http://www.genome.org/cgi/content/full/16/3/316

Ecologists studying microbial life in the environment have recognized
the enormous complexity of microbial diversity for many years, and the
development of a variety of culture-independent methods, many of them
coupled with high-throughput DNA sequencing, has allowed this
diversity to be explored in ever-greater detail. Despite the
widespread application of these new techniques to the characterization
of uncultivated microbes and microbial communities in the environment,
their application to human health and disease has lagged. Because DNA-
based techniques for defining uncultured microbes allow not only
cataloging of microbial diversity but also insight into microbial
functions, investigators are beginning to apply these tools to the
microbial communities that abound on and within us, in what has aptly
been called "the second Human Genome Project." In this review we
discuss the sequence-based methods for microbial analysis that are
currently available and their application to identify novel human
pathogens, improve diagnosis of known infectious diseases, and advance
understanding of our relationship with microbial communities that
normally reside in and on the human body.

It has long been recognized that standard culture methods fail to
adequately represent the enormous microbial diversity that exists in
nature because of the fastidious growth requirements of many
microorganisms. Even when growth conditions are altered to mimic
environmental nutrient composition, up to 80% of organisms identified
by culture-independent methods fail to grow in culture (Connon and
Giovannoni 2002Go). To avoid reliance on cultivation, many culture-
independent methods have been developed to search for novel bacterial
species, including pathogens. These methods include screening of
expression libraries with immune serum, nucleic acid subtractive
methods, and small molecule detection with mass spectroscopy, among
others, and these methods have been reviewed elsewhere (Relman
2002Go). This review will focus primarily on sequence-based methods
because of their general applicability and the continued expansion of
high-throughput, low-cost sequencing capacity.

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identification of uncultivated organisms that cause human disease.

Because DNA can be extracted from any potentially infected material
and used as a substrate for 16S rRNA gene amplification, Fredericks
and Relman (1996Go) predicted that a rash of claims for disease
causation by new pathogens would follow application of this method to
human tissues and laid out a strategy for proving disease causation
for organisms that might be difficult or impossible to grow. Reiman
and Falkow (2001) later amplified these criteria. Remarkably, this has
not come to pass and only a few new pathogens have been identified
through 16S rRNA gene amplification. This may be because present
methods for cultivation are sufficient for the vast majority of
pathogens capable of growth on human tissues, or it may be that we
have yet to apply culture-independent methods to many clinical
conditions that have an infectious component**. Whatever the reason,
these new infectious agents are notable and worthy of review here.

**  I think the authors are just being diplomatic**

The first novel pathogen to be identified by sequence-based methods
was Rochalimaea henselae, the organism responsible for bacillary
angiomatosis (BA). The hallmark of BA is abnormal proliferation of
small blood vessels in the skin and visceral organs of
immunocompromised patients. Although bacteria had been found in tissue
sections by Warthin-Starry staining, they could not be cultured
because of their fastidious growth requirements (Perkocha et al.
1990Go). In 1990, Relman et al. amplified a partial 16S rRNA gene
sequence from tissue samples obtained from bacillary angiomatosis
patients, but not from normal tissues, using broad-range PCR (Relman
et al. 1990Go). Analysis of this 16S sequence suggested a novel
species most closely related to Rochalimaea spp. Further evidence for
causation was provided by the isolation of a slow-growing, Rochalimaea-
like bacillus from a single BA patient in an independent study (Slater
et al. 1990Go). Two years later, genotypic analysis of the complete
16S rRNA gene and other genomic loci further confirmed the novelty of
the isolated BA agent, Rochalimaea henselae (later moved into the
genus Bartonella) (Regnery et al. 1992Go).

The same strategy was soon applied to other potentially infectious
diseases and led to the identification of Ehrlichia chaffeensis, a new
species associated with tick bites that causes a febrile illness.
Ehrlichiosis is clinically similar to Rocky Mountain spotted fever,
another tick-borne disease caused by the intracellular parasite,
Rickettsia rickettsii. Although testing of the index patient's serum
for antibodies against R. rickettsii was negative, patient serum
contained antibodies reactive to E. canis, a well-described canine
pathogen (Maeda et al. 1987Go). This suggested that E. canis or a
related species was responsible for disease, and 16S rRNA gene
amplification from infected macrophages led to identification of E.
chaffeensis (Anderson et al. 1991Go; Dawson et al. 1991Go). Causation
is supported by concordance of E. chaffeensis 16S rRNA and serologic
findings in patients with fever, leukocyte inclusions, and history of
a tick bite, as well as a salutary clinical response to appropriate
antibiotics accompanied by loss of E. chaffeensis 16S rRNA gene from
leukocytes.

