The Identification of Uncultured Microbial Pathogens

The Identification of Uncultured Microbial Pathogens David A. Reiman

Departments ofMedicine and ofMicrobiology and Immunology, Stanford University School of Medicine, Stanford. and Palo Alto VA Medical Center, Palo Alto. California

Awareness of microbial pathogens has depended heavily on their cultivation or propagation in the laboratory. Even the development ofdiagnostic assays based on antigen detection or serology requires specific antisera, purified microbial cells, or components thereof. Years of clinical observation and tissue examination suggest, however, that some diseases may be caused by microorganisms that fail to be characterized by these traditional methods. Whipple's disease is one example of human pathology associated with a visible but uncultured and unidentified bacillus. Furthermore, there may be grounds for postulating a microbial etiology for a number of human diseases without even visible evidence ofa microorganism, due to the insensitivity of visual detection methods. These circumstances have suggested the need for a culture-independent approach for the identification and detection of microbial pathogens. The following discussion describes the development of one such approach that is rooted in the field of molecular phylogenetics and evolution. Whipple's disease provides a useful example of its application.

Whipple's Disease: A Disorder Associated with a Visible but Uncultured Bacillus The features of Whipple's disease reflect a constellation of clinical and histologic findings first elucidated in 1907 [1] and later clarified with larger case studies and improved mi-

Received 9 March 1993. Presented in part: annual meeting of the Infectious Diseases Society of America, October 1992, Anaheim, California. Grant support: Lucille P. Markey Charitable Trust (D.R. is a Lucille P. Markey Scholar) and Director's Research Fund, sponsored by SmithKline Beecham. Reprints or correspondence: Dr. David A. Reiman, Palo Alto VA Medical Center 154T, 3801 Miranda Ave., Palo Alto, CA 94304. The Journal of Infectious Diseases 1993;168:1-8 © 1993 by The University of Chicago. All rights reserved. 0022-1899/93/6801-000 I$0 1.00

crographic and tissue-staining techniques [2, 3]. Migratory arthralgias, fever, abdominal pain, diarrhea, and weight loss remain among the most common signs and symptoms of this disease. George Whipple's initial observations and those of subsequent pathologists suggest primary involvement of the small intestine and mesenteric lymph nodes with a prominent mononuclear cell (macrophage) infiltrate and fat deposition. The characteristic vacuoles within these macrophages and their dramatic reaction with periodic acid-Schiff (PAS) stain provide a nearly pathognomonic diagnostic clue to this disease, especially when seen within the duodenum and upper small intestine [4]. Whipple's disease is a systemic disorder and can be manifest by syndromes involving the central nervous system, heart, lungs, peripheral lymphatics, and joints as well as by fever of unknown origin. Pathology similar to that seen within the small intestine has been reported in all of these extraintestinal sites; however, the PAS stain is nonspecific and PAS-positive, vacuolated macrophages in these other sites may suggest other diseases [2]. In the early 1960s, several groups of investigators published clear evidence of bacillary structures within the duodenallamina propria and other affected tissues from patients with Whipple's disease [5-7]. In fact, Whipple described what he thought were "rod-shaped organisms" in a mesenteric lymph node from his original case [l]. We now recognize that these are extra- and intracellular bacilli whose unusual walls react with PAS reagent. Accumulations of partially degraded bacilli may explain the PAS staining properties of the macrophage vacuoles [8, 9]. Questions about the nature of this bacillus have been frustrated by numerous unsuccessful efforts to cultivate the organism on laboratory media or in an animal or to purify the organism. Because traditional methods for identifying microorganisms rely on their cultivation or purification, the identity of the Whipple bacillus, until recently, has remained unknown. The situation posed by Whipple's disease may be generalized [10]. Visible but uncultured microorganisms have been

Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on May 4, 2014

Clinicians have long been aware of human diseases that are associated with visible but uncultured microorganisms. Without the ability to cultivate these organisms, they have remained unidentified. Environmental microbiologists have also discovered on the basis of recent advances in the field of molecular phylogeny that culture-based methods for detecting microorganisms are biased and insensitive. A culture-independent experimental approach is described for the identification of microbial pathogens. This approach incorporates fundamental aspects of 168 rRNAbased molecular phylogeny as well as nucleic acid amplification technology. From its application to Whipple's disease, one can speculate as to the potential insights a highly sensitive, cultureindependent method may provide into the diversity and natural ecology of human microbial pathogens.

2

Reiman

associated with a number of human diseases. For example, until 1990 the silver-staining bacilli seen within the lesions of bacillary angiomatosis had not been propagated and, as a result, had not been identified [11]. Furthermore, an infectious etiology has been postulated for a number of syndromes without visual evidence of a microorganism. How might one develop an approach for the identification of microbial pathogens that does not rely on the traditional requirement for cultivation?

