A phylogenetic tree of the prevalent species and phylotypes detected on the surface of the tongue dorsum for each of the subjects is shown in Fig. . Collectively, the overall bacterial diversity of the tongue dorsum is striking: 92 different bacterial taxa or phylotypes belonging to six bacterial phyla. Only 38, or about 40%, of the total number were identified as known species. Consequently, about 60% of the total were identified as phylotypes. As shown in Fig. , 29 of these phylotypes were unique to the tongue dorsum, in that they were not found from the sequence analysis of over 6,000 clones from other oral sites, including the subgingival plaque of healthy subjects and subjects with, periodontitis, acute necrotizing ulcerative gingivitis, and refractory periodontitis (22
); the supragingival plaque of children with rampant caries (1
); or advanced noma lesions (24
); nor were they found on or in crevicular epithelial cells of healthy subjects and subjects with periodontitis (13
). In our ongoing studies, we have detected over 300 novel phylotypes and 200 known species in oral sites. At present, we estimate that over 700 bacterial species are present in the oral cavity, over half of which we cannot presently cultivate. At the time of this publication, a list of 630 species or phylotypes of the oral cavity was compiled. The creation of a website is in progress; however, an updated version of this list can be obtained from the corresponding author.
The number of species or phylotypes that were detected in each subject ranged from 12 to 29, with 16 to 21 species or phylotypes in tongue samples from subjects without malodor and from 12 to 29 species or phylotypes in tongue samples from subjects with malodor (Table ). The bacterial profiles for each of these subjects are depicted in the colored columns of boxes in Fig. . Those species most associated with health were Streptococcus salivarius, Rothia mucilaginosa (Stomatococcus mucilaginosus), and an uncharacterized, cultivable species of Eubacterium (strain FTB41) (Fig. and Table ). The 15 most prevalent species or phylotypes are listed in Table , where they comprise 60 to 85% of the total clones in subjects without malodor and 20 to 88% of the total clones in subjects with malodor. A prevalent species is defined as a species that was detected in at least three subjects. It is noteworthy that S. salivarius was by far the most predominant species detected in healthy subjects: in one subject (subject H1), S. salivarius represented more than 40% of the detectable species. In contrast, S. salivarius was detected in only one of the subjects with halitosis and was detected at very low levels.
Percentage of prevalent species or phylotypes on tongue dorsum
Those species most associated with halitosis were Atopobium parvulum
, Eubacterium sulci
, Fusobacterium periodonticum
, a phylotype (clone BS095) of Dialister
, a phylotype (clone BW009) of Streptococcus
, a phylotype (clone DR034) of the uncultivated phylum TM7 (8
), and Solobacterium moorei
(Fig. and Table ). Note that in most of the samples, several species or phylotypes represented a significant proportion of the total (Table ). Although some species were not detected in all subjects, they were the predominant species in one or more samples. For example, Cryptobacterium curtum
was detected in only one of the samples from a subject with halitosis, but it represented about 20% of the clones analyzed in that subject. Other species, such as Granulicatella
, Streptococcus parasanguis
, Streptococcus infantis
, and Veillonella
spp., were commonly detected in most of the samples (Table ).
The tongue dorsum harbors a highly diverse, yet characteristic, bacterial population. In healthy subjects, S. salivarius
was by far the predominant species. In contrast, S. salivarius
was typically absent from subjects with halitosis. Although bacteria other than S. salivarius
appeared to be associated with halitosis, it is not known if they are directly involved in oral malodor. Cultural studies have associated R. mucilaginosa
with malodor (7
) and S. salivarius
and Veillonella parvula
or Veillonella dispar
as common healthy tongue organisms (C. E. Kazor and W. J. Loesche, unpublished data).
On the basis of our sequence analyses, the tongue dorsum possesses a unique microbiota: about one-third of the bacterial population was found only on the tongue and not in or on the surfaces of other oral sites. However, a sample of sufficient size (e.g., in a large clinical trial) is necessary to provide the power to detect differences in microbial compositions to identify more precisely those species that are associated with halitosis and health. Such studies will be accomplished by using 16S rRNA-based oligonucleotide probes in checkerboard DNA-DNA hybridization assays (1
) or eventually by using oligonucleotide microarrays.
It has been suggested that the majority of cases of oral malodor are due to bacterial proteolytic activity in the mouth, such as might be measured by the BANA test (16
). Since known BANA test-positive oral species typically found in subgingival plaque, i.e., P. gingivalis
, T. denticola
, T. forsythensis
, and various Capnocytophaga
species, were not detected (15
), other tongue bacterial species are likely responsible for the BANA reaction of the tongue coating. Now that we have identified additional predominant bacterial species present on the tongue dorsa of individuals with halitosis, it would be of interest to examine the ability of these other cultivable species to hydrolyze the BANA substrate and to produce VSCs and other by-products that may contribute to the clinical presentation of malodor.