Int J Curr Pharm Res, Vol 15, Issue 1, 54-58Original Article


A STUDY ON BACTERIOLOGICAL IDENTIFICATION OF SUB-GINGIVAL PLAQUES IN PERIODONTITIS PATIENTS

PURIMITLA USHARANI*

*1Department of Microbiology, Dr. Patnam Mahender Reddy Institute of Medical Sciences, Rangareddy, Telangana, India
Email: purimitlausha2@gmail.com

Received: 15 Oct 2022, Revised and Accepted: 22 Dec 2022

ABSTRACT

Objective: The current study's objectives were to isolate and characterize the periodontal pathogens from subgingival plaque obtained from patients with chronic periodontitis using conventional microbiological techniques.

Materials: In the Department of Microbiology at a Tertiary Care Teaching Hospital was where this study was carried out. Over the course of six months, from January to June 2019, samples were taken from subgingival pockets in patients with chronic periodontitis who visited the periodontology outpatient department at our institute of dental sciences. There were 21 cases in the research. For the purpose of preventing saliva contamination, tooth surfaces were dried using sterile gauze. Subgingival plaque samples were taken from the majority of diseased sites using a sterile periodontal Gracy curette, put in a test tube with fluid thioglycollate medium, and taken to the microbiology lab where they were processed right away using conventional microbiological procedures.

Results: E. coli grew in the majority of samples (9), followed by Pseudomonas species and Staphylococci in 7 samples. Only sample number 7 out of 21 showed no growth for Candida albicans; the others all exhibited development. Samples like Sample 1, 5, 6, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, and 20 produced isolates that were multidrug resistant.

Conclusion: Therefore, while developing a treatment plan for adult patients with periodontitis, the microbial diversity discovered in the current study should be taken into account. Variations in the quantities and species of cultivable bacteria have been found in investigations of periodontitis patients from different geographic regions, including developed and developing countries.

Keywords: Periodontitis, Yeast, Aerobes, AST, Culture

© 2023 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijcpr.2023v15i1.2073 Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr

INTRODUCTION

Periodontitis is an oral cavity disease that is characterized by ongoing inflammation of the periodontal tissues. Long-term accumulation of a lot of tooth plaque is what causes the sickness [1]. In order to prevent severe and irreparable damage to the tooth's protective and stable structures, persistent periodontitis must be diagnosed early on [2]. However, since chronic periodontitis is a disease that advances easily, very few people would seek dental care in the early stages. Periodontitis that is mild to moderately advanced can be managed with the proper mechanical removal of the biofilm and subgingival analytics. Regular monthly periodontal checkups and thorough dental hygiene are essential for controlling the severity of the disease. Given their [3] high prevalence in both the adult and paediatric populations, oral depression contaminations are a serious general medical concern generally. There are more than one billion cases of serious periodontal diseases worldwide, which are estimated to affect 14% of adults [4, 5]. The main causes of periodontal disease are poor dental hygiene and tobacco smoking [6]. Periodontitis was generally reported to start after the age of 15, and by the time they were 17 y old, 10% of Indian young men were affected [7]. To determine the prevalence of periodontal disease in the Konda Reddy clans residing in Bhadrachalam, Khammam District, a fresh report was initiated. Additionally, it was discovered that the majority of them (93.60%) used twigs to brush their teeth, while only 6.20% used a combination of a toothbrush, finger, and twig together with toothpaste and charcoal [8]. The oral cavity is home to several different types of facultative bacteria Streptococcus species, as well as Granulicatella, Gemella, and Veillonella, while most microscopic organisms are localised to certain areas [9]. The biological locality of commensal, cooperative, and harmful bacteria present in the oral cavity is known as the human oral microbiota. The majority of oral microbiota occurs as a biofilm, and it plays a crucial role in maintaining oral homeostasis, protecting the mouth cavity, and preventing the spread of infection. The present study aimed to isolate and identify the periodontal pathogens using standard microbiological techniques from subgingival plaque collected from patients suffering with Chronic Periodontitis.

