excluded from the study. The single colony was found maximum as compared to others (60.74%) [Table 3]. Among 112 colonies, S. mutans was found 22.22%. Apart from S. mutans, other bacteria were also associated with dental caries. They were Enterococcus sp. (6.66%), CONS sp. (20%), and Bacillus sp. (4.44%) were found as Gram-positive bacteria [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d and [Figure 3] Acinetobacter sp. (2.22%), Pseudomonas (4.44%), Shigella sp. (15.55%), Citrobacter freundii (4.44%) were identified as GNB [Figure 2]e,[Figure 2]f,[Figure 2]g,[Figure 2]h. There were 3 Candida sp. were identified [Figure 3]a,[Figure 3]b,[Figure 3]c; C. glabrata (4.44%), C. albicans (2.22%) and C. tropicalis (2.22%) [Table 4].
Table 3: Frequency of cultured colony

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Figure 2: (a) Acinetobacter on nutrient agar, (b) Pseudomonas on nutrient agar, (c) Shigella species on nutrient agar, (d) Citrobacter freundii on nutrient agar, (e) Enterococcus sp. on blood agar, (f) Coagulase negative Staphylococcus aurius on nutriant agar, (g) Gram positive Bacillus on nutrient agar and (h) Streptococcu mutans on blood agar

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Figure 3: (a) Candida glabrata on Candida differential agar. (b) Candida albicans on Candida differential agar. (c) Candida tropicalis on Candida differential agar

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Table 4: Frequency of organisms

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Antibiotic susceptibility test for Gram-positive bacteria revealed that Bacillus sp. remains resistant to many groups of antibiotic. S. mutans having the highest percentage of occurrence in this study showed resistant to β-lactams, cephalosporins, aminocoumarins, and macrolides, a group of antibiotics. Other organisms showed less resistant than these two species [Table 5]. At an average, penicillin was the most resistant antibiotic and chloramphenicol and gentamicin were the most sensitive antibiotics for Gram-positive bacteria in dental caries.
Table 5: Resistance percentage of GPC

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In case of GNB, C. freundii showed resistant to many groups as compared to Shigella, Pseudomonas, and Acinetobacter species [Table 6]. Clarithromycin is the most resistant and colistin is the most sensitive antibiotic for GNB.
Table 6: Resistance percentage of GNB

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Antifungal susceptibility test for all dental carries isolated Candida sp. were carried out and it was revealed that fluconazole had the highest resistance than others and itraconazole is most sensitive for fungal infection in dental caries [Table 7].
Table 7: Resistace percentage of Candida species