110 1993;168 (July)

Ribosomal rRNA genes present in all living cells Function of encoded molecule is highly conserved Accumulates mutations at a slow, consistent rate (Evolutionary distance between two organisms can be inferred from number of rRNA sequence differences between them) Contains: regions of highly conserved sequence (useful for design of broad range PCR and sequencing primers)

*

Ribosomal RNA as a Molecular Clock *~

regions of highly variable sequence (useful for design of specific primers and probes and for phylogenetic analysis)

Figure 1. Reasons why rRNA sequence is useful for determining evolutionary relationships. peR, polymerase chain reaction.

sequence and secondary structure. With the 16S rRNA sequence of a previously uncharacterized microorganism in hand, it is a straightforward procedure to determine its evolutionary relationships to all other characterized organisms and place it within a phylogenetic tree. In the initial years of rRNA-based phylogenetic analysis, rRNA sequence was obtained from RNase T 1 oligonucleotide catalogs of bulk rRNA and, in later years, from reverse transcriptase sequencing of this RNA [18]. More recently, investigators have used the polymerase chain reaction (PCR) to amplify 16S rRNA gene sequences from purified bacterial cultures, even in the presence of human DNA [19-22]. The key has been to recognize the presence of 16S rRNA sequences conserved within only the domain Bacteria, from which "broad-range" primers can be designed [18]. To set up a PCR, the only prerequisite information about the target is some knowledge about the nucleotide sequences that flank each end of the target. From these sequences, the short oligonucleotides (primers) that catalyze the copying of the target by the DNA polymerase are designed [23, 24]. Nothing need be known about the target sequence itself. Thus, divergent and diagnostic regions of the 16S rRNA gene can be amplified by selecting conserved areas of the gene as flanking sequences (figure 4). The resultant amplified DNA contains internal regions ofvariable sequence that form the basis for specific phylogenetic analysis. In general, consensus primers can be designed for any discrete group of organisms with common ancestry; for example, genus-specific primers have been used to identify Mycobacterium species from culture material [26]. Thus, the features of the 16S rRNA molecule provide the basis for an approach to the identification of an uncultured bacterial pathogen.

An Approach to the Identification of Uncultured Microbial Pathogens The task of identifying or characterizing an uncultured, unpurified bacterial pathogen using infected human tissue is


In general terms, one would be interested in any molecular feature with the following two characteristics: The feature should be highly conserved, so that one might reasonably assume it to occur in a previously uncharacterized microorganism. On the other hand, this feature should provide highly specific information about the nature of this or any organism. In an effort to establish a more reliable means of determining evolutionary relationships among species, a new field emerged during the late I 960s and 1970s, known as molecular phylogeny [12-14]. This field was based on the realization that certain widely shared genetic sequences could be viewed as "molecular clocks." The analysis of these sequences proved to be more reliable than phenotypic analysis and led to the increasing reliance on this kind ofapproach for evolutionary and taxonomic investigations. To understand how the principles of molecular phylogeny have led to the identification of previously uncharacterized microbial pathogens, one must first appreciate some of the features ofa molecular clock. Since all genetic sequences accumulate mutations over time, one can view evolutionary distance as proportional to the number of nucleotide differences among two copies of the same gene. To be able to establish evolutionary relationships among distantly related microorganisms, a molecular clock must accumulate random mutations at a slow rate and in a reliable manner [14]. Genetic sequences are most useful as molecular chronometers when they encode molecules with a highly conserved and essential biologic function. At present, the most widely used molecular clock is the small subunit (16S or 16S-like) rRNA (figure 1). This molecule, together with the large subunit rRNA, folds in a precise fashion to form ribosomes. These are highly conserved structures, found in all living cells, that perform the crucial task of protein synthesis. The constrained structure and function of ribosomes ensure reliable clock-like behavior on the part of rRNA sequence. On the basis of small subunit rRNA (and rRNA gene, rONA) sequence comparisons, the evolutionary relationships among all known extant species can be represented in a universal phylogenetic tree (figure 2) [14]. Large sequence differences separate the three domains-Archaea, Eukarya, and Bacteria, the last of which contains all known human prokaryotic pathogens (figure 3) [15, 17]. Each domain can be defined by certain regions of conserved rRNA

JID 1993;168 (July)

Identification of Uncultured Microorganisms

Bacteria Figure 2. Universal phylogenetic tree based on comparison of small subunit rRNA sequences. All known extant species fall into three domains: Bacteria, Archaea, and Eucarya. Most gram-positive bacteria cluster within one bacterial division (by that name). Modified from Woese et al. [15], Woese [14], and Turner et al. [16].

