Diane R. Stothard

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1‐T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5‐T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.

Evolutionary analysis of the spotted fever and thyphus groups of Rickettsia using 16S rRNA gene sequences

Summary The bacterial genus Rickettsia is traditionally divided into three biotypes, the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) based on vector host and antigenic cross-reactivity. DNA sequence data were gathered from the 16S ribosomal RNA gene of several SFG and TG species. Comparative sequence analysis shows that: i) all species of Rickettsia are closely related, exhibiting 0.3–2.6% sequence divergence; ii) although there are identifiable clusters corresponding to the SFG and TG, species of Rickettsia fall into more than two distinct phylogenetic groups; iii) the tick-borne species Rickettsia bellii and Rickettsia canada diverged prior to the schism between the spotted fever and typhus groups; iv) the newly described AB bacterium is clearly a member of Rickettsia , but its phylogenetic placement within the genus is problematic; v) the mite-borne Rickettsia akari , the tick-borne Rickettsia australis and the recently described flea-borne ELB agent form a loose cluster that cannot be definitively associated with either the TG or the traditional SFG cluster. This latter Glade may represent a unique group(s) distinct from the main cluster of spotted fever and typhus group species. The divergence of Rickettsia was an ancient event within the α-subclass of the proteobacteria. The sequence divergence between Rickettsia and Ehrlichia , the closest known genus to Rickettsia , is nearly equal to the sequence divergence between Rickettsia and all other α-subclass proteobacterial taxa included in the analysis. When Rickettsia was compared to a representative group of the α-subclass, twenty-eight nucleotide sites were identified which uniquely characterize the 16S rRNA sequences of all species of Rickettsia . The approximate time of divergence between the various species of Rickettsia , estimated from the bacterial 16S rRNA molecular clock, coincides with the approximate divergence time of the hard body ticks which are the arthropod hosts of many Rickettsia . Thus, the possibility of coevolution between these intracellular bacteria and their tick hosts exists.

Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus Rickettsia

The eubacterial genus Rickettsia belongs to the alpha subgroup of the phylum Proteobacteria. This genus is usually divided into three biotypes on the basis of vector host and antigenic cross-reactivity characteristics. However, the species Rickettsia bellii does not fit into this classification scheme; this organism has characteristics common to both the spotted fever group and the typhus group biotypes and also exhibits some unique features. Sequences of the 16S rRNA and 23S rRNA genes from Rickettsia rickettsii (spotted fever group), Rickettsia prowazekii (typhus group), and R. bellii were studied to determine the position of R. bellii in the rickettsial classification scheme. The 23S rRNA gene sequences described in this paper are the first 23S rRNA sequences reported for any member of the Rickettsiaceae. The 23S rRNA gene contains substantially more phylogenetic information than is contained in the 16S rRNA sequences, and the 23S rRNA gene sequence has diverged about 1.9 times faster in the three Rickettsia species which we studied. Taken together, the molecular data obtained from the two genes indicate that R. bellii is not a member of either the spotted fever group or the typhus group; rather, this organism appears to be the product of a divergence which predates the separation of the genus into the spotted fever group and the typhus group. Consequently, different combinations of the ancestral characteristics retained by R. bellii have been retained in the more derived lineages of the genus.(ABSTRACT TRUNCATED AT 250 WORDS)

16S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus Ehrlichia

Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, > 95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

Use of a reverse dot blot procedure to identify the presence of multiple serovars in Chlamydia trachomatis urogenital infection.

ABSTRACT Epidemiologic research requires identification of Chlamydia trachomatis serovars and detection of mixed infection. Antibody-based serotyping is unworkable when specimens are urine or vaginal swabs. We developed a reverse dot blot (RDB) to screen for multiple serotypes in these specimens. RDB yielded the predicted results on all artificially mixed samples and on seven of eight clinically mixed samples.