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1.
J Clin Microbiol ; 39(7): 2655-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11427588

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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Chlamydia Infections/microbiology , Chlamydia trachomatis/classification , Female Urogenital Diseases/microbiology , Oligonucleotides/genetics , Polymerase Chain Reaction/methods , Porins , Base Sequence , Chlamydia trachomatis/genetics , Female , Humans , Infant , Molecular Sequence Data , Sensitivity and Specificity , Serotyping
3.
Int J Syst Evol Microbiol ; 51(Pt 2): 339-347, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11321078

ABSTRACT

In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5,513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, polA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed trans-stadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes.


Subject(s)
Boutonneuse Fever/microbiology , Cats/parasitology , Rickettsia/classification , Siphonaptera/microbiology , Animals , Bacterial Outer Membrane Proteins/genetics , Intestinal Mucosa/microbiology , Molecular Sequence Data , Rickettsia/genetics , Rickettsia/ultrastructure , Terminology as Topic
4.
Am J Kidney Dis ; 36(6): 1122-5, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11096035

ABSTRACT

Significant evidence suggests that Chlamydia pneumoniae has a major role in occlusive vascular disease. Vascular access thrombosis in chronic hemodialysis patients is a frequent problem; the underlying pathological state is stenosis caused by endothelial hyperplasia. There is presently no literature concerning C pneumoniae in vascular access thrombosis. We embarked on a study to evaluate the possible role of C pneumoniae in access failure. Ten consecutive patients with thrombosed polytetrafluoroethylene (PTFE) conduit arteriovenous fistulae undergoing surgical thrombectomy and revision were studied. We sought to detect C pneumoniae using both culture and polymerase chain reaction (PCR) methods. An excisional biopsy of the stenotic vein segment just above the anastomosis with the PTFE graft was obtained at surgery. Vein samples weighing at least 30 mg were aseptically placed in transport media and stored at 4 degrees C for up to 24 hours. The samples then were sonicated, inoculated in Hep-2 cell culture vials containing confluent monolayers, and passaged three times over 2 weeks. Detection was by direct fluorescent antibody staining. Both culture and PCR were performed in an active chlamydia research laboratory. None of the 10 samples was positive for C pneumoniae by culture or PCR. Based on our preliminary pilot study, we do not believe C pneumoniae has a major role in endothelial hyperplasia and consequent graft loss in the hemodialysis patients we studied.


Subject(s)
Arteriovenous Shunt, Surgical/adverse effects , Blood Vessel Prosthesis/adverse effects , Catheters, Indwelling/microbiology , Chlamydia Infections/microbiology , Chlamydophila pneumoniae/isolation & purification , Renal Dialysis/adverse effects , Thrombosis/microbiology , Aged , Blood Vessel Prosthesis/microbiology , Chlamydia Infections/etiology , Endothelium, Vascular/microbiology , Endothelium, Vascular/surgery , Female , Fluorescent Antibody Technique, Direct , Humans , Hyperplasia/microbiology , Hyperplasia/surgery , Male , Middle Aged , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction , Polytetrafluoroethylene/adverse effects , Thrombosis/etiology , Thrombosis/surgery
5.
J Clin Microbiol ; 37(8): 2687-93, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10405422

ABSTRACT

The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoeba are described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific for Acanthamoeba as shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba 18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformis and Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosa and Escherichia coli). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba 18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely with Acanthamoeba keratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoeba in 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genus Acanthamoeba in both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.


Subject(s)
Acanthamoeba/genetics , Acanthamoeba/isolation & purification , Genes, rRNA , Oligonucleotide Probes , RNA, Ribosomal, 18S/genetics , Animals , Genes, Protozoan , Humans , RNA, Ribosomal, 18S/analysis
6.
Mol Biol Evol ; 16(7): 987-95, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10406115

