Arbitrarily primed polymerase chain reaction fingerprinting for the genotypic identification of mutans streptococci from humans

Abstract

Determining whether two strains of bacteria are unique, identical or clonally related depends upon comparisons of phenotypic and/or genotypic traits. Individual isolates can then be grouped according to differences or similarities among those traits. One method of genotyping strains of bacteria is commonly referred to as chromosomal DNA fingerprinting. Previously, we generated chromosomal DNA fingerprints of mutans streptococci to study the transmission of this organism within families. Here, we developed and evaluated an arbitrarily primed polymerase chain reaction (AP-PCR) method for the genotypic characterization of mutans streptococci. Results were compared to those derived from the more conventional chromosomal DNA fingerprinting method. First, we showed that randomly selected clinical isolates displayed a unique banding profile by both methods; the mean similarity indices between DNA fragment patterns were 0.69 for chromosomal DNA fingerprinting and 0.74 for AP-PCR. This indicated that AP-PCR demonstrated less diversity than chromosomal DNA fingerprinting. Subsequently, we tested the agreement between chromosomal DNA fingerprinting and AP-PCR in determining genotypic similarities among 21 mutans streptococci strains obtained from 10 mother-child pairs, and 5 mutans streptococci strains from 5 fathers. The Kappa value for agreement was 0.88. We conclude that AP-PCR, which generates patterns of 8 to 12 amplicons, is capable of distinguishing strains of mutans streptococci among non-related individuals. Moreover, AP-PCR can discern both homogeneity and heterogeneity of mutans streptococci genotypes among mother and child pairs. Overall, we found that AP-PCR gave results comparable to those of chromosomal DNA fingerprinting.

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

“…It has been shown that the polymorphism in length of the amplified sequences (amplicons) of RAPD fingerprints obtained by AP-PCR can be used to compare bacterial strains [22][23][24], including the groups of mutans streptococci [28,29] and mitis streptococci [30]. In the present study, this application of RAPD analysis to genotyping of oral streptococci confirmed that the RAPD assay can distinguish the species S. mutans and S. sobrinus from each other.…”

“…As the RAPD profiles have only a small number of bands and, consequently, are much easier to read than the corresponding REA patterns, they should be considered helpful for epidemiological and transmission studies of mutans and other oral streptococci, as already shown with P. gingivalis [27] and suggested for mutans streptococci [28,29].…”

“…They include DNA-DNA hybridisation [1,12,14], restriction endonuclease analysis (REA) [16][17][18][19], multilocus enzyme electrophoresis (MLEE) [20], ribotyping, PCR and colony hybridisation with DNA probes [21]. A PCR-based procedure of DNA fingerprinting, called arbitrarily primed PCR (AP-PCR) [22] or randomly amplified polymorphic DNA (RAPD) analysis [23], has been applied to strain identification and typing of oral bacteria [24][25][26][27], oral streptococci [28][29][30] and non-oral streptococci [31][32][33][34]. The present study used RAPD analysis for typing S. mutans and S. sobrinus strains and to explore the potential of this typing method to distinguish these from the other taxa that comprise the group of mutans streptococci.…”

Horizontal Transmission of Mutans Streptococci in Children

“…Characterization of oral bacteria by AP-PCR or DNA fingerprinting has become well-accepted (Saarela et al, 1996;Li and Caufield, 1998;Gronroos and Alaluusua, 2000;Li et al, 2001;Toi et al, 2003;Ersin et al, 2004;Klein et al, 2004;Liu et al, 2004;Napimoga et al, 2004;Nascimento et al, 2004;Kamiya et al, 2005;Li et al, 2005;Cogulu et al, 2006;Liu et al, 2007;Hames-Kocabas et al, 2008). Moreover, AP-PCR has been shown to have good discriminating ability for mutans streptococci differentiation (Saarela et al, 1996).…”

“…Others have reported transmission between family members, such as spouse-spouse, father-child, and sibling-sibling (Saarela et al, 1993;Li and Caufield, 1998;Redmo Emanuelsson and Wang, 1998;Kozai et al, 1999;Emanuelsson and Thornqvist, 2000;van Loveren et al, 2000;Redmo Emanuelsson and Thornqvist, 2001;Nie et al, 2002;Tedjosasongko and Kozai, 2002;Ersin et al, 2004;Hames-Kocabas et al, 2008). Although there have been three reports of transmission between unrelated young infants in nursery schools in China, Brazil, and Japan (Mattos-Graner et al, 2001;Tedjosasongko and Kozai, 2002;Liu et al, 2007), these children ranged in age from 2 mos to 4 yrs, when salivary transfer is more likely than in older children.…”