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Applied and Environmental Microbiology, August 1999, p. 3287-3292, Vol. 65, No. 8
Department of Molecular Genetics, Forsyth
Dental Center, Boston, Massachusetts 021151;
Division of Comparative Medicine, Massachusetts Institute
of Technology, Cambridge, Massachusetts
021392; and Biomedical Research
Associates, Frederick, Maryland 217023
Received 22 January 1999/Accepted 6 May 1999
The "altered Schaedler flora" (ASF) was developed for
colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA
sequence analysis. Three strains were previously identified as
Lactobacillus acidophilus (strain ASF 360),
Lactobacillus salivarius (strain ASF 361), and
Bacteroides distasonis (strain ASF 519) based on phenotypic
criteria. 16S rRNA analysis indicated that each of the strains differed
from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains
of Lactobacillus murinis and Lactobacillus
animalis (both isolated from mice), and all of these strains
probably belong to a single species. Strain ASF 360 is a novel
lactobacillus that clusters with L. acidophilus and
Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis,
[Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3.
This unnamed genus is in the
Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The
spiral-shaped strain, strain ASF 457, is in the Flexistipes
phylum and exhibits sequence identity with rodent isolates of
Robertson. The remaining four ASF strains, which are extremely
oxygen-sensitive fusiform bacteria, group phylogenetically with the
low-G+C-content gram-positive bacteria (Firmicutes,
Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al.
Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium
plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to
that of E. plexicaudatum. Since the type
strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a
neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any
organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present
study should allow rapid identification of ASF strains and should
permit detailed analysis of the interactions of ASF organisms during
development of intestinal disease in mice that are coinfected with a
variety of pathogenic microorganisms.
The gastrointestinal tracts of
mammals, including mice and rats, contain a diverse microecosystem. The
ceca of normal mice contain numerous species, and the concentration of
bacteria can be as great as 1011 bacteria/g of feces
(41, 42). These microorganisms not only provide essential
nutrients (e.g., vitamin K) for their hosts but also colonize mucosal
niches, which in part helps protect the hosts against microbial
pathogens (22, 25, 42, 49, 53, 55). For example, numerous
studies have demonstrated the increased susceptibility of germfree mice
to a variety of infectious agents compared to that of mice with the
normal complement of microorganisms (17).
Gnotobiotic animals colonized with known microbiota have been used to
great advantage as models for biomedical research (17). For
certain studies, it is particularly desirable to colonize germfree mice
with a defined microbiota.
In the mid-1960s, Russell W. Schaedler was the first researcher to
colonize germfree mice with selected bacteria isolated from normal mice
(40). He subsequently supplied animal breeders with this
group of microorganisms (2) for use in colonizing their
rodent colonies. These defined bacteria included aerobic bacteria that
were easy to grow and some less-oxygen-sensitive anaerobic organisms.
The so-called extremely oxygen-sensitive (EOS) fusiform bacteria, which
make up the vast majority of the normal microbiota of rodents, were not
included due to technical difficulties in isolating and cultivating EOS
bacteria (26, 27). Of the defined microbiotas later used for
gnotobiotic studies, the one known as the "Schaedler flora" was the
most popular. This flora contained eight bacteria, which were
designated Escherichia coli var. mutabilis,
Streptococcus faecalis, Lactobacillus
acidophilus, Lactobacillus salivarius, group N
Streptococcus, Bacteroides distasonis, a
Clostridium sp., and an EOS fusiform bacterium.
In 1978, the National Cancer Institute (NCI) decided to revise the
Schaedler flora or "cocktail" consisting of eight bacteria in order
to standardize the microbiota used to colonize axenic (germfree)
rodents, including mice, at all NCI contractors, as well as mice used
at NCI. Roger Orcutt, therefore, developed the new defined microbiota
now known as the "altered Schaedler flora" (ASF), which consisted
of four members of the original Schaedler flora (the two lactobacilli,
B. distasonis, and the EOS fusiform bacterium), a
spiral-shaped bacterium, and three new fusiform EOS bacteria
(30).
Although very important, it is very difficult to monitor a gnotobiotic
mouse colony with a defined microbiota. Not only is it necessary to
demonstrate that the colony is free of any adventitious microorganisms,
but it must also be demonstrated that the microorganisms of the
specified microbiota are present. In the past, workers monitoring
gnotobiotic animals relied on examining the morphology of the
microorganisms and performing a limited evaluation of the biochemical
traits and growth characteristics of the organisms.
The goals of this study were, therefore, to identify the bacteria in
the ASF by 16S rRNA sequence analysis and to characterize the
phylogenetic positions of these organisms relative to those of known
bacteria. The long-term goal of our studies is to develop sensitive and
specific molecular techniques for monitoring the microbiotas of
gnotobiotic animals.
Bacterial strains and cultivation.