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  Discussion Top


Dental caries is the most studied oral disease in the world, the majority of studies concentrated in school children, with not enough research on the situation of the disease in young adults and old ages. The prevalence of dental caries was observed in many studies. Adhikari et al.[17] reported the prevalence of dental caries in the study population to be 47.1%. Similarly, the prevalence of dental caries was 60.3% in the study conducted by Bhagat and Shrestha.[18] The present study showed that the incidence of dental caries was found to be 52.8%, which shows as the time goes on, the increasing pattern of caries patients among the population also raises as per the research work. In this study, the occurrence of dental caries in females (60.83%) was found to be higher than in males (39.2%). It may be due to females' dental eruption 6 months earlier than males, so they are exposed to cariogenic factors or may be due the increasing sugar consumption, low exposure to fluorides containing toothpaste, and poor access to oral health care. Dikshit and Limbu[19] showed the prevalence of dental caries in males with 55% and in females with 44% which is different from this study which may be due to the high number of male patients. The present study showed that the frequency of dental caries was found to be higher in the age group of 16–20 years with 32.75%, which is the pinpoint of adolescence period, followed by 0–5-year-old children with 55.5%. As the age advances, there was a rise in proportion of population affected by caries. The majority of adolescence (10–19 years) and children (1–9 years) had dental caries due to consumption of sugary foods such as chocolate, candy, jellies, and soft drinks approximately 3–5 times daily, which may be considered one of the most important factors in high caries experience in these groups. Similar findings were obtained in other studies as well. According to Subedi et al.,[20] the prevalence of dental caries in 12–13 age groups was 53.23%. A study conducted by Khanal et al.[21] reported that 85.25% dental caries was found in children of 1–6 years old in Jorpati, Kathmandu. Caries was significantly higher in children with mothers of low education and low family income. Similarly, Adhikari et al.[17] reported that caries prevalence in the age group of 11–14 years was 52.46% in a tertiary care center in the western region of Nepal. Dikshit and Limbu[19] recognized that caries prevalence in 12–13-year-olds was 41% in Chepang school children of Chitwan district which is not concurring with this study which may be due to the least number of patients of 11–15 age groups. The present study reported the pattern of tooth-wise distribution of caries among the study population, which showed that the most severely affected teeth were maxillary molars (53.6%), followed by mandibular molars (27.9%) and maxillary incisors (18.5%). The mandibular incisors were the least affected teeth (7.3%). This may be due to the anatomical structures of molar teeth which have lots of grooves, pits, and crannies that collect food particles. As a result, they are harder to keep clean than front teeth where plaque can build and bacteria can thrive between back teeth as molars, producing the acids that destroy tooth enamel. The similar findings were also obtained in the study conducted by Dikshit and Limbu[19] who reported maxillary molars (32.11%), mandibular molars (36.92%), and maxillary incisors (22.55%) being predominantly affected teeth by caries while mandibular incisors (1.12%) were least affected. Both the studies showed that maxillary and mandibular molars and maxillary incisors were more affected whereas the same studies showed that mandibular incisors were least affected. This may be due to mandibular incisors are protected by the tongue and opening of major salivary ducts near the incisors which helps it to more resistance toward caries. Saliva maintains the supersaturation of calcium in plaque and also neutralizes acids, raises pH, and reverses the diffusion rate of calcium and phosphate toward the tooth surface. The present study showed that a greater number of isolates were Gram-positive bacteria (53.32%) whereas the contribution of GNB in dental caries was 26.65% and fungus isolate was (8.88%). The incidence rate of Gram-positive bacteria was found higher in caries than GNB because the most common bacteria that are found in the supragingival plaque are Gram-positive cocci (GPC) (53.32%). These prominent bacteria are responsible for the plaque formation due to their interactions with each other and the tooth surface. Similar results were obtained in a study conducted by Olajokun et al.[22] in which 100% of bacterial isolates were Gram positive associated with caries. Among all the bacterial isolates, S. mutans was a predominant bacterium (43.77%) associated with caries. S. mutans collects around the teeth and gums in a sticky, creamy-colored mass called plaque, which serves as a biofilm. Bacteria in a person's mouth convert sugars (glucose and fructose and most commonly sucrose) into acids such as lactic acid through a glycolytic process called fermentation. If left in contact with the tooth, these acids may cause demineralization, which is the dissolution of its mineral content. The similar finding was also obtained in a study conducted by Olajokun et al.[22] who reported that 45.6% S. mutans was related to caries which is in accord with this study. The present study showed that 6.66% Enterococcus sp., 20% CONS sp., and 4.44% Bacillus sp. were found as GPC and 2.22% Acinetobacter sp., 4.44% Pseudomonas sp., 15.55% Shigella sp., and 4.44% C. freundii sp. (4.44%) were identified as GNB in fungus 4.44% C. glabrata and 2.22% C. albicans were revealed. The rest were followed by 2.22% C. tropicalis. Omolaja et al.[23] found that 53.13% S. mutans and only 6.25% P. aeruginosa and P. vulgaris were isolated from caries. Similarly, Olajokun et al.[22] reported that 45.6% S. mutans, 41.2% Lactobacillus spp., and 13.2% S. aureus were isolated from carious lesion. In a study of Fayaz et al.,[24] S. aureus in the etiology of oral dysesthesia and mucositis is complicated by the diversity of the normal oral flora and by healthy carriage of S. aureus in some patient groups. However, the high rates of S. aureus are suspected in patients presenting with symptoms of oral mucosa pain, burning, erythema, and swelling. Isolates of S. aureus are capable of producing a wide range of exfoliative toxin and enterotoxin.[25] The present study showed that almost all bacterial isolates were fermenting sucrose fermenter except Pseudomona s spp. and Klebsiella pneumoniae. Because these bacteria do not possess extracellular enzyme capable of cleaving the α-1 and α-2 glycosidic bond of sucrose. Sucrose is a fermentable carbohydrate which has been shown to be caries initiation and development. K. pneumoniae is classically thought of as community acquired and occurring in elderly and debilitated population with underlying alcoholism, chronic lung disease, or diabetes. Pseudomonas spp. is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and its pathogenicity especially relies on the production of cellular and extracellular virulence factors associated with root caries. In this study, all Gram-positive bacterial isolates were found more sensitive toward gentamicin (79.2%), followed by chloramphenicol 79.17% and norfloxacin with 70.8%, whereas all Gram-negative isolates were found to be more sensitive toward ciprofloxacin, followed by imipenem and gentamicin of 89.28% and ceftriaxone with 50%, respectively. The present study also revealed that antibiotic susceptibility testing of the isolates showed a high degree of resistance toward different antibiotics. The key factor influencing the emergence of resistance to an antibiotic is due to overuse of antibiotics in humans. In some developing countries, antibiotics are available without prescription, and this potentially facilitates overuse. The use of closely related drugs for other condition also plays a role in the spread of resistance 27.9%.[26] In the present study, a substantial resistance was observed to a number of commonly used antibiotics. This may be due to the indiscriminate and inappropriate use of antibiotics that is rampant in India. Hence, it is important to periodically monitor the antibiotic resistance pattern in different regions.


  Conclusion Top


India is one of the developing countries facing a high prevalence of dental caries in all age groups which is a life-threatening infectious disease. The present study concluded that most commonly S. mutans, S. aureus, and P. aeruginosa were directly associated with the establishment of dental infections. It was further concluded that the prevalence of dental caries was found to be in an increasing trend, compared to previous studies. Moreover, abuse of antibiotics has led to the emergence of multidrug-resistant bacteria which are difficult to control as these bacteria are resistant to most of the antibiotics. However, multiple drug resistance patterns of some strains of bacterial isolates to gentamicin should be of interest. Thus, the current study pointed out that there is a great need to monitored antimicrobial resistance of bacterial isolates at time interval regularly. In turn, this study will allow the development of novel diagnostic and treatment methods in reducing periodontitis, gingivitis, cellulitis, malocclusion, and other life-threatening clinical conditions as the adverse pregnancy outcomes, increased risk of myocardial infarction, cardiovascular, respiratory, erectile, diabetes complications, cavernous sinus thrombosis, and Ludwig angina globally.

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Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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