green nonsullur bacteria

3

Archaea Eucarya

Methanomicrobiales animalsciliates Methanobacteriales green plants Methanococcales . Thermoproteus fungi flagellates Thennotogales

micros poridia

tissue digestion material, broad-range bacterial 16S rRNA PCR primers catalyze the amplification of the 16S rRNA gene from the released pathogen chromosomal DNA. These primers will not anneal to and amplify small subunit rRNA genes from members of other domains and, in particular, the human rRNA genes. Control tissue samples are processed in parallel with the experimental samples so that they may reveal problems with environmental 16S rONA cross-contamination and indicate the specificity of sequences associated with the experimental tissues. The DNA sequence of the amplified products may be determined directly or by first cloning the products in a recombinant plasmid vector. Direct sequencing generates a consensus sequence more readily and is less labor-intensive; however, the process of sequencing cloned products reveals sequence heterogeneity, which may reflect the presence of multiple strains or species. With either approach, the new consensus sequence is then aligned to known bacterial 16S rRNA secondary structures and analyzed for relatedness to other 16S rRNA sequences in current data bases [13,3134]. PCR primers, designed from regions of hypervariable 16S rRNA sequence may help to confirm the specific associ a-

green sulfur bacteroidesdeinoeocci spirochetes bacteria fIavobacteria and relatives

Figure 3. Phylogenetic tree of domain Bacteria. All currently characterized human prokaryotic pathogens belong to this domain, and majority belong to Gram-positive or Proteobacteria divisions. Modified from Woese [14].

cyanobacteria

PROTEOBACTERIA


essentially that of obtaining the 16S rRNA sequence from this organism. Without the means for in vitro cultivation or purification of the bacterium, one must resort to an alternative strategy to acquire enough material for DNA or RNA sequence determination. As suggested in the previous discussion, PCR is an attractive method. One can design PCR primers from sequences that are known to occur in all previously studied bacterial 16S rRNA genes, with the expectation that these primers will find complementary targets within the 16S rRNA gene ofany previously uncharacterized member of the bacterial domain. One starts with infected host tissue for this type of investigation (figure 5) [10, 27]. Proteinase K and nonionic detergent are used to disrupt tissue stroma and lyse host and pathogen cells, releasing a mixture of nucleic acids [28]. The tissue anatomic site of origin and the type of original tissue preservation are crucial. Cutaneous or mucosal sites are often contaminated with host commensal bacterial flora, any of which may serve as the source of 16S rRNA gene targets for PCR amplification. Fresh-frozen samples are preferable to formalin-fixed tissues because DNA is better preserved by the former process [29, 30]. In reactions containing crude

Reiman

4

JIO 1993;168 (July)

conserved regions divergent regions 200

600

400

800

1000

1200

1400

~

~

1542

~

8FPL

1492RPL

~

~

8FPL

S06R ~

~

515FPL

~

515FPL

138

...

~

91E

806R

~

............ 138

11E

Application of This Approach to Bacillary Angiomatosis Bacillary angiomatosis (BA) is an angioproliferative disorder involving the skin and numerous internal organs, usually of immunocompromised persons [11, 35]. The consistent response of patients to antibiotics and the finding of clumps of pleomorphic bacilli in affected tissues have strongly suggested a bacterial etiology. However, prior to 1990 these bacilli could not be cultivated in the laboratory, and their identity remained unknown. The availability of fresh-frozen tissues from an immunosuppressed patient with disseminated visceral BA motivated the development of this PCR-amplified, 16S rRNA-based approach and led to the identification of the BA causative agent [36]. Phylogenetic analysis demonstrated this was an o-proteobacterium (later named Rochalimaea henselae) [36a, 36b] and was most closely related to Rochalimaea quintana, the cause of trench fever.

Digest infected tissue, releasing total DNA Amplify 165 ribosJmal RNA (rRNAl genes of pathogen with polymerase chain reaction and "broad-range" primers

+

...----- ----....

Determine sequence of amp I"f" I Ied gene From variable regions design Is this rRNA sequence pathogen-specific pcR' primers - - . . specifically associated with disease in question? and oligonucleotide probes

~ Determine evolutionary relationships of pathogen to other known organisms based on 165 rRNA sequence analysis

Figure 5. Polymerase chain reaction (PCR)-amplified, 16S rRNA-based approach to identification of uncultured pathogens.

138

Several aspects of the BA investigation bear further mention because they illustrate potential benefits of this experimental approach. Specific 16S rRNA PCR primers can be extremely useful reagents for detecting a single or a group of related organisms in contaminated tissue samples. Rochalimaea-specific primers have helped to incriminate R. henselae in bacillary peliosis, in a syndrome of persistent or relapsing fever, and in some cases of classic cat-scratch disease. Early observations of 16S rRNA sequence microheterogeneity within BA tissues [36] may now be explained in part by the isolation and molecular detection of R. quintana from some cases of BA [37]. Sequence microheterogeneity has emphasized the possibility of multiple microbial etiologies for a given syndrome. Finally, the phylogenetic analysis ofR. henselae and R. quintana has revealed a particularly close evolutionary relationship between these organisms and Bartonella bacilliform is, the only other known bacterial cause of human angioproliferative tissue pathology [38]. This would lead one to speculate that these organisms share a similar mechanism for inducing this type of host response [11, 39].