ASF bacteria, including
four EOS fusiform anaerobes (Taconic stock culture strains ASF 356, ASF
492, ASF 500, and ASF 502), a spiral-shaped bacterium (Taconic strain
ASF 457), two previously identified lactobacilli (Taconic strains ASF
360 and ASF 361), and a Bacteroides sp. (Taconic strain
519), were obtained from Taconic, Germantown, N.Y. The bacteria were
cultured anaerobically on Schaedler agar (Difco Laboratories, Detroit,
Mich.) supplemented with 5% sterile fetal calf serum (Summit
Biotechnology, Ft. Collins, Colo.) in an anaerobic glove chamber
containing a 10% CO2-10% H2-80%
N2 atmosphere (Coy Laboratory, Grass Lakes, Mich.).
Anaerobiosis was monitored with a resazurin indicator. The media were
prereduced by placing them inside the chamber 2 days prior to
inoculation of bacteria. The temperature in the chamber was maintained
at 33 to 35°C.
Extraction of DNA for sequence determination.
Bacteria were
harvested, washed twice with 1 ml of sterile phosphate-buffered saline,
and then collected by centrifugation at 8,000 × g. The pellets
were used for extraction of DNA templates that were used to amplify 16S
rRNA by PCR. DNA was extracted from the cell pellets by using a
commercial kit (High Pure PCR template preparation kit; Boehringer
Mannheim) according to the manufacturer's instructions.
Amplification of 16S rRNA cistrons by PCR and purification of PCR
products.
The 16S rRNA cistrons were amplified with bacterial
universal primers F24 and F25 (Table 1).
PCR was performed in thin-walled tubes with a Perkin-Elmer model 9700 thermocycler. One microliter of the DNA template was added to a
reaction mixture (final volume, 50 µl) containing 20 pmol of each
primer, 40 nmol of deoxynucleoside triphosphates, and 1 U of
Taq 2000 polymerase (Stratagene, La Jolla, Calif.) in buffer
containing Taqstart antibody (Sigma Chemical Co.). In a hot-start
protocol, samples were preheated at 95°C for 8 min, and this was
followed by amplification in which the following conditions were used:
denaturation at 95°C for 45 s, annealing at 60°C for 45 s, and elongation for 1.5 min with an additional 5 s for each
cycle. A total of 30 cycles were performed, and then a final elongation
step consisting of 72°C for 10 min was performed. The PCR
amplification results were examined by electrophoresing preparations in
a 1% agarose gel. The DNA was stained with ethidium bromide and
visualized under short-wavelength UV light.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Phylogeny of the Defined Murine Microbiota: Altered
Schaedler Flora
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
PCR and sequencing primers
16S rRNA sequencing. Purified DNA obtained from the PCR was sequenced by using an ABI prism cycle sequencing kit (BigDye terminator cycle sequencing kit with AmpliTaq DNA polymerase FS; Perkin-Elmer). The primers in Table 1 were used for sequencing. Quarter dye chemistry was used with primers at a concentration of 80 µM and 1.5 µl of PCR product in a final volume of 20 µl. Cycle sequencing was performed by using a model ABI 9700 apparatus and 25 cycles consisting of denaturation at 96°C for 10 s, annealing, and extension at 60°C for 4 m. Sequencing reactions were performed with a model ABI 377 DNA sequencer.
16S rRNA data analysis. Sequence data were entered into RNA, a program set for data entry, editing, sequence alignment, secondary-structure comparison, similarity matrix generation, and dendrogram construction for 16S rRNA in Microsoft QuickBasic for use with PC computers, and sequences were aligned as previously described (31). Our database contains more than 1,000 sequences obtained in our laboratory and more than 500 sequences obtained from GenBank. Sequences were first checked by BLAST analysis versus all entries in the GenBank database (1). Neighboring sequences for the ASF organisms not already in our database were downloaded and added to our database. Dendrograms were constructed by the neighbor-joining method (37).
Nucleotide sequence accession numbers.
The GenBank accession
numbers for the strains examined in this study or used as reference
strains are given in Table 2. The 16S
rRNA sequences of ASF strains determined in this study have been
deposited in the GenBank database under the following accession numbers: ASF 360, AF157050; ASF 361, AF157049; ASF 519, AF157056; ASF
457, AF157055; ASF 356, AF157052; ASF 492, AF157054; ASF 500, AF157051;
and ASF 502, AF157053.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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An essentially complete 16S rRNA sequence (length, 1,500 bases) was determined for each of the ASF strains. A neighbor-joining phylogenetic tree that included the closest neighbors of each ASF strain was constructed by using the sequences listed in Table 2 (Fig. 1). The strains previously presumptively identified as L. acidophilus (strain ASF 360), L. salivarius (strain ASF 361), and B. distasonis (strain ASF 519) based on phenotypic criteria were not members of these species but rather were members of neighboring species. The sequence of strain ASF 361 differed from the L. salivarius sequence but was essentially identical to the sequences of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice). Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an as yet unnamed genus along with [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. The spiral-shaped strain, strain ASF 457, falls in the Flexistipes phylum and is most closely related to Geovibrio ferrireducens and an organism isolated from the stomach of a Colobus monkey (9). The remaining four ASF strains, which are EOS fusiform bacteria, grouped phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Germfree mice and rats that are monoassociated with a bacterium or a particular microbiota are commonly used in biomedical research. The ASF has been widely used since the 1980s as a group of defined bacteria for colonizing the gastrointestinal tracts of commercially available mice and rats used for biomedical research. In this report we provide an initial taxonomic description of these bacteria based on a 16S rRNA analysis.
