A rapid DNA extraction procedure and random primer labelling of Escherichia coli ribosomal RNA with cloned reverse transcriptase have been used to establish a simple and highly sensitive method for studying ribosomal DNA polymorphism in bacteria. Examples of inter- and intraspecies differentiation of bacteria are given and potential applications in bacterial epidemiology and taxonomy are discussed.
Ribosomal DNA (rDNA) polymorphism was compared with electrophoretic enzyme polymorphism for the intraand interspecies differentiation of Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. ddovae, Y. frederiksenii and Y. kristensenii DNA from 90 strains previously classified into six zymotypes (Y. enterocolitica and Y. frederiksenii) and into distinct enzyme electrophoretic patterns (the four other species) was digested with EcoRI or Hind111 and analysed by Southern blotting. The six species were clearly differentiated from each other.In Y. enterocolitica, the subclassification of biotype 1 into zymotypes 1A and 1B was also reflected in the rDNA and the four other bio-zymotypes gave four Merent classes of restriction pattern. In Y. frederiksenii, both EcoRI and Hind111 gave five distinct riboclasses which correlated with the zymotypes. In the four other species, the phenotype polymorphism appeared to be better correlated with the restriction fragment length polymorphism data in some enzymes than others. The data demonstrate that the inter-and intraspecies classification by rDNA polymorphism using two restriction enzymes is similar to that based on electrophoretic enzyme polymorphism. The analysis could be refined for taxonomic and epidemiological purposes by using other restriction enzymes.
~~~Eschevichia cofi strains causing human extra-intestinal infections may be divided into two groups, B, and B, according to the electrophoretic patterns of carboxylesterase B. This study compares the restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) for 45 B, strains and 45 B, strains to examine the genetic structure of B, strains and to distinguish them from B, strains. The isolates were chosen for diversity in their allozymes of esterases, B, A, C and I, their production of virulence factors (a-haemolysin, mannose resistant haemagglutinin and cytotoxic necrotizing factor) and certain 0 antigens, and their pathological and geographical origins. DNA was digested with Hind111 and BamHI restriction enzymes and analysed by Southern blotting. The resulting rDNA RFLP patterns of B, strains were distinct from those of the B, strains. Moreover, the B, strains appeared to be less heterogeneous than the B, strains. The B, strains gave 13 ribotypes (resulting from the combination of the rDNA RFLP patterns obtained with Hind111 and BamHI digestions) while the B, strains gave 32 ribotypes. Correspondence analysis of the data showed that several clusters of strains were identified in the B, strains by particular ribotypes, certain associations of esterase B and A electrophoretic variants, 0 serotypes and virulence factor production. In contrast, these parameters appeared to be unrelated in the B, strains, reflecting their heterogeneity. These findings, which differentiate two levels of genetic heterogeneity within E. cofi pathogenic isolates, indicate that the B, strains constitute a phylogenetically distinct group within the species.
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