Evolutionary Relationships of the Whipple's Disease Bacillus The identification of the uncultured bacillus of Whipple's disease has followed from the same PCR-amplified, 16S rRNA-based experimental approach described above. A complete 16S rRNA gene sequence was amplified from duodenal mucosal biopsy tissue using a variety of broad-range bacterial primer pairs with overlapping 16S rRNA gene targets [40]. Experiments using more specific PCR primers confirmed that this novel sequence was found in all 5 Whipple's disease patients and in none of 14 control tissues. The latter included small intestinal mucosal samples and gastric mucosa from a patient with gastritis. (Helicobacter pylori 16S rONA sequence was detected in this latter tissue.) The novel Whipple's disease-associated sequence was nearly identical to a partial 16S rRNA sequence identified by Wilson et al. [41] in a patient with Whipple's disease.


tion of the new sequence with only tissues involved by the disease in question. By constructing a phylogenetic tree using the specific disease-associated 16S rRNA sequence, the evolutionary relationships of the pathogen are deduced.

"

Figure 4. Schematic diagram of bacterial 16S rRNA gene. Broad-range primers are indicated by arrows; dotted lines refer to parts of gene that are amplified by various pairs of these primers. Broadrange primer sequences are highly conserved within domain Bacteria. Approximate 16S rDNA nucleotide positions are labeled (Escherichia coli numbering). Modified from Reiman [25].

JID 1993;168 (July)


The Whipple's disease bacillus is a previously uncharacterized actinomycete on the basis of analysis of the diseaseassociated 16S rRNA sequence [40, 41]. Many bacteria that belong within this group are soil or water saprophytes. Some are skin or oral commensal organisms found in warmblooded animals and are occasional causes of disease. Although the Whipple's disease bacillus appears to share common ancestry with the actinobacteria (Dennatophilus. Arthrobacter. Terrabacter species), it is not closely related to any other known genus [40]. The name Tropherynia whippeIii has been proposed for the Whipple's disease bacillus [40].

Infections of Insects: Identifying Uncultured Bacterial Endosymbionts

from cat fleas after observing a rickettsia-like organism within these arthropods that was resistant to cultivation. Polymorphisms within these sequences have suggested the presence of a previously unknown organism related to R. typhi. This organism has been detected within flea populations collected from regions in Los Angeles County where there have been documented cases of human murine typhus [45]. It is unclear whether this organism might be responsible for a mild, typhus-like illness in the local human population.

The Diversity of Environmental Microbial Communities Environmental microbiologists have postulated that the majority of extant microorganisms remain unidentified because of the insensitivity of culture-based methods. A series of studies published during the past 3 years has confirmed this [46-50]. All of these studies relied on cloned or amplified 16S rRNA molecules using broad-range oligonucleotides. From a variety of natural habitats, including Atlantic and Pacific Ocean water, Yellowstone National Park hot spring, and Australian subsurface soil, cultured organisms constitute only a small fraction « I0%) of the total extant species that are identified by using amplified 16S rRNA sequences. The same general concusions may apply to the human endogenous commensal microbial flora. There is certainly every reason to think that a sizable porportion of the human commensal flora has been ignored by culture methods [10]. One study of selected human subgingival plaque microflora compared the detection sensitivity of anaerobic culture with bacterial genomic DNA probes, ELISA, and immunofluorescence assay [51]. Not surprisingly, laboratory culture was significantly less sensitive than the other techniques. With the substantially greater sensitivity of PCR, one would expect that broad-range 16S rRNA amplification of subgingival plaque will surely reveal microbial species that were previously unknown. These "new" species will probably fall within multiple bacterial divisions. Although there are no known human pathogens within the domain Archaea. a proper search for archaea in humans has probably not yet been conducted.