ABSTRACT

It has previously been observed that Rickettsia prowazekii has an unusual arrangement of the rRNA genes. In this species, the three rRNA genes, 16S (rrs), 23S (rrl), and 5S (rrf), are not linked in the typical arrangements for bacteria. Rather, the 16S rRNA gene has been separated from the 23S and 5S rRNA gene cluster, and the 23S rRNA gene is preceded by a gene which codes for methionyl-tRNAf(Met) formyltransferase (fmt). In this study, we screened the genus Rickettsia for the fmt-rrl motif in order to examine the phylogenetic depth of this unusual rRNA gene organization. A rearranged operon structure was observed in Rickettsia conorii, Rickettsia parkeri, Rickettsia sibirica, Rickettsia rickettsii, Rickettsia amblyomii, Rickettsia montana, Rickettsia rhipicephali, Rickettsia australis, Rickettsia akari, Rickettsia felis, Rickettsia canada, and Rickettsia typhi. There is also evidence for a divided operon in Rickettsia belli, but in this species, the fmt gene could not be identified upstream of the 23S rRNA gene. In order to place the rearrangement event in the evolutionary history of the Rickettsia, phylogenetic analyses were performed based on the fmt-rrl spacer regions and the 23S rRNA genes. Based on these phylogenies, we suggest that the genomic rearrangement of the rRNA genes preceded the divergence of the typhus group and the spotted fever group Rickettsia. The unique organization of the 23S rRNA genes provides a simple diagnostic tool for identification of Rickettsia species.


Subject(s)
Gene Rearrangement , Phylogeny , RNA, Ribosomal, 23S/genetics , Rickettsia/genetics , Base Sequence , DNA Primers , Gene Deletion , Hydroxymethyl and Formyl Transferases/genetics , Mutation , Rickettsia/classification , Species Specificity
7.
J Clin Microbiol ; 36(12): 3686-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9817897

ABSTRACT

Previous epidemiological studies of Chlamydia trachomatis frequently have required expansion of isolates in tissue culture. The possibility that C. trachomatis omp1 might undergo mutation during such expansion has not been examined systematically. We found no differences in the omp1 sequences from 10 clinical specimens before and after 20 in vitro passages.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Chlamydia trachomatis/genetics , Porins , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Base Sequence , Culture Techniques , Humans , Molecular Sequence Data , Mutation
8.
Infect Immun ; 66(12): 6010-3, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9826386

ABSTRACT

Our laboratory recently recovered Chlamydia trachomatis in tissue culture from a urogenital specimen which tested negative by commercial plasmid-based PCR. Immunotyping and omp1 sequencing identified the isolate as a serovar E isolate. Further investigation by PCR and Southern hybridization indicated that this isolate lacks the chlamydial cryptic plasmid.


Subject(s)
Chlamydia Infections/diagnosis , Chlamydia trachomatis/isolation & purification , Male Urogenital Diseases/diagnosis , Plasmids , Adult , Bacterial Outer Membrane Proteins , Bacterial Typing Techniques , Chlamydia trachomatis/classification , Chlamydia trachomatis/genetics , False Negative Reactions , Humans , Male , Polymerase Chain Reaction , Reagent Kits, Diagnostic
9.
Infect Immun ; 66(8): 3618-25, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9673241

ABSTRACT

Phylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains of Chlamydia trachomatis. Nucleotide sequences of omp1, the gene encoding the major outer membrane protein (MOMP) of C. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with available omp1 sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Chlamydia Infections/microbiology , Chlamydia trachomatis/genetics , Genes, Bacterial , Porins , Bacterial Outer Membrane Proteins/classification , Bacterial Outer Membrane Proteins/physiology , Base Sequence , Binding Sites , Chlamydia trachomatis/classification , Chlamydia trachomatis/pathogenicity , DNA, Bacterial , Humans , Molecular Sequence Data , Phylogeny , Sequence Analysis
10.
J Eukaryot Microbiol ; 45(1): 45-54, 1998.
Article in English | MEDLINE | ID: mdl-9495032

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.


Subject(s)
Acanthamoeba/classification , Evolution, Molecular , RNA, Ribosomal, 18S/genetics , Acanthamoeba/genetics , Alleles , Animals , Base Sequence , Genes, Protozoan , Genetic Heterogeneity , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 18S/classification , Sequence Alignment/methods , Sequence Homology, Nucleic Acid
11.
Int J Syst Bacteriol ; 45(2): 207-11, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7537055

ABSTRACT

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.


Subject(s)
Ehrlichia/classification , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Rickettsiaceae/classification , Animals , Base Sequence , DNA, Bacterial , Dogs , Ehrlichia/genetics , Molecular Sequence Data , Phylogeny , Rickettsiaceae/genetics , Salmon/microbiology
12.
Int J Syst Bacteriol ; 44(4): 798-804, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7981106

ABSTRACT

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)


Subject(s)
Rickettsia prowazekii/classification , Rickettsia rickettsii/classification , Rickettsia/classification , Base Sequence , DNA, Ribosomal/chemistry , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Rickettsia/genetics
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