Lactobacilli are common colonizers of the gastrointestinal mucosal and squamous epithelia of mice (20, 34, 38, 39, 41). Historically, most of the indigenous lactobacilli either have not been identified to the species level or have been identified as minor variants of human species by using a limited number of biochemical tests (20, 34, 39). Unfortunately, different vertebrate species often contain unique bacterial species that are distinct from phenotypically similar human-associated species. Thus, ASF 360 and ASF 361 were identified as minor phenotypic variants of L. acidophilus and L. salivarius, respectively. With the emerging use of molecular techniques, such as restriction endonuclease fingerprinting, DNA-DNA hybridization, plasmid content analysis, and 16S rRNA sequencing, classification and identification of lactobacilli have been greatly improved (6, 7, 16, 33, 36, 43, 47, 52, 58). 16S rRNA sequence analysis has clearly demonstrated that ASF 360 and ASF 361 are distinct from each other and distinct from L. acidophilus or L. salivarius. However, as shown in Fig. 1, the 16S rRNA sequence of ASF 361 appears to be essentially identical to the 16S rRNA sequences of the previously described species L. murinus and L. animalis. L. murinus strains have been isolated from the intestinal tracts of mice and rats (21). L. animalis strains have been isolated from the dental plaque and alimentary tracts of animals (8). The type strains of L. murinus and L. animalis appear to belong to a single species. While L. animalis strains isolated from mice may belong to the same species as L. murinus strains, strains isolated from other mammalian sources may belong to different species. Therefore, a thorough examination of L. murinus and L. animalis strains is necessary to resolve these taxonomic issues. The possibility that these species are identical was suggested previously (24). L. murinus was named 2 years before L. animalis was named, and therefore the name L. murinus has priority according to the rules of nomenclature.
Bacteroides spp. are microbes that are commonly found in the intestinal tracts of mammals. Many Bacteroides species, including B. distasonis, have been isolated from the ceca of conventional mice and characterized (39, 50, 51). These bacteria were included in the genus Bacteroides because they are nonmotile, gram-negative, strictly anaerobic, non-spore-forming rods which do not produce butyric acid (50, 51). However, after many of the early studies were performed, it was recognized that the genus Bacteroides contained species representing several genera. A majority of the species previously included in the genus Bacteroides have been placed in the genera Porphyromonas, Prevotella, and Bacteroides sensu stricto (44-46). [B.] distasonis is not a true member of the genus Bacteroides but rather falls in a novel genus closely related to the genus Porphyromonas (32). Strain ASF 519 is related to [B.] distasonis but is clearly a distinct species. [B.] distasonis, [B.] merdae, [B.] forsythus, CDC group DF-3 (54), and strain ASF 519 comprise a novel unnamed genus in the Cytophaga-Flavobacterium-Bacteroides phylum.
Strain ASF 457, a spiral-shaped obligately anaerobic bacterium, was described as a spirochete by Orcutt et al. (30). Bacteria with spiral-shaped morphology are commonly found in large numbers mixed with tapered rods in the mucus layers of the ceca and colons of mice (18, 39). As determined by 16S rRNA analysis, this bacterium is related to G. ferrireducens, a dissimilatory, Fe(III)-reducing bacterium (3), Deferribacter thermophilus (19), and Flexistipes sinusarabici (10, 28) in the Flexistipes phylum (23). Within the level of sequencing error, the sequence of strain ASF 457 is identical to sequences of rodent isolates described by B. R. Robertson (35) and deposited in the GenBank database (accession no. AF059186 to AF05988). ASF 457 and the Robertson strains probably are isolates of the same species. Two other Robertson strains (accession no. AF059189 and AF05990) and a strain isolated from the stomach of a Colobus monkey (9) belong to related species. It appears that the Flexistipes phylum contains species that inhabit mammalian gastrointestinal tracts, as well as iron-reducing environmental isolates.