MATERIALS AND METHODS

This study was conducted in the Department of Microbiology in a Tertiary Care Teaching Hospital. Samples were collected from subgingival pockets in patients with chronic periodontitis attending the Periodontology Outpatient Department at our Institute of Dental Sciences, over a period of 6 mo from January to June 2019. The study comprised of 21 cases. Clinically diagnosed cases of chronic periodontitis were included in the study and patients with history of systemic conditions such as diabetes mellitus, nutritional deficiencies, pregnant woman, antibiotic usage in the last 2 mo and patients with a history of undergoing any dental procedures in the last 2 mo were excluded.

Sample collection

Tooth surfaces were dried with sterile gauze to avoid contamination by saliva. Subgingival plaque sample was collected from most pathological site using sterile periodontal Gracy curette and placed in fluid thioglycollate medium in a test tube and brought to the microbiology laboratory and processed immediately.

Laboratory methods

In the lab, a fluid thioglycollate medium sample was added to 1 ml of trypticase soy broth and vortexed for 1 minute. Direct smears of the samples were made, and modified Fontana staining and Hucker's modification of the Gram-stain for anaerobes was used. A standard loop was used to take a loopful of the sample from the vortexed solution and inoculate it onto the plates of nutrition agar, Mac Conkey agar (MA), and brain heart infusion agar. As per the guidelines outlined in Mackie and McCartney's Practical Medical Microbiology, all aerobic isolates were recognized and biochemically characterized. The antimicrobial susceptibility testing was done for aerobic isolates by disc diffusion method as described by Kirby and Bauer on Mueller-Hinton agar. The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the antimicrobial. AST was performed according to CLSI guidelines using Mueller-Hinton agar (MHA) plates using the concentration of antibiotics per well, recommended by the WHO experts committee on biological standardization. The plates were incubated at 37 °C for 16-18h h.

RESULTS

Majority of the samples 9 showed the growth of E. coli followed by Pseudomonas species and Staphylococci in a total of 7 samples. Only one sample showed the growth of Proteus and one sample with no growth (table 1). All the samples showed growth for Candida albicans except sample number 7 out of 21 samples showed no growth. The growth was seen in the MCA plates (table 2) for culture confirmation. The results of the antibiotic susceptibility pattern of isolated pathogens were depicted in table 3. The samples like Sample 1, 5, 6,8,10,11,12,13,15,16,17,18,19 and 20 yielded multidrug-resistant isolates.

Table 1: Culture identification from sub-gingival samples

Patient sample Colony morphology Organism isolated
Sample 1 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 2 Smooth, cream or white colonies with entire edges Enterococci species
Sample 3 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 4 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 5 Smooth, cream or white colonies with entire edges Enterococci species
Sample 6 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 7 No Growth -
Sample 8 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 9 Flat and smooth colonies that are between 2 Pseudomonas species
Sample 10 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 11 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 12 Flat and smooth colonies that are between 2 Pseudomonas species
Sample 13 Flat and smooth colonies that are between 2 Pseudomonas species
Sample 14 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 15 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 16 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 17 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 18 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli
Sample 19 Smooth, cream or white colonies with entire edges Enterococci species
Sample 20 Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2 mm Staphylococci species
Sample 21 Appear large, circular, low convex, grayish, white, moist, smooth, and opaque E. coli

Table 2: Growth on MacConkey agar

Patient sample Colonies Organisms isolated
Sample 1 Pink colour colonies-Lactose Fermenter E. coli
Sample 2 Pink colour colonies-Lactose Fermenter E. coli
Sample 3 Pink colour colonies-Lactose Fermenter Klebsiella species
Sample 4 Pink colour colonies-Lactose Fermenter E. coli
Sample 5 Lactose Fermenter E. coli
Sample 6 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 7 No Colonies No Growth
Sample 8 Pink colour colonies-Lactose Fermenter E. coli
Sample 9 Pink colour colonies-Lactose Fermenter Klebsiella species
Sample 10 Pink colour colonies-Lactose Fermenter E. coli
Sample 11 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 12 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 13 Colourless colonies, Non-Lactose Fermenter Proteus species
Sample 14 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 15 Pink colour colonies-Lactose Fermenter E. coli
Sample 16 Pink colour colonies-Lactose Fermenter Klebsiella species
Sample 17 Pink colour colonies-Lactose Fermenter E. coli
Sample 18 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 19 Colourless colonies, Non-Lactose Fermenter Pseudomonas species
Sample 20 Pink colour colonies-Lactose Fermenter E. coli
Sample 21 Colourless colonies, Non-Lactose Fermenter Pseudomonas species

