Identification of mutans and other oral streptococci by random amplified polymorphic DNA analysis

Abstract

The identification and classification of the non-haemolytic or viridans group of streptococci have long been recognised as difficult and unsatisfactory. Phenotypic and genotypic heterogeneity have resulted in ambiguous speciation, particularly with mutans streptococci and other oral streptococci. This study was done to determine whether random amplified polymorphic DNA (RAPD) analysis is useful to identify and even classify oral and other streptococci. DNA was prepared and purified from 25 strains of mutans streptococci including 11 reference strains of Streptococcus mutans, seven of S. sobrinus, three of S. rattus and one each of the four other species of the mutans group, together with 20 other reference species, mostly streptococci, and from 49 fresh isolates of mutans streptococci and of S. mutans from human saliva and dental plaque. DNA amplification was primed with each of three arbitrarily selected primers nine or 10 nucleotides in length. The amplified DNA fragments (amplicons) obtained were compared by agarose gel electrophoresis. Species-and strain-specific RAPD fingerprints were obtained not only from pure genomic DNA, but also from the supernates of crude cellular or colony extracts. Pending the analysis of numerous other strains, the data suggest that RAPD may be of value: (i) to distinguish the species S. mutans and S. sobrinus from each other and potentially from other species of oral streptococci, (ii) to differentiate and possibly classify oral streptococci and (iii) as a valuable tool in mutans streptococci epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of streptococcal isolates.

Typing and Subtyping of 83 Clinical Isolates Purified from Surgically Implanted Silicone Feeding Tubes by Random Amplified Polymorphic DNA Amplification

“…Additional studies are using plasmid analysis to investigate the transfer of antibiotic resistance genes in order to link RAPD fingerprints to antibiotic resistance and sensitivity. This investigation supports previous reports that RAPD analysis is efficient, reproducible, and capable of detecting genomic polymorphisms among various microbial species without previous knowledge of the nucleotide sequence on the target DNA (22), and the technique has been shown to be valuable in studies dealing with biofilm (22,25). RAPD is also preferable for gram-positive species, where chromosomal preparations are difficult and low concentrations of nucleic acid are achieved.…”

“…The B. brevis isolates were purified from two specific locations on the same tube from one patient, the inner portion of the internal stabilizer and the outer portion of the shaft. A hypothesis, supported by several reports (1,22), can be drawn that the isolates came from different environmental sources and were not from one organism proliferating through various locations of the tube. B. licheniformis was found in biofilms isolated from three different patients, and all of the RAPD profiles were different, leading to the hypothesis that each patient encountered different environmental sources of the contaminant that produced biofilm proliferation.…”

Genetic variability of Streptococcus mutans isolated from low-income families, as shown by RAPD markers

“…Interestingly, Truong et al (38) analyzed reference strains of oral streptococci using RAPD for the identification and subclassification of all the studied strains and suggested that RAPD can be valuable to discriminate the species S. mutans and S. sobrinus, both from each other and from other oral streptococci. However, similarly to those authors, the present study indicated that the RAPD markers discriminated the strains ATCC 13540 and BAA572 of S. pyogenes, forming a separate group (Group II, Fig.…”

“…In addition, several markers based on the PCR method have been utilized to identify bioserotypes of the mutans group and their relative occurrence within the oral microbiota (14,16,21,25,31,32). Among those, RAPD markers have been widely used in the study of the genetic polymorphism and in the distribution of populations of S. mutans (13,18,19,29,34,38).…”

Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan

“…eDNA was isolated from the biofilm by the same procedure as in the quantification of eDNA. RAPD was carried out using the random primers that were published previously: OPA02 (5=-TGCCGAGCTG-3=) and OPA18 (5=-AGGTGACCGT-3=) (49,50). The PCR was conducted using the PrimeSTAR premix (TaKaRa Bio, Inc., Shiga, Japan) under the following cycling conditions: 94°C for 1 min, 32°C for 1 min, and 72°C for 2 min for 40 cycles.…”