Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleasesEcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with anEcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (±1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.

Streptococcus pyogenes strains were genotyped by a combination of molecular methods for high- resolution epidemiologic studies of disease outbreaks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the emm gene is reported. Alone or in conjunction with other molecular techniques (16S ribotyping, pulsed-field gel electrophoresis, and detection of exotoxin genes), PCR-RFLP could differentiate outbreak-related strains from contemporaneous background strains of the same M serotype. Three outbreaks were studied: pharyngitis in a boarding school (serotype M5), cross-infection in a hospital burn unit (serotype M76), and severe invasive disease in two elderly care homes (serotype R28). It was possible, for example, to identify within serotype R28 a clone with particular potential for invasive disease. In all cases, the four molecular methods yielded complementary results that were hierarchically related. Strains could be assigned to the outbreak or the background in a precise, reproducible, and rapid manner.

Reproducible methodologies and a scheme for high-resolution genotyping of Streptococcus pyogenes were defined with respect to a study of six predominant M serotypes causing invasive group A streptococcal disease in the United Kingdom. Serotype reference strains were compared with nine clinical isolates of each serotype from patients with diseases such as pneumonia, puerperal sepsis, toxic shock-like-syndrome, cellulitis, or necrotizing fasciitis. Four enzymes were evaluated for their discriminatory power in 16S rRNA gene-specific ribotyping. Discriminatory power was greatest with EcoRI, which generated serotype-specific ribotypes, and with SacI, which could subdivide strains of the same M serotype. Twenty-five combined ribotypes were found among the 60 strains, and the indices of discriminatory power (D values) of this method varied from 0.51 within serotype M1 to 0.98 within strains of serotype M5. Macrorestriction with the rarely cutting endonuclease SmaI and pulsed-field gel electrophoresis gave D values varying from 0.37 within serotype M1 to the maximal 1.0 within serotype M5. Comparison of macrorestriction profiles revealed various degrees of genetic heterogeneity within M serotypes. Strains of M1, M3, M6, and M11 exhibited clonally related macrorestriction profiles, while those of R28 and M5 strains were consistent with polyphyletic origin.