Table 3: Antibiotic susceptibility of periodontal pathogens

Sample Antibiotic symbol Antibiotic Zone of inhibition Senstive/Resistant S/R
Sample 1 C Chloramphenicol No zone R
TEI Teicoplanin No zone R
LZ Linezolid No zone R
NIT Nitrofurantoin No zone R
CPM Cefepime No zone R
Sample 2 HLG Gentamicin 30 mm S
AK Amikacin 25 mm S
IMP Imipenam 30 mm S
COT Co-Trimoxazole 30 mm S
AMP Ampicillin 20 mm S
MRP Meropenem 20 mm S
AMC Amoxicillin 30 mm S
Sample 3 AMC Amoxicillin 25 mm S
AMP Ampicillin 20 mm S
IMP Imipenam 30 mm S
AK Amikacin 25 mm S
MRP Meropenem 25 mm S
HLG Gentamicin 25 mm S
COT Co-Trimoxazole 15 mm S
Sample 4 AK Amikacin 20 mm S
HLG Gentamicin 25 mm S
COT Co-Trimoxazole 25 mm S
AMP Ampicillin 25 mm S
MRP Meropenem 25 mm S
IMP Imipenam 30 mm S
AMC Ampicillin 20 mm S
Sample 5 IMP Imipenam 25 mm S
MRP Meropenem 15 mm R
COT Co-Trimoxazole 10 mm R
HLG Gentamicin 20 mm S
AMC Amoxicillin 15 mm R
AMP Ampicillin 10 mm R
AK Amikacin 25 mm S
Sample 6 C Chlorophenicol 20 mm S
TEI Teicoplanin 15 mm R
LZ Linezolid 30 mm S
NIT Nitrofurantoin 15 mm R
CPM Cefepime 5 mm R
Sample 8 AMP Ampicillin 05 mm R
AMC Amoxicillin 10 mm R
AK Amikacin 15 mm S
MRP Meropenem 10 mm R
HLG Gentamicin 20 mm S
COT Co-Trimoxazole 15 mm S
IMP Imipenam 25 mm S
Sample 9 AMP Ampicillin 22 mm S
AMC Amoxicillin 26 mm S
AK Amikacin 15 mm S
HLG Gentamicin 20 mm S
COT Co-Trimoxazole 25 mm S
IMP Imipenam 25 mm S
MRP Meropenem 23 mm S
Sample 10 AMP Ampicillin 10 mm R
AMC Amoxicillin 14 mm R
MRP Meropenem 10 mm R
AK Amikacin 15 mm S
HLG Gentamicin0 28 mm S
COT Co-Trimoxazole 25 mm S
IMP Imipenam 25 mm S
Sample 11 AMP Ampicillin 08 mm R
AMC Amoxicillin 10 mm R
MRP Meropenem 10 mm R
AK Amikacin 25 mm S
HLG Gentamicin 24 mm S
COT Co-Trimoxazole 15 mm R
IMP Imipenam 30 mm S
Sample 12 AMP Ampicillin 05 mm R
AMC Amoxicillin 08 mm R
MRP Meropenem 08 mm R
AK Amikacin 10 mm R
HLG Gentamicin 15 mm R
COT Co-Trimoxazole 05 mm R
IMP Imipenam 30 mm S
Sample 13 AMP Ampicillin 10 mm R
AMC Amoxicillin 20 mm S
AK Amikacin 10 mm R
MRP Meropenem 10 mm R
HLG Gentamicin 26 mm S
COT Co-Trimoxazole 08 mm R
IMP Imipenam 25 mm S
Sample 14 AMP Ampicillin 15 mm R
AMC Amoxicillin 27 mm S
MRP Meropenem 20 mm S
AK Amikacin 20 mm S
HLG Gentamicin 25 mm S
COT Co-Trimoxazole 25 mm S
IMP Imipenam 25 mm S
Sample 15 AMP Ampicillin 13 mm R
AMC Amoxicillin 25 mm S
MRP Meropenem 20 mm R
AK Amikacin 24 mm S
HLG Gentamicin 18 mm S
COT Co-Trimoxazole 15 mm R
IMP Imipenam 30 mm S
Sample 16 AMP Ampicillin 15 mm R
AMC Amoxicillin 20 mm S
MRP Meropenem 18 mm R
AK Amikacin 17 mm R
HLG Gentamicin 13 mm R
COT Co-Trimoxazole 19 mm R
IMP Imipenam 30 mm S
Sample 17 AMP Ampicillin 10 mm R
AMC Amoxicillin 10 mm R
MRP Meropenem 18 mm R
AK Amikacin 18 mm R
HLG Gentamicin 30 mm S
COT Co-Trimoxazole 20 mm S
IMP Imipenam 27 mm S
Sample 18 AMP Ampicillin 08 mm R
AMC Amoxicillin 18 mm R
MRP Meropenem 10 mm R
AK Amikacin 15 mm R
HLG Gentamicin 10 mm R
COT Co-Trimoxazole 15 mm R
IMP Imipenam 25 mm S
Sample 19 AMP Ampicillin 08 mm R
AMC Amoxicillin 10 mm R
MRP Meropenem 10 mm R
AK Amikacin 15 mm R
HLG Gentamicin 20 mm S
COT Co-Trimoxazole 10 mm R
IMP Imipenam 15 mm R
Sample 20 AMP Ampicillin 08 mm R
AMC Amoxicillin 17 mm R
MRP Meropenem 15 mm R
AK Amikacin 15 mm R
HLG Gentamicin 30 mm S
COT Co-Trimoxazole 08 mm R
IMP Imipenam 17 mm R
Sample 21 AMP Ampicillin 25 mm S
AMC Amoxicillin 10 mm R
MRP Meropenem 25 mm S
AK Amikacin 20 mm S
HLG Gentamicin 25 mm S
COT Co-Trimoxazole 10 mm R
IMP Imipenam 24 mm S

