Genetic and physiological study on cariogenic Streptococcus species.

number: 
759
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Ranya Abbas al-Fatlawi
Supervisor: 
Dr. Subhi J. Hamza Al-Zubaidi
Dr. Falah A. Attawi
year: 
2003
Abstract:

Seventy five plaque, soft caries and calculus samples were collected. Oependhg on Iheir growth in MS-agar, thirty eight samples were considered as true toLteoma (about 104 bacteria /ml). Using biochemical identification system, only eighteen isolates were considered belongs to species of the genus Streptococcus. Depending on colonial morphology, five isolates were characterized as Streptococcus sanguis, while eight isolates were related to Streptococcus mutans, however, the other two isolates appear related to Streptococcus milleri, and three isolates considered as Streptococcus salivarius. According to their ability for production of dextran and levan, sixteen isolates were capable of producing dextran and considered as either related to Streptococcus mutans or Streptococcus sanguis, which allows separation of the isolates from those belongs to Streptococcus salivarius as producing of levan. Further conformation of the results were done using API 20 strip test and characterized as Streptococcus mutans, Streptococcus sanguis and Streptococcus milleri. Growth characteristics of Streptococcus mutans was studied, lag phase take about 3hrs, followed by log phase when the cell number reach about 9 x 105 cell/ml at the mid log phase which take about 6.5hrs. Then at stationary phase the cell number reaches 15 x 109 cell/ml. The results of utilization of various sugars indicate that a vast variation among isolates and sugars used was found, but large stimulation in growth of Streptococcus mutans was noticed with sucrose and glucose, while fructose utilized weakly. Acidogenicty of culture medium during growth and utilization of carbohydrates was also tested. Results show that isolates belongs to Streptococcus mutans cause higher reduction in pH of liquid culture reaching about 2.7 as compared to that of Streptococcus sanguis which stop to pH of 5.6. Productivity of selected isolates for glucan was determined, an amount of 120jig and 70jig of glucan per milliliter of liquid culture was determined for isolate R22 (Streptococcus mutans) and isolate rig (Streptococcus sanguis) respectively. Bacterial isolates were also tested for the production of protease; all isolates were found able to produce the enzyme and R^was chosen as the best producer. The inhibition of growth and acid production for the isolates by inhibitors like sodium fluoride, SDS and antibiotics was studied. Sodium fluoride was found to be the most effective agent. Results also indicate that isolates belong to Streptococcus mutans were resistant to bacitracin, streptomycin, tetracycline, amoxicillin, and carbenicilline while most isolates show sensitivity to penicillin, ampicillin, erythromycin, cephaloxin, vancomycin, methicillin, and kanamycin. Determinants of various virulence factors in Streptococcus mutans were studied depending on plasmid curing, isolation of plasmid DMA and transformation; was studied. Curing agents such as acridine orange and ethidium bromide were found not effective in curing plasmid DMA. Isolation of plasmid DMA from the two isolates R?2 ancTR24 was successful. Two plasmids bands were characterized presumably related to the two markers fluoride tolerance and streptomycin resistance. Transformation of plasmid DNA isolated from Streptococcus mutans in Streptococcus sanguis was also successful with a frequency of (2.5x107cell/ml) and from these results one could assume that a genetic linkage between them may be present and localized on the same plasmid. Attempts for transfer of genetic marker for extracellular polysaccharide synthesis, the characteristic of the cariogenic Streptococcus mutans, to Streptococcus sanguis was done without success.