A third example of success with 16S rRNA gene amplification is
Whipple's disease. Whipple's disease is a rare disease first described
in 1907 in a missionary who died of an illness marked by chronic joint
pain, weight loss, and severe abdominal pain. In the report of this
patient, "rod-like bacilli in a small node" were noted (Whipple
1907Go). Eighty-four years passed before the identification of the
etiologic agent, despite its consistent observation in affected
tissues (Chears Jr. and Ashworth 1961Go; Yardley and Hendrix 1961Go)
and patients' improvement with antibiotic treatment (Trier et al.
1965Go). In 1991, a partial 16S rRNA gene sequence was amplified from
a small-bowel biopsy specimen taken from a patient with Whipple's
disease (Wilson et al. 1991Go), and the complete 16S rRNA gene
sequence was determined a year later, revealing it to be an
actinomycete not closely related to any known genus. It was therefore
given a new genus and species name, Tropheryma whipplei, based on the
unusual features of the disease and the distinct morphological
characteristics of the bacillus (Relman et al. 1992Go). It is worth
noting that T. whipplei was particularly recalcitrant to cultivation
(Raoult et al. 2000Go). The complete genome sequence of T. whipplei
predicted deficiencies in amino acid synthesis (Bentley et al. 2003Go;
Raoult et al. 2003Go), and with this information, Renesto et al.
(2003Go) successfully designed a complete medium that allowed cell-
free cultivation of T. whipplei. This was the first demonstration that
genomic information could guide rational design of media for axenic
cultivation of fastidious bacteria.

Whereas most human tissues are normally devoid of cultivable
microorganisms, many epithelial-lined cavities of the human body in
contiguity with the environment harbor microbial communities, the
complexities of which are just beginning to be understood. These
include the skin, mouth, ear, gastrointestinal tract, and vagina.
Identifying pathogens within this complex bacterial background is more
difficult than identifying them in normally sterile compartments. One
of the most successful examples of this involves the study of dental
plaque. Because of their known role in dental caries and periodontal
disease, human oral flora have been studied intensively through both
culture-dependent and culture-independent techniques. About 500
bacterial species have been found in the human oral cavity (Thoden van
Velzen et al. 1984Go; Meyer and Fives-Taylor 1998Go; Paster et al.
2001Go) and 40%-60% of these species are uncultivated
"phylotypes" (Kroes et al. 1999Go; Paster et al. 2001Go). Studies
using conventional culture methods have established that early
colonizing streptococci play a key role in initiating the formation of
dental caries (Kolenbrander et al. 1990Go; Whittaker et al. 1996Go),
while Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis
and Treponema denticola contribute to the development of periodontal
disease (Meyer and Fives-Taylor 1998Go). Recent 16S rDNA sequence
analysis uncovered that in addition to these known pathogens, several
additional organisms, including organisms assigned to uncultivated
phyla OP11 and TM7, are strongly associated with periodontitis or
acute necrotizing ulcerative gingivitis in humans (Choi et al. 1994Go;
Dewhirst et al. 2001Go; Paster et al. 2001Go; Brinig et al. 2003Go;
Hutter et al. 2003Go; Ouverney et al. 2003Go).

Recently, methanogenic Archaea have also been linked to periodontal
disease based on 16S rRNA sequencing and FISH analysis (Kulik et al.
2001Go; Lepp et al. 2004Go), providing the first example of archaeal
disease association in humans. In the latter study, the relative
abundance of methanogenic Archaea was associated with disease severity
and decreased with effective treatment (Lepp et al. 2004Go). However,
Archaea were not uniformly found in the subgingival space of severely
affected individuals, leaving open the question of whether the Archaea
are playing a causative role in this polymicrobial disease.
Interestingly, the abundance of Archaea and T. denticola were
inversely correlated, suggesting that they might compete for the same
niche in the community. It was hypothesized that both organisms serve
as "hydrogen sinks" in the highly reduced environment of the
subgingival space, allowing acid-producing members of the community to
grow to higher density than they might in the absence of Archaea or
Treponemes. While additional genomic and metabolic studies will be
required to fully understand the role of methanogenic Archaea in
periodontal disease, this example clearly illustrates how culture-
independent methods may do more than increase our appreciation of the
number of known pathogens. These methods also stand to teach us a
great deal about the mechanisms of disease, especially in
circumstances where the paradigm of a single causative agent seems not
to hold. Other clinical circumstances that may benefit from similar
analyses include sinusitis, ventilator-associated pneumonia, small-
bowel overgrowth syndromes, inflammatory bowel disease, and bacterial
vaginosis.