The majority of the members of the gastrointestinal microbiota of mice and rats are fusiform bacteria or tapered rods and are referred to in broad terms as EOS bacteria. These bacteria outnumber facultatively anaerobic bacteria by as much as 100 to 1 and aerobic bacteria by thousands to one (39). Although large numbers of the EOS fusiform bacteria or tapered rods are present (18, 20), only a few of these organisms have been cultivated, and fewer still have been named and extensively studied (48, 50, 57). Because it is difficult to identify these organisms at the species and genus levels, older taxonomic studies often grouped these bacteria on the basis of morphological criteria and growth characteristics (18) and in many cases considered them members of the genera Eubacterium, Fusobacterium, and Clostridium (20, 50, 57). The four EOS fusiform ASF strains belong in the low-G+C-content gram-positive bacterial group (Firmicutes, Bacillus-Clostridium group). Strain ASF 356 is most closely related to Clostridium propionicum. Strain ASF 492 possesses a subpolar tuft of flagella that is inserted subterminally, an unusual morphological characteristic shared by Roseburia cecicola (48) and Eubacterium plexicaudatum (57). The ASF 492 sequence clearly differentiates this organism from R. cecicola, but unfortunately, the type strain and other viable strains of E. plexicaudatum have been lost (56). The American Type Culture Collection still had vials of ATCC 27514T that were never released because they were found to be nonviable. The complete 16S rRNA sequence of ATCC 27514T (a nonviable strain kindly provided by the American Type Culture Collection) was determined, and this sequence was identical to the 16S rRNA sequence of strain ASF 492. Elsewhere, we will propose that ASF 492 should become the neotype strain for E. plexicaudatum. Our results demonstrate that 16S rRNA sequence analysis is an ideal tool for determining the molecular identities of archival or reference organisms which are no longer viable. Strain ASF 502 is most closely related to Ruminococcus gnavus. Strains ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. (5). Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organism currently in the GenBank database.
Our findings again highlight the pitfalls of placing human and animal isolates with similar phenotypic characteristics in a single species. Taxonomic analysis of the family Pasteurellaceae (32), as well as many other organisms, has indicated that individual mammalian organisms have their own unique associated species. Molecular techniques, such as 16S rRNA sequencing, easily detect the existence of polyphyletic groups and can be used to prevent misclassification based phenotypic similarity.
Our findings also illustrate the taxonomic complexities of the normal
flora of the mouse. Clearly, most mouse floras are much more diverse
than the ASF in mice maintained under strict germfree conditions to
prevent introduction of other bacterial species adept at colonizing the
murine lower bowel. It is common for investigators to stipulate that
mice have been maintained under specific-pathogen-free conditions.
Unfortunately, this term is misinterpreted in most scientific
publications and is commonly used to mask a lack of detailed
information regarding the microbial pathogen status of the animals
being studied. Because mice are housed in a "pathogen-free" environment and are periodically screened by viral serology and/or intestinal culture methods for known pathogenic bacteria and parasites, it is frequently assumed that infectious agents are not present and do
not contribute to the pathogenesis of the disease being studied.
Invariably, in these studies the intestinal flora is considered the
normal flora. Indeed, a number of newly recognized murine enterohepatic
helicobacters, which are fastidious microaerobes, were placed in this
category; they were ignored because they are difficult to culture and
because there were no previous data attributing any importance to the
presence of large numbers of these spiral organisms in the crypts of
the lower intestines of mice. It is now known that several of these
helicobacters, most notably Helicobacter hepaticus, can
cause serious gastrointestinal disease in a number of inbred and mutant
mice (4, 11-13, 15). Also, it is important to recognize
that certain members of the microflora of the intestine may be
protective. This was clearly illustrated in young neonatal IL-10
/ mice susceptible to inflammatory bowel disease
at a young age. These mice were protected against the development of
colitis by oral administration of Lactobacillus sp.
(29). The authors hypothesized that the
Lactobacillus sp. prevented bacterial adherence to gut mucosa and subsequent bacterial translocation.
This research provided an unambiguous molecular approach to identify the AFS organisms. Our information should allow workers who utilize ASF-colonized mice to more precisely monitor the microbiota of these gnotobiotic animals by using 16S rRNA-based probe or PCR techniques. The availability of the PCR probes should also result in more accurate quality control of the defined murine microbiota and prevent infections of mice with microbial pathogens (11, 14).
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ACKNOWLEDGMENTS |
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This work was supported in part by NIH grants R01DE-10374 (to F.E.D.), R01DE-11443 (to B.J.P.) R01DK52413 (to D.B.S. and J.G.F.), R01CA-67529 (to J.G.F. and D.B.S.), P01CA 26731 (to J.G.F.), and RR01046 (to J.G.F.).
We thank the American Type Culture Collection for providing a vial of the nonviable type strain of E. plexicaudatum.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Comparative Medicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Bldg. 16, Rm. 825C, Cambridge, MA 02139. Phone: (617) 253-1757. Fax: (617) 258-5708. E-mail: jgfox{at}mit.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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