475

organisms by at least 3 bases. ’ro amplify as rDNA from the WABO, a 16-base possible specifically oligonucleotide complementary to this site was used as a PCR primer, and a primer directed at the eubacterial kingdom was also used. The more WABO-specific PCR was done with serial tenfold dilutions of DNA from the biopsy specimen, starting with a 375th sequences of related as

part of the total DNA. In the PCR done with

eubacterial-specific primer, the much product of the appropriate patient’s specimen yielded molecular size (ie, about 721 bases long). (The sequence information has been submitted to Genbank and is available from the corresponding author.) The reagent-control reactions did not generate a PCR product, indicating that there was no laboratory contamination. The organism detected in our patient belonged to the phylogenetic branch of gram-positive bacteria that possess a high G-C content,1 and was most closely related to Rhodococcus (91 % identical), Streptomyces (89%), and Arthrobacter (90%) species. It was less related (84-86% identical) to Mycobacterium, Corynebacterium, Actinomyces, and Propionibacterium species. We also compared the 16 S rRNA sequence of the WABO with the sequences of intracellular pathogens of the Mycoplasma, Rickettsia, Chlamydia, and Rochalimaea species, and, because attempts to culture the Whipple’s bacillus have sometimes yielded streptococci, we aligned the WABO sequence with those of two streptococci. None of these genera were similar to the bacterium detected in the patient with Whipple’s disease. As little as 2-5 x 10" of the DNA purified from the biopsy specimen consistently yielded a PCR band of the appropriate molecular size in the PCR reaction designed to be specific for the WABO. Although the proximal small bowel is not sterile, it is unlikely that the sequence we obtained was from anything other than the bacilli seen on electron microscopy. The WABO sequence could be detected by PCR in as small a fraction as 2-5 x 10-e of the DNA from the biopsy specimen. Assuming a typical copy number of five for the rRNA gene, the results would indicate that the biopsy contained at least around 107 cells of the organism. This exceeds by many orders of magnitude the number of bacteria associated with the mucosa of human proximal small bowel. Comparison of the sequence of WABO with other 16 S rRNA sequences indicated that the organism belongs to a genus not represented in the Genbank or EMBL databases. This result is not surprising. Similar approaches to studies of natural environments indicate that most bacteria may belong to genera that have not been cultured or described previously.3 It is possible that, despite the fact that human flora and pathogens have been intensively studied for many years, not all relevant genera have been found. Furthermore, even if only known bacteria are considered, there are important gaps in the 16 S rRNA sequence collections of Genbank and EMBL. The sequence data indicate that the organism associated with Whipple’s disease in our patient was most closely related to Rhodococcus, Arthrobacter, and Streptomyces species. These genera are predominantly soil organisms, although some Rhodococcus species are known for their

pathogenecity.

R

equi typically

causes a

granulomatous

reaction and is found within macrophages at the site of infection.4 Thus, the relation between the organism studied here, Rhodococcus species, and mycobacteria is consistent with the role of the Whipple’s bacillus as an intracellular pathogen. Knowledge of the phylogenetic placement of the WABO may aid in developing methods to cultivate this

organism.

We thank David Stahl for available to us before its release This work

was

the sequence of Rhodococcus Genbank.

making on

equi

supported by the US Department of Veterans Affairs. REFERENCES

1. Woese CR. Bacterial evolution. Microbiol Rev 1987; 51: 221-71. 2. Wilson KH, Blitchington R, Greene RC. Amplification of bacterial 16 S rRNA sequences with polymerase chain reaction. J Clin Microbiol 1990; 28: 1842-46. 3. Ward DM, Weller R, Bateson MM. 16 S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature

1990; 345: 63-65. 4. Van Etta LL, Filice RM, Ferguson RM, Gerding DN. Corynebacterium equi: a review of 12 cases of human infection. Rev Infect Dis 1983; 5: 1012-17.

ADDRESSES Division of Infectious Diseases (K. H. Wilson, MD, R Frothingham, MD) and Division of Gastroenterology (J. A. P Wilson, MD), Duke University Medical Center, Durham, North Carolina, USA; and Durham Veterans Affairs Medical Center, Durham, North Carolina (K. H. Wilson, R. Blitchington, BS, R. Frothingham). Correspondence to Dr Kenneth H. Wilson, Infectious Diseases Section, VA Medical Center, 508 Fulton St, Durham, NC 27705, USA.

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