Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions.
The utility of PCR analysis of transfer DNA intergenic spacer length polymorphism (tDNA-ILP) for the identification to the species level of clinically relevant viridans streptococci was evaluated with a collection of reference strains of 15 species of the salivarius, anginosus, mitis and mutans rRNA homology groups. PCR products generated by using fluorescent, outwardly directed, consensus tDNA primers were analysed by electrophoresis on denaturating polyacrylamide gels and by laser fluorescence scanning. Eleven species showed specific and distinct tDNA patterns: Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus anginosus, Streptococcus mutans, Streptococcus criceti and Streptococcus ratti. Indistinguishable patterns were obtained among two groups of species: Sreptococcus vestibularis and Streptococcus salivarius on the one hand and Streptococcus constellatus and Streptococcus intermedius on the other. S. mitis strains produced heterogeneous patterns that could be separated into three groups: a group containing S. mitis biovar 1 and two S. mitis biovar 2 groups, one of which clustered with S. parasanguinis strains while the other showed patterns unrelated to other species. These results agree in part with protein electrophoretic analysis showing that S. mitis biovar 2 strains belong to several streptococcal taxa. In conclusion, PCR analysis of tDNA-ILP holds promise for rapid identification of viridans streptococci that are difficult to identify by phenotypic tests.
AFLP is a genomic fingerprinting technique based on the selective amplification of restriction fragments from a total double-digest of genomic DNA. The applicability of this method to differentiate between species and genomovars of the genus Burkholderia was tested, with particular emphasis on taxa occurring in cystic fibrosis patients. In this study, 78 well-characterized strains and field isolates were investigated by two methods of AFLP fingerprinting. In the manual procedure, a radioactively labelled primer was used, amplified fragments were separated by conventional PAGE and the patterns were revealed by autoradiography. In the automated procedure, a fluorescently labelled primer was used in combination with electrophoresis and on-line data collection by means of an automated DNA sequencer. Overall, there was good agreement between the two AFLP procedures and the data were mostly consistent with results obtained from SDS-PAGE of whole-cell proteins and DNA-DNA hybridization experiments. The automated AFLP procedure has considerable technical advantages compared with the manual AFLP procedure, but a thorough visual analysis of the DNA profiles was required to avoid misidentification of some Burkholderia cepacia genomovar III strains.
The 16S-23S rDNA intergenic spacer region of organisms identical with or closely related to ‘Tropheryma whippelii’, the uncultivated causative agent of Whipple's disease, was analysed directly from 38 clinical specimens of 28 patients using a specific nested PCR followed by direct sequencing. As compared to the reference sequence in public databases, two novel ‘T. whippelii’ spacer types were recognized. In the absence of DNA-DNA hybridization data it is uncertain whether the three types found represent subtypes of a single species or three different but closely related species. Methods were developed to detect all three variants by single-strand conformation polymorphism analysis and by type-specific PCR assays, thus allowing the screening of large numbers of specimens. Further studies may provide a clue to the possible associations between the type of infecting strain and the various clinical presentations of Whipple's disease.
Borrelia duttonii, the cause of East African tick-borne relapsing fever, has until now been refractory to growth in laboratory media. This spirochaete has only be propagated in mice or by tissue culture, restricting both yield and purity of cells available for research. The successful isolation of five clinical isolates of B. duttonii from patients in Central Tanzania and their comparison with Borrelia recurrentis is reported. Electron microscopy revealed spirochaetal cells with pointed ends, a mean wavelength of 1·8 μm with an amplitude of 0·8 μm, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted at each end of the spirochaete, again comparable with the counts of 8-10 flagella found in B. recurrentis. PFGE revealed a chromosome of approximately 1 Mb, a large plasmid of approximately 200 kb, and a small plasmid of 11 kb in all strains of B. duttonii and in B. recurrentis. B. duttonii possessed a further 7-9 plasmids with sizes ranging from 20 to 90 kb. In two isolates of B. duttonii, the profiles were identical. In contrast, all 18 isolates of B. recurrentis fell into one of five plasmid patterns with 3-4 plasmids ranging from 25 to 61·5 kb in addition to those of 11 and 200 kb described above. Analysis of the SDS-PAGE profiles of B. duttonii strains revealed a highmolecular-mass band of 33·4-34·2 kDa in four strains (variable large protein, VLP) and a low-molecular-mass band of 22·3 kDa in the remaining strain (variable small protein, VSP). This resembles the protein profiles found in B. recurrentis. The G+C ratio of B. duttonii was 27·6 mol%. Nucleotide sequence of the rrs gene (16S rRNA) from four B. duttonii isolates revealed 100% identity among these strains and 99·7% homology with three strains deposited by others in GenBank. The rrs gene of eight representative clinical isolates of B. recurrentis confirmed their close similarity with B. duttonii.
Genetic variation among 35 strains representing the four currently recognized species of Saccharomyces sensu stricto (Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianuslcarlsbergensis and Saccharomyces paradoxus) was estimated by analysing the electrophoretic mobilities of nonspecific esterases, acid phosphatase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes. Twenty-two electrophoretic types were identified, a result in agreement with the phenotypic and genetic polymorphisms reported for this group of yeasts. However, the four species were clearly distinguishable based on the patterns obtained using three of the enzymes assayed, the resolving power not being improved by the introduction of data correspondent to lactate dehydrogenase. The overall diversity was higher among S. cerevisiae isolates, in contrast with S. paradoxus which showed only two patterns, one of which was common to four of the five strains studied. Concordant results from the application of the method and DNA hybridization experiments demonstrate its value for identification purposes.