DISCUSSION

Chronic periodontitis is an uncontrollable infection that irritates the mouth and destroys the bond between the gums and teeth's supporting tissue. Around 14% of people globally are estimated to be affected by severe chronic periodontitis. Less frequent dental visits and cigarette use are the two main causes of chronic periodontitis. In the entire world, 51% of people had periodontal disease, and 46.6% had gum disease. 26.2% of people had direct periodontitis, whereas 19% had severe periodontitis. Males predominate more than females. Heteogeneity is another possibility. In India, 17.3% of the population suffers from chronic periodontitis. Periodontitis' aetiology has been linked to a number of bacterial species or groups of species [10-12]. A particular bacterial strain is linked to adult periodontitis, and several complexes have been identified in subgingival plaque samples [13-15]. Adult patients with periodontal disease were examined clinically, epidemiologically, and microbiologically by Daniluk et al. [16], who discovered that no Candida yeasts were recovered from these individuals. Aerobic and anaerobic bacteria cultures from subgingival and supragingival plaque samples were found to be present in 19 out of 21 patients. Forty-two bacterial strains in all, 24 (57.1%) of which belonged to 7 anaerobic species and 18 (42.9%) to 12 aerobic species, were recovered from subgingival plaques. Streptococci and Staphylococci made up the majority of the isolated aerobic organisms. However, E. coli, Staphylococci, and Pseudomonas were the most frequently isolated species in the current investigation. Candida growth was seen in the majority of the samples. C. albicans, however, may play a part in the formation of periodontal microbial plaque and its adherence to the periodontal tissues, according to Jarvensivu et al. [17]. Additionally, according to these authors' findings, fungal components could not be seen in the epithelium, whereas hyphal germination begins in the gingival pocket [17]. Pseudomonas aeruginosa and periodontal pathogens in the oral cavity and lungs of cystic fibrosis patients were examined by Caldas et al. in 2015. In this study, 16 P. aeruginosa strains were identified [18]. The results presented in the study were somewhat correlated from the previous studies and most of the predominant organisms isolated were Pseudomonas, Staphylococci and E. coli.