Whipple's Disease Bacillus as a Pathogen: More than Meets the Eye? Many of the organisms that share a common evolutionary ancestor with the Whipple bacillus are widely prevalent in the environment. If this were true of T. whippelii. one would expect that humans are frequently exposed to this organism. The relatively high proportion of farmers among the Whipple's disease patient population [2] is consistent with acquisition ofinfection through soil exposure. This raises some interesting questions. Might T. whippelii be found in soil DNA samples? Might other clinical syndromes be ascribed to this


An increasing number of microbial endosymbionts have been detected within higher organisms. Often, by nature, an endosymbiotic relationship satisfies highly specific and welldeveloped nutritional requirements for the participants. Many of these requirements remain obscure, precluding in vitro cultivation or further characterization of these microorganisms. Two examples of molecular phylogenetic microbial identification under these circumstances are provided. For a number of years, mating incompatibility among certain insect species has been associated with the presence of a monomorphic rickettsia-like microorganism within insect gonad tissues. In 1936, this organism was first observed and named Wolbachia pipientis on the basis ofmorphology. However, the organism was never cultivated outside of its natural insect hosts, presumably because of unrecognized growth requirements. O'Neill et al. [42] have now characterized a number of these insect endosymbionts by amplification of their 16S rRNA genes directly from homogenized insect ovaries by using broad-range bacterial PCR primers. Analysis of endosymbiont sequences from 7 diverse insect strains and species has revealed a closely related group of a-proteobacteria with a shared recent ancestor that may all be assigned to a single species. This species is most closely related to Anaplasma (persistent, erythrocyte-associated pathogens of various vertebrates), Ehrlichia, and Rickettsia species. The latter organisms are also eukaryotic endosyrnbionts, as well as arthropodborne pathogens of mammals. A survey of other insects with specific 16S rRNA primers suggests that sex ratio distortion is associated with a single bacterial species and is more pervasive than was previously suspected [42]. Other genetic sequences, besides that of the rRNA small subunit, may be used to determine evolutionary relationships among groups of organisms. In most cases, it is only within a relatively restricted group of organisms that these alternative sequences are useful as evolutionary clocks. An example is the citrate synthase gene sequence, which has been proposed as a means for the identification and evolutionary classification of rickettsia-like organisms [43]. Azad et al. [44] have amplified citrate synthase sequences directly

5

Reiman

6

Sarcoidosis Crohn's Disease Malakoplakia Chronic recurrent multifocal osteomyelitis Figure 6. role.

Diseases in which microbial agents may play direct

Other Human Diseases in which Uncultured Microorganisms May Playa Role The possibility of a microbial etiology for a number of chronic inflammatory diseases has been debated for years. In the face of negative microbial cultures, circumstantial data such as visible organisms and a response to antimicrobial therapy strongly incriminate microbial agents in disease causation. BA and Whipple's disease are relatively rare examples of diseases for which these kinds of data exist. Nonetheless, the insensitivity of microbial visualization and the possibility of antibiotic failure with slowly growing or cell wall-deficient pathogens suggest that there may be a variety ofchronic inflammatory diseases with an unsuspected microbial etiology. The diseases listed in figure 6 are offered as examples, without necessarily any significant supportive data. Sarcoidosis has already been mentioned in the context of the Whipple bacillus. Two reports published in 1992 suggested a role for Mycobacterium tuberculosis or other mycobacteria in some cases of sarcoidosis [56, 57]. However, the absence of reliable diagnostic criteria for this disease precludes the exclusion of some cases of tuberculosis, and per-

haps other granulomatous diseases, when there are rare organisms and little necrosis. Sarcoidosis may have multiple etiologies involving microbial cells or parts thereof. Many investigators have speculated on an infectious etiology for Crohri's disease since its first description [58]. In particular, the similarity of this disease to a mycobacteriosis has prompted an intensive search for a Mycobacterium species [59]. Mycobacterium paratuberculosis. an animal pathogen, is the subject of much of this effort [60]; however, this organism has been incriminated in only a minority of'Crohri's disease patients, and it, like other mycobacteria, can be identified in the intestinal tract of individuals without any apparent abnormality. Despite the lack of evidence for an infectious etiology, an un biased (broad-range) search for bacterial 16S rRNA in extraintestinal Crohri's disease tissues would be of great interest. Preliminary investigations are underway. Malakoplakia is a chronic granulomatous disorder that causes tumor-like nodules most often involving the genitourinary tract. Histologically, macrophages predominate; these cells appear to contain intraphagosomal coliform bacteria. The identity of these bacteria is not known in most cases, but some patients are cured with antibacterial therapy [61]. With visible organisms and the possibility of tissues relatively free of commensal flora, this would seem to be an attractive opportunity for PCR-amplified 16S rRNA-based microbial identification. Chronic inflammatory osteolytic bone lesions, sometimes containing granulomata, characterize a rare pediatric disease known as chronic recurrent multifocal osteomyelitis (CRMO). As with sarcoidosis, there is usually no response to antibiotics nor any visible organisms [62]. However, for the same reasons discussed earlier, a microbial etiology for CRMO has not been ruled out [63]. Frozen bone samples would be appropriate targets for a PCR-amplified, 16S rRNA-based phylogenetic investigation.

