Genetic diversity within oral and blood isolates of Streptococcus mutans serotype c

• Main Authors: Wen-Chung Huang, 黃紋娟
• Other Authors: 賈景山
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/86953246184679568186

碩士 === 國立臺灣大學 === 微生物學研究所 === 92 === The broad spectrum of physiological and virulence properties of bacterial pathogens mirrors the existence of different subsets of genes enabling the pathogen’s different lifestyles. Whole genome sequence comparisons of related species had shown that specialization for an ecological niche is accompanied by the reorganization of the genomic structure. Close adaptation of parasites and intracellular pathogens (e.g. Mycoplasma spp., Chlamydia trachomatis, Mycobacterium leprae and Rickettsia prowazekii) to the physiologically stable environments of their host cells, a reductive genome evolution occurred that led to the loss of genes not essential for life within the host (a phenomenon termed ‘evolution by reduction’), but leaving genes responsible for functions needed for the performance of key metabolic steps and for interconversions of metabolites obtained from the host. The increasing number of available chromosome maps has prompted reconsideration of existing theories about the evolution of bacterial genome organization. There is increasing evidence that large genome rearrangement is not exceptional. The large inversions observed in bacterial genomes are symmetrical in relation to the origin of replication. Streptococcus mutans is a member of mutans streptocci, which consist of 9 serotypes (a, b, c, d, e, f, g, and k). S. mutans serotype c is the most predominant clinical isolates from human oral cavity. S. mutans is a primary pathogen for human dental caries and also an opportunistic pathogen for infective endocarditis. Previous studies suggested that S. mutans might be a heterogeneous group of bacterium exhibited distinct phenotypical characteristics, such as colony morphology, sugar fermentation, etc. The increased use of molecular typing method, such as pulsed field gel electrophoresis (PFGE) of chromosomal DNA cut with a restriction enzyme that recognizes rare sites in the DNA, provides a useful tool for the classification of clinically relevant bacteria. The purposes of the present study are, firstly, to evaluate the genetic diversity of the S. mutans serotype c strains isolated from healthy donors, patients with dental caries or endocarditis; secondly, to identify possible molecular mechanisms and genetic elements involved in the generation of genetic diversity. Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Streptococcus mutans strains UA159 and GS-5(S) revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, three specific probes are designed. S. mutans GS-5 have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons. In addition, comparative analysis of chromosomal macrorestriction polymorphism of Streptococcus mutans strains UA159 and GS-5(R) revealed the presence of gene insertions, and gbpA gene deletion. To determine what kind of genetic element could be implicated in this rearrangement, specific probes for insertion sequence are designed. Southern blot analysis suggested that ISs may be involved in the gene rearrangement. In this study, we have examined 65 strains of S. mutans by PFGE with restriction enzymes NotI or I-CeuI, which cuts a 26-bp site in the rrl gene that has been shown to be highly conserved in eubacteria. Both the size and the number of NotI and I-CeuI fragments exhibit considerable variation among all tested strains, indicated that S. mutans is genetically heterogeneous even within strains belonging to the same serotype c group. Based on the PFGE band patterns of I-CeuI digested chromosomal DNA, four pulsotypes were found, with different fragment of DNA insertion or deletions, when compared with UA159 strain. Strains exhibit large insertions in the first pulsotype (20%), smaller insertions in the second pulsotype (29.2%), large deletions in the third pulsotype (1.5%), and smaller deletions in the fourth pulsotype (18.5%). The percentage of small deletion was identified at a higher frequency in isolates from blood than in oral cavity from either healthy or patients with dental caries (37.5 Vs 8 and 20 %; respectively). In addition, clinical isolates, regardless of their origin, exhibited higher frequency of insertions (large or smaller) than the laboratory strains (53.4% Vs 14.3 %). Based on these data, we concluded that S. mutans is genetically heterogeneous with a high frequency of large chromosomal inversion (41.4%) and different length of insertion and/or deletions scattering beside the putative chromosomal replication origin.