Indian Journal of Dental Research

ORIGINAL RESEARCH
Year
: 2012  |  Volume : 23  |  Issue : 1  |  Page : 26--30

A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine


Girija AS Smiline, Suba K Pandi, P Hariprasad, R Raguraman 
 Department of Microbiology, Meenakshi Ammal Dental College, Madhuravoyal, Chennai, India

Correspondence Address:
Girija AS Smiline
Department of Microbiology, Meenakshi Ammal Dental College, Madhuravoyal, Chennai
India

Abstract

Background: Dental caries being the commonest unmet public health problem indicates its need to urge the dentists to overcome this problem globally. Caries exhibit in different types and is found to be associated with co-aggregation property of microbial flora with other oral hygienic factors. In spite of the surgical removals, excavations and administration of antimicrobials for carious dentine, there seems to be repeated infection and chronic prevalence of caries. A complete understanding of microbial etiology and prevention of emerging drug-resistant strains will aid in the eradication of this chronic dentine problem condition from the oral cavity. Aim: This study is aimed to isolate the predominant bacterial pathogens associated with caries and to screen for the emergence of drug resistance among the isolated caries pathogens. Materials and Methods: Carious dentine specimens were collected from 75 endodontic patients and the samples were processed microbiologically to isolate the caries pathogens. Identification of the strains was done by standard biochemical characterization studies. Statistical analysis of the isolates was done by Pearson Chi-square test and Fisher«SQ»s exact test. The predominant isolates were subjected to antimicrobial sensitivity test using Kirby Bauer«SQ»s method. The results were recorded and analyzed for drug resistance. Results: Carious dentine samples yielded a high percentage of Lactobacillus sp., and Candida albicans from different type of caries. Among the study population, dentinal caries was the most predominant type affecting most males with other associated risk factors. Nearly 47.3% of the isolated Lactobacillus sp. and 55.5% of the yeast C. albicans were screened to show resistance against the antimicrobials used for the study. Conclusion: This study concludes by stating that Lactobacillus sp., and C. albicans are mostly involved in the caries etiology and show resistance to the commonest antimicrobial agent. This implicates the need for periodical antimicrobial susceptibility examination of the caries pathogens that will aid to prevent the emergence of resistance property among the dentinal pathogenic organisms.



How to cite this article:
Smiline GA, Pandi SK, Hariprasad P, Raguraman R. A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine.Indian J Dent Res 2012;23:26-30


How to cite this URL:
Smiline GA, Pandi SK, Hariprasad P, Raguraman R. A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine. Indian J Dent Res [serial online] 2012 [cited 2021 Apr 11 ];23:26-30
Available from: https://www.ijdr.in/text.asp?2012/23/1/26/99033


Full Text

Dental caries continues to be a significant public health problem in many parts of the world. Although the bacteria responsible for caries initiation and early caries progression have been studied extensively, the microbiology of dentine caries has been reported to show considerable diversity and has not yet been fully characterized. Dissolution by acid of the surface enamel particularly the lactic acid produced by Streptococcus mutans is considered to be the primary event in caries development. [1] This exposes the underlying avascular mineralized connective tissue matrix of dentine, which is prone to invasion. This occurs by migration of bacteria into the network of tubules occupied by the processes of the pulpal odontoblasts. [2] Over the past 115 years, the scientific study of dental caries has further refined the processes. Today we know that dental caries is a multifaceted disease process that involves over­lapping circles of the host, bacteria and nutrients, which subsequently results in demineralization activity. [3]

Reports suggest that the early stage of invasion involves Lactobacillus sp., Actinomyces sp., and S. mutans. [4] This phase is followed by the invasion of more diverse group of microorganisms including Gram-negative anaerobes. [5] There is evidence that interspecies cooperation enhances the migration of the mixed bacterial flora through the dentinal tubules. [6] Streptococcus mutans is more pathogenic than others and they also vary in their potential to produce dental caries. The precise role of Lactobacilli sp., in the initiation and progression of dental caries cannot be defined clearly but studies suggest that it plays a vital role in the initiation stages to the progression and deep lesion stages. [7] Actinomyces sp., is a commonly found species on dental plaques and many species of Actinomyces play significant roles in different form of caries. [8] Similarly, yeasts have also been reported to aid the caries progression in the affected cases. Candida albicans, a Gram-positive budding yeast has been reported from primary endodontic infections and it is very much possible to isolate them from plaque, caries, subgingival microflora and active periodontal cavity. [9]

For the past 150 years, surgical treatment has been followed in many dental offices to remove the demin­eralized tooth structure. The flaw in this method is that the causative organisms are not removed completely. [10] So the next logical progression is to treat the remaining infection with effective antimicrobial agents; however, the emergence of drug resistance among the pathogenic population has worsened the treatment regimens in dentistry. This results in compelling evidence to progress with clinical assessments of antibiotic susceptibility and other microbiology laboratory efforts to understand the limits of this everlasting problem. So this study is aimed to evaluate the commonest caries pathogens from different type of caries and to screen for the emergence of drug resistance among the caries causing pathogenic bacteria.