Proof of Disease Causality without Microbial Cultivation Without a purified or cultivated organism, Koch's postulates are difficult to fulfill, and without antigenic characterization of a putative pathogen, it is nearly impossible to develop an assay for specific humoral or cellular immune responses [64]. One is left with several less-satisfying alternatives [10]. First, the presence of a specific 16S rRNA sequence from a putative pathogen should be statistically correlated with specific tissue pathology. Also, sequence detection results should correlate with disease recurrence and clinical cure. Because of the potential sensitivity of a PCR assay, positive detection results may persist after clinical resolution. PCR target quantitation would be preferable, so that one might calculate the equivalent of an infectious load or a gene dosage effect. Finally, the significance of an amplified 16S rRNA sequence can be enhanced by specific hybridiza-


organism, besides classic Whipple's disease? One clinical syndrome currently being investigated is chronic unexplained diarrhea in human immunodeficiency virus (HIV)-seropositive persons. About 10 years ago, several reports described a Whipple's disease-like syndrome in patients with AIDS. Mycobacterium aviuni complex and Rhodococcus equi were isolated from some of these patients [51a, 51 b]. (Both of these organisms, like T. whippelii, are actinomycetes). More recently, microsporidiosis has been diagnosed in a significant number of HI V-seropositive patients with culture-negative chronic diarrhea [52]. However, a significant number of patients remain without an identified pathogen; it may not be unreasonable to consider a role for T. whippelii in this clinical setting. Early Whipple's disease may mimic sarcoidosis [53-55]. In 1 patient, bacilli similar in appearance to T. whippelii were seen within a sarcoid-like granuloma [55]. On the basis of these reports, one might speculate that T. whippelii is one of multiple possible etiologies-if sarcoidosis is, in fact, caused by a microbial agent(s). This theory is under investigation.

JID 1993;168 (July)

JlD 1993;168 (July)


tion of this sequence with the visible microorganisms in the original tissue samples [65]. In situ peR with specific 16S rRNA primers would provide another means to the same goal [66). The combination of these experimental strategies may help to establish a causal relationship between the organism, whose presence is inferred from an amplified 16S rRNA sequence, and the disease under study.

Conclusions

Acknowledgments

I thank Tom Schmidt (Miami University, Oxford, OH) for invaluable assistance with rRNA-based phylogenetic analysis and with figures 2 and 3 and Stanley Falkow (Stanford University) for immeasurable advice and support. References I. Whipple GH. A hitherto undescribed disease characterized anatomically by deposits offat and fatty acids in the intestinal and mesenteric lymphatic tissues. Johns Hopkins Hosp Bull 1907; 18:382-91. 2. Dobbins WOo Whipple's disease. Springfield. IL: Charles C Thomas. 1987. 3. Fleming JL. Wiesner RH. Shorter RG. Whipple's disease: clinical. biochemical, and histopathologic features and assessment of treatment in 29 patients. Mayo Clin Proc 1988;63:539-51. 4. Black-Schaffer B. The tintoral demonstration of a glycoprotein in Whipple's disease. Proc Soc Exp Bioi Med 1949;72:225-7. 5. Cohen AS. Schimmel EM. Holt PRo Isselbacher KJ. Ultrastructural abnormalities in Whipple's disease. Proc Soc Exp Bioi Med 1960; 105:411-4. 6. Yardley JH. Hendrix TR. Combined electron and light microscopy in Whipple's disease: demonstration of "bacillary bodies" in the intestine. Bull Johns Hopkins Hosp 1961; I09:80-98. 7. Chears WCJ. Ashworth CT. Electron microscopic study of the intestinal mucosa in Whipple's disease: demonstration of encapsulated bacilliform bodies in the lesion. Gastroenterology 1961:41: 129-38. 8. Dobbins WOo Kawanishi H. Bacillary characteristics in Whipple's disease: an electron microscopic study. Gastroenterology 1981;80: 1468-75. 9. Silva MT. Macedo PM. Nunes JFM. Ultrastructure of bacilli and the bacillary origin of the macrophagic inclusions in Whipple's disease. J Gen Microbiol 1985; 131: 1001-13. 10. Reiman DA, Falkow S. lden tification of uncultured microorganisms: expanding the spectrum of characterized microbial pathogens. Infect Agents Dis 1992; I:245-53. I I. Cockerell C J. LeBoit PE. Bacillary angiomatosis: a newly characterized.

