Research - MS (Thesis)

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Title: Studies of Dental Plaque Forming Microorganisms and Their Sensitivity Assay against Commercially Available Antibiotics

Author: Md. Kamal Uddin

Supervisor: Dr. Md. Nural Anwar

Co-Supervisor: Md. Towhid Hossain

Research Field: Medical Microbiology

Batch: 11th

Session: 2010-2011


Abstract

Dental caries is a biofilm mediated disease which is the predominant cause of tooth decay. Antibiotics to treat dental caries infection are routinely prescribed which led to the increased resistance against bacteria. The present study was designed for the isolation and identification of dental plaque forming bacteria, optimization of their biofilm forming conditions, response to different sugars and nitrogenous compounds for biofilm formation. The purpose of this investigation was to perform antibiotic susceptibility assay on a panel of bacteria isolated from dental caries infection.
Dental plaques from the carious portion of teeth of patients were collected as sample and twenty-three isolates were isolated and subjected to screening the biofilm forming microorganisms. For screening three methods were followed such as cover slip assay, test tube assay, and microtiter plate assay. The results were quantitatively measured by absorbance or image processing technique. Based on the measurement of higher biofilm formation three bacterial isolates (KA13, KA22 and KA23) were finally selected for detailed study. The selected bacterial isolates were characterized based on their morphological, cultural, physiological and biochemical properties; and the isolates were provisionally identified as Staphylococcus aureus (KA13), Bacillus subtilis (KA22) and Streptococcus mutans (KA23) according to the description of Bergey’s Manual of Determinative Bacteriology (8th and 9th edition).
Different environmental and cultural conditions were optimized for biofilm formation by the selected isolates. Two media were used to observe their effect on biofilm formation. Among this Luria-Bertani broth induced better biofilm formation than nutrient broth. The optimum temperature for biofilm formation was found at 37ºC for all the bacterial isolates. The bacterial isolate KA23 exhibited maximum biofilm formation after 24 hours of incubation at media pH 6.5. On the other hand, the isolates KA13 and KA22 showed higher biofilm formation at 48 hours of incubation time with medium pH 7.5.
The effect of various sugars at different concentrations (180 to 1800µg/200µL/well in microtiter plate) was observed. In this experiment, biofilm formation was induced in the presence of glucose at 1350µg/200µL by the isolate KA13 and KA23. On the other hand, the bacterial isolate KA22 showed highest biofilm formation at 900µg/200µL of sucrose. The other three sugars (fructose, lactose and starch) showed less effect on biofilm formation.
The effect of different nitrogen sources (yeast extract, beef extract, peptone, tryptone, ammonium nitrate and potassium nitrate) at different concentration (180 to 1800µg/200µL) was also observed. In all case, glucose at 1350µg/200µL was used as carbon source. The presence of yeast extract in the medium greatly induce biofilm formation.
The effect of NaCl (180 to 1800 µg/200µL per well) on biofilm formation was investigated and found that the isolate KA13 and KA23 was showed better biofilm formation at 900µg/200µL per well whereas, the isolate KA22 exhibited better biofilm formation at 1350 µg/200µL.
The antibiotic sensitivity assay of the selected isolates was performed against nine commercially available antibiotic disks by disk diffusion method. The MIC and MBC values of six antibiotics and one mouthwash solution (chlorohexidine gluconate) were determined. Two pathogenic bacteria a) Staphylococcus aureus ATCC 25923 and b) Bacillus subtilis IFSTIM 22 were used as standard test organism. The results of sensitivity assay, MIC and MBC determination were interpreted according to the recommended chart mentioned in “Performance standards for antimicrobial susceptibility testing’’ Vol. 31(1), 2011. It was found that, the standard test organisms and the selected isolates were resistant to amoxicillin, ampicillin, penicillin, and cefixime. The isolate KA13 and KA23 were found resistant to erythromycin. The standard test organism Bacillus subtilis IFSTIM 22 was also found resistant to ciprofloxacin, gentamicin and chloramphenicol. Both of the test organism and our isolate KA22 and KA23 were susceptible to tetracycline, though our isolate KA13 was found intermediate sensitivity to it. Chlorohexidine gluconate has a good cidal effect to all of these bacterial isolates and its MIC and MBC value was very low ranging from 0.005 to 0.5µg/µL and 0.005-0.032 µg/µL respectively. From the results of our research experiment, it has been shown that the above organisms acquire much resistance to the used antimicrobial agents.