CONCLUSION

In conclusion, the microbial diversity discovered in the present study should be taken into account in the periodontitis treatment plan for adult patients. Variations in the quantities and species of cultivable bacteria have been found in investigations of periodontitis patients from different geographic regions, including developed and developing countries. For oral infections, there are many different microbiological diagnostic techniques available, and the treatment of these infections depends on the relationship between the doctor and the microbiologist.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Rams TE, Flynn MJ, Slots J. Subgingival microbial associations in severe human periodontitis. Clin Infect Dis. 1997;25Suppl 2:S224-6. doi: 10.1086/516248, PMID 9310686.

  2. Slots J, Rams TE, Listgarten MA. Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis. Oral Microbiol Immunol. 1988;3(2):47-52. doi: 10.1111/j.1399-302x.1988.tb00080.x, PMID 3268751.

  3. Dahlen G, Wikström M. Occurrence of enteric rods, staphylococci and Candida in subgingival samples. Oral Microbiol Immunol. 1995;10(1):42-6. doi: 10.1111/j.1399-302x.1995.tb00116.x, PMID 7644272.

  4. Dymock D. Detection of microorganisms in dental plaque. In: Jass J, Surman S, Walker J. editors. Medical biofilms. John Wiley & Sons Ltd; 2003;4(2):199-220.

  5. Meurman JH. Dental infections and general health. Quintessence Int. 1997;28(12):807-11. PMID 9477871.

  6. Haffajee AD, Socransky SS. Microbial etiological agents of destructive periodontal diseases. Periodontol 2000. 1994;5:78-111. doi: 10.1111/j.1600-0757.1994.tb00020.x, PMID 9673164.

  7. Kamma JJ, Nakou M, Manti FA. Predominant microflora of severe, moderate and minimal periodontal lesions in young adults with rapidly progressive periodontitis. J Periodontal Res. 1995;30(1):66-72. doi: 10.1111/j.1600-0765.1995.tb01254.x. PMID 7722848.

  8. 8. Naheeda, Asif SM, Padma M, Paul A. Assessment of periodontal status of Konda Reddy tribe in Bhadrachalam, Khammam District, India. J Chlin Diagn Res 2015:9zc23.

  9. Cao CF, Aeppli DM, Liljemark WF, Bloomquist CG, Bandt CL, Wolff LF. Comparison of plaque microflora between Chinese and Caucasian population groups. J Clin Periodontol. 1990;17(2):115-8. doi: 10.1111/j.1600-051x.1990.tb01072.x, PMID 2303572.

  10. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25(2):134-44. doi: 10.1111/j.1600-051x.1998.tb02419.x, PMID 9495612.

  11. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA. Bacterial diversity in human subgingival plaque. J Bacteriol. 2001;183(12):3770-83. doi: 10.1128/JB.183.12.3770-3783.2001, PMID 11371542.

  12. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998 Feb;25(2):134-44. doi: 10.1111/j.1600-051x.1998.tb02419.x. PMID 9495612.

  13. Piovano S. Bacteriology of most frequent oral anaerobic infections. Anaerobe. 1999;5(3-4):221-7. doi: 10.1006/anae.1999.0204.

  14. Meurman JH. Dental infections and general health. Quintessence Int. 1997;28(12):807-11. PMID 9477871.

  15. Darveau RP, Tanner A, Page RC. The microbial challenge in periodontitis. Periodontol. 1997;14:12-32. doi: 10.1111/j.1600-0757.1997.tb00190.x, PMID 9567964.

  16. Daniluk T, Tokajuk G, Cylwik Rokicka D, Rozkiewicz D, Zaremba ML, Stokowska W. Aerobic and anaerobic bacteria in subgingival and supragingival plaques of adult patients with periodontal disease. Adv Med Sci. 2006;51Suppl 1:81-5. PMID 17458065.

  17. Järvensivu A, Hietanen J, Rautemaa R, Sorsa T, Richardson M. Candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo. Oral Dis. 2004;10(2):106-12. doi: 10.1046/j.1354-523x.2003.00978.x, PMID 14996281.

  18. Rivas Caldas R, Le Gall F, Revert K, Rault G, Virmaux M, Gouriou S. Pseudomonas aeruginosa and periodontal pathogens in the oral cavity and lungs of cystic fibrosis patients: a case-control study. J Clin Microbiol. 2015 Jun;53(6):1898-907. doi: 10.1128/JCM.00368-15. PMID 25854483, PMCID PMC4432057.