pseudoneoplastic, infectious, cutaneous vascular disorder. J Am Acad Dermatol 1990;22:501-12. 12. Fox GE, Stackebrandt E. Hespell RB. et al. The phylogeny of the prokaryotes. Science 1980;209:457-63. 13. Olsen GJ. Lane OJ. Giovannoni SJ, Pace NR. Stahl DA. Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol 1986;40:337-65. 14. Woese CR. Bacterial evolution. Microbiol Rev 1987;51:221-71. 15. Woese CR, Kandler O. Wheelis ML. Towards a natural system oforganisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990;87:4576-9. 16. Turner S, Burger WT, Giovannoni SJ. Mur LR. Pace NR. The relationship of a prochlorophyte Prochlorothrix hollandica to green chloroplasts. Nature 1989;337:380-2. 17. Winker S, Woese CR. A definition of the domains Archaea, Bacteria, and Eucarya in terms of small subunit ribosomal RNA characteristics. System Appl Microbiol 1991; 14:305-10. 18. Lane OJ. Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 1985;82:6955-9. 19. Medlin L. Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 1988;71:491-9. 20. Chen K, Neimark H, Rumore P, Steinman CR. Broad range DNA probes for detecting and amplifying eubacterial nucleic acids. FEMS Microbiol Lett 1989;48: 19-24. 21. Wilson KH, Blitchington RB, Greene RC Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbioi 1990;28: 1942-6. 22. Weisburg WG, Barns SM. Pelletier DA. Lane OJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991; 173:697-703. 23. Saiki RK. Scharf S. Faloona F. et al. Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230: 1350-4. 24. Eisenstein BI. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med 1990;322: 178-83. 25. Reiman DA. Universal bacterial 16S rONA amplification and sequencing. In: Persing DH. Smith TF. Tenover FC, White TJ. eds. Diagnostic molecular microbiology: principles and applications. Washington, DC: American Society for Microbiology, 1993 (in press). 26. Boddinghaus B, Rogall T, Flohr T. Blocker H, Bottger EC Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol 1990;28: 1751-9. 27. Schmidt TM, Reiman DA. Phylogenetic identification of uncultured pathogens using ribosomal RNA sequences. In: Clark VA, Bavoil PM, eds. Methods in enzymology: bacterial pathogenesis. A. Orlando. FL: Academic Press. 1993 (in press). 28. Wright DK, Manos MM. Sample preparation from paraffin-embedded tissues. In: Innis MA. Gelfand DH. Sninsky JJ, White TJ. eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press. 1990: 153-8. 29. Shibata OK. Arnheim N. Martin WJ. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 1988;167:225-30. 30. Paabo S. Higuchi RG. Wilson AC Ancient DNA and the polymerase chain reaction. The emerging field of molecular archaeology. J Bioi Chern 1989;264:9709-12. 31. Fitch WM. Smith TF. Optimal sequence alignments. Proc Natl Acad Sci USA 1983;80: 1382-6. 32. Gutell RR, Weiser B, Woese CR, Noller HF. Comparative anatomy of 16S-like ribosomal RNA. Prog Nucleic Acid Res 1985;32: 155-216. 33. Olsen GJ. Earliest phylogenetic branchings: comparing rRNA-based


The nature of several previously uncharacterized microbial pathogens has been clarified by a non-culture-based experimental approach. Among these pathogens are the agents ofBA (and some cases of cat-scratch disease) and Whipple's disease. The potential usefulness of this and related molecular phylogenetic approaches may be considerable. There is a new perspective from which to study the diversity of human microbial pathogens and endogenous commensal microbial flora. From this perspective may come a more accurate understanding of the complexities of host-microbe interactions.

