Correspondence
Isolation of Rochalimaea Species
N Engl J Med 1993; 328:1422-1423May 13, 1993
- Article
To the Editor:
The in vitro cultivation of rochalimaea species from lesions of bacillary angiomatosis by Koehler and colleagues (Dec. 3 issue)1 is an exciting development in the efforts to understand the etiologic process and pathogenesis of bacillary angiomatosis. The 16S ribosomal RNA (rRNA) gene sequence previously amplified directly from diseased tissues with a culture-independent approach2 is 99.8 percent similar3 to that of the cultivated Rochalimaea henselae,4 strengthening the evidence that the latter is an etiologic agent of bacillary angiomatosis. The isolation and identification of R. quintana by Koehler et al. and by previous investigators4 also implicate this organism in the causation of bacillary angiomatosis and raise a number of interesting questions.
R. henselae and R. quintana can be distinguished by their 16S rRNA gene sequences (approximately 25 differences at 1462 nucleotide positions3), restriction-fragment-length polymorphisms (RFLPs) of the citrate synthase gene, chromosomal DNA-DNA hybridization, and cellular fatty-acid profiles4. My colleagues and I were reluctant to suggest the presence of R. quintana in one of our original tissue samples (from Patient 3 in our study) on the basis of a 241-base 16S rRNA gene sequence alone, given the probability of the microheterogeneity of the 16S rRNA sequence associated with formalin-fixed tissues2. In fact, one of the two cloned partial 16S rDNA products amplified from the formalin-fixed tissue of our Patient 4 differed from the BA-TF (R. henselae) and R. quintana sequences at two nucleotide positions. Might this have represented a third species or strain variant? In contrast, the full 16S rRNA sequence and citrate synthase RFLP data of Koehler et al. on isolates from their Patients 1 and 2 more strongly support the identification of R. quintana.
Even 16S rRNA sequence identity between two organisms may be an insufficient basis for their assignment to the same species5. Therefore, it is possible that the isolates identified as R. quintana by Koehler and colleagues and the R. quintana isolates from the American Type Culture Collection (VR358) and from persons with trench fever during World War I are genotypically dissimilar. The association of only some isolates with angiogenesis might reflect these bacterial genotypic differences -- perhaps because of the horizontal transmission of virulence-associated genes -- as well as host-specific differences. (It is also possible that R. quintana requires a cofactor to cause bacillary angiomatosis.) To sort out these issues we shall need to study further cases, expand current 16S rRNA sequence data bases, and try to correlate 16S rRNA sequence data with microbial virulence-associated characteristics. The findings of Koehler et al. emphasize the importance of determining full 16S rRNA sequences and the usefulness of sequencing multiple amplification products from the same tissue sample. Polymicrobial disease processes may be more common than we assume.
5 ReferencesDavid A. Relman, M.D.
Stanford University, Stanford, CA 943051
Koehler JE ,Quinn FD ,Berger TG ,LeBoit PE ,Tappero JW . Isolation of rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis. N Engl J Med 1992;327:1625-1631
Full Text | Web of Science | Medline2
Relman DA ,Loutit JS ,Schmidt TM ,Falkow S ,Tompkins LS . The agent of bacillary angiomatosis -- an approach to the identification of uncultured pathogens. N Engl J Med 1990;323:1573-1580
Full Text | Web of Science | Medline3
Relman DA ,Lepp PW ,Sadler KN ,Schmidt TM . Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria. Mol Microbiol 1992;6:1801-1807
CrossRef | Web of Science | Medline4
Welch DF ,Pickett DA ,Slater LN ,Steigerwalt AG ,Brenner DJ . Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:275-280
Web of Science | Medline5
Fox GE ,Wisotzkey JD ,Jurtshuk P Jr . How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 1992;42:166-170
CrossRef | Medline
Author/Editor Response
The authors reply:
To the Editor: Dr. Relman has illuminated several important points regarding the identity of the rochalimaea species isolated from cutaneous and osseous lesions of bacillary angiomatosis. As reported in our paper, we sequenced the entire 16S ribosomal DNA (rDNA) of the R. quintana isolates from multiple clones of several isolates from Patients 1 and 2, and subsequently also performed DNA-DNA hybridization studies. We believed that this was necessary because the identification of partial 16S rDNA sequences could be misleading in closely related species, and we were reporting the association of a second rochalimaea species with bacillary angiomatosis. Although the complete DNA sequence of a bacterial genome would provide the ideal basis of phylogeny and thus taxonomy, this information is currently unavailable, and the gold standard for defining a species remains DNA-DNA hybridization1. A species is currently defined as encompassing strains with 70 percent or greater DNA-DNA relatedness1; the isolates from all three of our patients with R. quintana infection met this criterion and, in fact, were 99 to 100 percent related to the trench-fever isolate of R. quintana (American Type Culture Collection VR358). This confirms the speciation we determined from 16S rDNA sequencing and analysis of the RFLPs of the citrate synthase gene and demonstrates that the isolates of R. quintana causing trench fever and bacillary angiomatosis are indeed the same species.
Pathogenic bacteria often have nonpathogenic counterparts within the same genus and species, with pathogenicity conferred by the transfer of extrachromosomal genetic material or differential expression of one or several chromosomal genes. These pathogenic characteristics do not constitute the basis for designating a genus or species. Regardless, virtually nothing is known about how R. quintana causes the unusual angiogenic response in some persons and only bacteremia (trench fever) in others; certainly, host factors may play a major part. R. henselae also appears to cause two different syndromes: vascular proliferative lesions in immunocompromised patients with bacillary angiomatosis and isolated bacteremia in immunocompetent and immunocompromised patients2.
As Fox et al.3 have reported in one case, the 16S rDNA sequence may not be sufficiently distinct to distinguish closely related species. We agree that future isolates must be studied carefully and that additional species of rochalimaea should be sought assiduously. We, too, emphasize the importance of carefully examining partial 16S rDNA sequences that deviate from known sequences, by sequencing additional clones in duplicate. The isolates with sequence differences that cannot be resolved should then be subjected to more rigorous testing. Speciation can then be done with a high degree of certainty by sequencing the entire 16S rDNA, and with total certainty by performing DNA-DNA hybridization. Either method may be appropriate, depending on the expertise within a particular laboratory, and with the caveat that in rare instances, the 16S rDNA sequence may not provide the definitive answer.
3 ReferencesJane E. Koehler, M.D.
University of California, San Francisco, San Francisco, CA 94143Donald J. Brenner, Ph.D.
Centers for Disease Control and Prevention, Atlanta, GA 303331
Wayne LG ,Brenner DJ ,Colwell RR , et al. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 1987;37:463-464
CrossRef2
Welch DF ,Pickett DA ,Slater LN ,Steigerwalt AG ,Brenner DJ . Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:275-280
Web of Science | Medline3
Fox GE ,Wisotzkey JD ,Jurtshuk P Jr . How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 1992;42:166-170
CrossRef | Medline
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