 Materials and Methods



A six-month study was conducted in the Department of Microbiology among the patients visiting the Department of Endodontics of Meenakshi Ammal Dental College. The source of material for the isolation of the caries pathogens was carious dentine removed from the carious tooth during endodontic restorative procedures. The specimens were received after obtaining the informed consent from the patients. A proforma was recorded for each study case to analyze the age, sex, occupation, marital status, risk factors such as drinking, smoking, tobacco chewing, tattooing and intravenous drug (IV) users, food habits viz., vegetarian/non-vegetarian, economical status and detailed clinical examination. After the removal of superficial plaque and debris overlying the lesion, the carious zone of decalcified and partially decalcified dentine was washed with sterile saline. The carious dentine was then excavated with sterile dental explorers and was transferred in to vials of sterile thioglycollate broth and brain heart infusion broth to a concentration of approximately 10 mg (wet weight) of dentine per ml prior to processing. [11]

The samples were brought to the microbiology laboratory and were processed within 3 h of collection. Samples were dispersed in the transport medium by using a Vortex mixer and the broth was incubated at 37°C/10% CO 2 for 2 h. After incubation the broth was inoculated onto sterile brain heart infusion blood agar (BHIBA), thioglycollate agar (TGA) and Sabouraud's dextrose agar (SDA). Incubation was performed at 37°C/10% CO 2 for 48 h. SDA was incubated at 37°C/24 h aerobically. After incubation the isolates were identified by colony morphology, gram's staining and was characterized biochemically. [12] The isolation rate was analyzed statistically using Pearson Chi-square test and Fisher's exact test. The isolates were stored at 4°C until subjected for the antimicrobial susceptibility test.

Preliminary screening of the emergence of drug resistance among the isolated caries pathogens was studied by conventional Kirby-Bauer method. [13] Briefly, a minimum of four colonies of the test organisms were touched with a sterile loop and transferred into sterile Mueller Hinton broth under aseptic conditions and was incubated for two hours at 37°C/2 h. After incubation the density of each microbial suspension was adjusted equal to that of 10 6 CFU/ml (standardized to that of 0.5 McFarland standard) and was used as the inoculum for performing the disc diffusion test. One hundred microliter of the inoculum of each test organism was spread as lawn cultures onto sterile Mueller Hinton agar plates using L-rods to achieve a confluent growth. For the yeast isolates, amphotericin B (100 U) discs [Hi-media] were placed on the surface of the cultures using sterile forceps. Similarly for the caries bacterial pathogens, chlorhexidine (0.2%) discs were used. Standard ATCC/MTCC organisms were included as controls. All the plates were incubated at 37°C/18 h and the zone of inhibition was measured using antibiotic sensitivity scale [Hi-media] and was recorded. Absence of the inhibition zone indicated the resistance property of the caries pathogens.

 Results



Out of the 75 study cases, dentinal type of caries was more predominant, constituting 89.3% of the total study population followed by 8% of root caries and 2.6% of smooth surface caries. In this study, caries was predominant among 25 to 35 years of age group. Economically poor status males were more affected than females [Graph 1]. Smoking and drinking were the prime risk factors associated with the study cases followed by tobacco chewing and IV users [Graph 2].

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A total of 38 isolates were biochemically characterized from the carious dentine [Table 1]. Lactobacillus sp., was identified as white, irregular, mucoidal growth on thioglycollate agar and showed Gram-positive bacilli in chains upon staining. Minute, α-hemolytic colonies on BHIBA showed the Gram-positive cocci in chains and were speciated biochemically as S. mutans . Actinomyces sp., was observed as white, irregular, transparent colonies on BHIBA, with Gram-positive branching short filaments upon staining. White, creamy, moist colonies on SDA agar were subjected to germ tube test and the formation of germ tubes indicated the virulent strains of C. albicans. {Table 1}

From the dentinal caries type, Lactobacillus sp., was the predominant isolate constituting nearly 27%, followed by 11.9% of germ tube positive Candida albicans and 9% of Streptococcus mutans. From the root caries type, Actinomyces sp., was isolated from 3 cases (50%), followed by Candida sp., (16.6%) and Lactobacilli sp., (16.6%). Smooth surface caries yielded S. mutans from a single specimen. Among all these isolated pathogens, Lactobacilli sp., and C. albicans were selected for the sensitivity studies as these were the predominant isolates. Statistical analysis of the isolation rate among the study population by Pearson Chi-square test and Fisher's exact test did not show any statistical difference among the isolates. Thus the caries pathogens isolated remain a common cariogenic microbial etiology in the study population without the disparity of age, gender and other associated factors.