7

8

Reiman

49. Fuhrman JA, McCallum K, Davis AA. Novel major archaebacterial group from marine plankton. Nature 1992;356: 148-9. 50. Liesack W, Stackebrandt E. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol 1992; 174:5072-8. 51. Loesche WJ, Lopatin DE, Stoll J, vanPoperin N, Hujoel PP. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard? J Clin Microbioi 1992;30:418-26. 51a. Strom RL, Gruninger RP. AIDS with Mycobacterium avium-intracellulare lesions resembling those of Whipple's disease [letter]. N Engl J Med 1983;309: 1323-4. 51b. Wang HH, Tollerud 0, Danar 0, HanffP, Gottesdiener K, Rosen S. Another Whipple-like disease in AIDS? [letter]. N Engl J Med 1986;314: 1577-8. 52. Molina JM, Sarfati C, Beauvais B, et al. Intestinal microsporidiosis in human immunodeficiency virus-infected patients with chronic unexplained diarrhea: prevalence and clinical and biologic features. J Infect Dis 1993;167:217-21. 53. Cho C, Linscheer WG, Hirschkorn MA, Ashutosh K. Sarcoidlike granulomas as an early manifestation ofWhipple's disease. Gastroenterology 1984;87:941-7. 54. Southern JF, Moscicki RA. Magro C, Dickersin GR, Fallon JT, Bloch KJ. Lymphedema, lymphocytic myocarditis, and sarcoidlike granulomatosis. Manifestations of Whipple's disease. JAMA 1989;261: 1467-70. 55. Spapen HDM, Segers O. DeWit N. et al. Electron microscopic detection of Whipple's bacillus in sarcoidlike periodic acid-Schiff-riegative granulomas. Dig Dis Sci 1989;34:640-3. 56. Saboor SA, Johnson NM, McFadden J. Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction. Lancet 1992;339: 1012-5. 57. MitchelllC, Turk JL, Mitchell ON. Detection of mycobacterial rRNA in sarcoidosis with liquid-phase hybridisation. Lancet 1992;339: 1015-7. 58. Crohn B, Ginzburg l, Oppenheimer G. Regional ileitis, a pathological and clinical entity. JAMA 1932;99: 1323-9. 59. Chiodini RJ. Crohns disease and the mycobacterioses: a review and comparison of two disease entities. Clin Microbiol Rev 1989;2:90117. 60. McFadden JJ, Butcher PO, Chiodini R, Hermn-Taylor J. Crohri's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis. as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol 1987;25:796-80 I. 61. van Furth R, Wertheimer PA. VantWout JW, Zwartendijk J. Ciprofloxacin for treatment of malakoplakia. Lancet 1992;339: 148-9. 62. King SM, Laxer RM, Manson 0, Gold R. Chronic recurrent multifocal osteomyelitis: a noninfectious inflammatory process. Pediatr Infect Dis J 1987;6:907-11. 63. Rahav G. Sacks TG, Bar-Ziv J. Chronic recurrent multifocal osteomyelitis: report of a case. Clin Infect Dis 1992;14:587-8. 64. Evans AS. Causation and disease: the Henle-Koch postulates revisited. Yale J Bioi Med 1976;49:175-95. 65. Stoler MH. In situ hybridization. Clin Lab Med 1990;10:215-36. 66. Haase AT. Retzel EF, Staskus KA. Amplification and detection oflentiviral DNA inside cells. Proc Natl Acad Sci USA 1990;87:4971-5.


evolutionary trees inferred with various techniques. Cold Spring Harb Symp Quant Biol 1987;52:825-37. 34. Olsen GJ, Larsen N. Woese CR. The ribosomal RNA database project. Nucleic Acids Res 1991;19(suppl):2017-21. 35. Stoler MH, Bonfiglio T A. Steigbigel RT. Pereira M. An atypical subcutaneous infection associated with acquired immune deficiency syndrome. Am J Clin Pathol 1983;80:714-8. 36. Reiman 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: 1573-80. 36a. Slater IN, Welch OF, Hensel D. Coody OW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990;323: 1587-93. 36b. Regnery RL, Anderson BE, Clarridge JE III. Rodriguez-Barradas Me. Jones DC, Carr JH. Characterization of novel Rochalitnaea species, R. henselae ssi. nov., isolated from blood ofa febrile, human immunodeficiency virus-positive patient. J Clin Microbiol 1992;30:265-74. 37. 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: 1625-31. 38. Reiman DA. Lepp PW. Sadler KN. Schmidt TM. Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacillifortnis. and other alpha-proteobacteria. Mol Microbiol 1992;6: 1801-7. 39. Garcia FU, Wojta J, Broadley KN. Davidson JM, Hoover RL. Bartonella bacilliforinis stimulates endothelial cells in vitro and is angiogenic in vivo. Am J Pathol 1990; 136: 1125-35. 40. Reiman DA. Schmidt TM. MacDermott RP. Falkow S. Identification of the uncultured bacillus of Whipple's disease. N Engl J Med 1992;327:293-30 I. 41. Wilson KH, Blitchington R, Frothingham R, Wilson JA. Phylogeny of the Whipple's-disease-associated bacterium. lancet 1991;338:4745. 42. O'Neill SL, Giordano R. Colbert AME. Karr TL, Robertson HM. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci USA 1992;89:2699-702. 43. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 1991; 173: 1576-89. 44. Azad AF, Sacci JB, Nelson WM, Dasch GA. Schmidtmann ET, Carl M. Genetic characterization and transovarial transmission of a typhuslike rickettsia found in cat fleas. Proc Natl Acad Sci USA 1992; 89:43-6. 45. Williams SG, Sacci JB, Schriefer ME, et al. Typhus and typhuslike rickettsiae associated with opossums and their fleas in los Angeles County, California. J Clin Microbiol 1992;30: 1758-62. 46. Giovannoni SJ, Britschgi TB, Moyer CL, Field KG. Genetic diversity in Sargasso Sea bacterioplankton. Nature 1990;345:60-3. 47. Ward DM, Weller R, Bateson MM. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 1990;345:63-5. 48. Schmidt TM, Delong EF, Pace NR. Analysis ofa marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol 1991; 173:4371-8.

JID 1993;168 (July)