Out of the 19 Lactobacillus sp., 47.3% were found to be resistant to chlorhexidine. Out of the 9 C. albicans, 55.5% showed resistance to chlorhexidine and 22.2% showed resistance to both chlorhexidine and Amphotericin B and 11.1% showed resistance to Amphotericin B alone. All the standard control organisms were sensitive for the chlorhexidine and amphotericin B.

To summarize, dentinal type of caries was the most predominant type of caries when compared to smooth surface and root caries. Middle-aged adults are mostly prone to dental caries. The commonest risk factors associated with dental caries were smoking and drinking while other minor factors like dietary habits, marital status and economic levels also had played minor roles. Lactobacillus sp., were the commonest isolate followed by the yeast C. albicans. Among the isolates, emergence of drug resistance was screened against chlorhexidine and amphotericin B. These results suggested the need for empirical antibiotic susceptibility testing regimen to be followed in all dental offices to create awareness about resistance among the dentists to prevent major dentine threatening chronic problems.

 Discussion



Dental caries is the most common chronic disease of childhood, and is the biggest unmet health care need among the human population. For disease initiation and progression, many factors have been interlinked, and removal of any one element ostensibly leads to the interception of the disease process. [14] Prevalence of dentinal caries in our community results due to poor oral hygiene. Socioeconomic disparities in both rates of disease and treatment are a major public health issue. [15] To date, effective biological interventions to prevent caries have not been developed. Surgical excavation being the major treatment procedure in caries, the use of antimicrobials like chlorhexidine, povidone iodine, fluorides, penicillin or other antifungal agents are also in use to kill a broad spectrum of organisms. They may be semi selective, in that medicaments can be prescribed that preferentially affect Gram-positive, Gram-Negative, anaerobic or aerobic organisms, but they still kill an array of like organisms. Thus it is concluded that broad spectrum antibi­otics or antimicrobials are not effective long-term unless their application is periodically repeated. This repeated suppression can be effective as long as resistant strains of the bacterial pathogens do not develop and also due to the suppression of the normal flora. [16]

In this study, the dentinal samples have showed a high incidence rate of two important pathogens viz., Lactobacillus sp., and C. albicans. As previous findings, [17] this study has proved that Lactobacilli sp. is the commonest pathogenic carious strain followed by other pathogens like C. albicans, S. mutans and Actinomyces sp. This study has showed that Lactobacillus sp. is the commonest pathogenic carious strain isolated under microaerophilic conditions. [18] The isolation rate of S. mutans is very low in this study, which might be due to two major reasons: 1. The microaerophilic method of cultivation followed in the study instead of anaerobiosis. [19] 2. Most of the study cases have been affected with the pit and fissure type of caries where the role of S. mutans is low when compared with L. acidophilus. [20] Root caries has yielded a high C. albicans isolates that indirectly indicates its role in the progression of the lesion to endodontic infections. [21] The isolation of A. viscosus from the root caries cases also suggests its role in the co-aggregation property of the microbes in the establishment of caries. [22] Statistical analysis of the caries pathogens among the study population in relation with the age, gender, marital status and other risk factors by the Pearson Chi-square test and Fisher's exact test did not show any significant differences suggesting the common prevalence of these pathogens in the etiology of dental caries.

Chlorhexidine being considered as the gold standard oral antibiotic of choice, an attempt has been made in this study to screen for the emergence of resistance against it, thus excluding the other antibiotics. Similarly, amphotericin B was selected for its susceptibility property against the fungal pathogens. Due to the limitation of maintaining the viability of S. mutans and Actinomyces sp., these pathogens were not subjected for the antimicrobial susceptibility tests. Due to the high prevalence rate of Lactobacillus sp., and C. albicans these pathogens were considered for the same. Thus, the antimicrobial susceptibility test reports have screened the property of emerging drug resistance among Lactobacillus sp., and C. albicans against chlorhexidine and amphotericin B. This has been assessed by the standard disc diffusion methodology. However, other advanced antimicrobial studies and molecular screening methods have to be performed to analyze the nature of drug resistance.

To conclude, there are different ways of accomplishing the removal of the pathogens by developing targeted antimicrobials. [23] Regardless of which of these or other strategies emerge as a winner in the war on caries, it is more interesting to solve the problem of this specific infection in the heavy bio-burden of the oral cavity virtually. This study suggests that periodical antimicrobial and antifungal sensitivity tests must be performed in all dental offices to find the nature of drug resistance. This periodical empirical approach will provide the data of the associated caries pathogens in dental caries and its susceptibility pattern to the antimicrobial agents. This approach will assist the dentist in the treatment of various threatening dental infections and to eradicate the emergence of drug-resistance property among the pathogenic microorganisms.

 Acknowledgments



We sincerely thank the help rendered by Dr. Kandaswamy, Former Professor and Head of Conservative Dentistry, Dr. Velmurugan. N, Professor and Head, Department of Conservative Dentistry, Meenakshi Ammal Dental College, Dr. Priya and Dr. Nandhini, Staff members, Department of Conservative Dentistry towards the collection of carious dentine specimens and for their expertise suggestions throughout the period of the study.

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