Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA1
Department of Medical Microbiology, University of Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands2
Department of Cell Biology, Histology and Immunology Section, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands3
Author for correspondence: Nita H. Salzman. Tel: +1 414 456 4244. Fax: +1 414 456 6535. e-mail: nsalzman{at}mcw.edu
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ABSTRACT |
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Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; MIB, mouse intestinal bacteria; OTU, operational taxonomic unit; SFB, segmented filamentous bacteria
b The GenBank accession numbers for the clone sequences reported in this paper can be found in Table 1; the accession number for isolate MIB-CB3 is AJ418059.
a Present address: Medical College of Wisconsin, Department of Pediatrics, 8701 Watertown Plank Rd, Milwaukee, WI 53226, USA.
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INTRODUCTION |
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Molecular methodologies relying on 16S rRNA gene sequences are now commonly used for the identification and classification of bacterial species within mixed microbial populations (Amann et al., 1991 ; Amann et al., 1995
; Suau et al., 1999
; Ward et al., 1990
). Oligonucleotide probes that are specific for the 16S rRNA of distinct bacterial species have been developed for use in fluorescence in situ hybridization (FISH) studies (Amann et al., 1990
, 1995
; Giovannoni et al., 1988
; Harmsen et al., 1999
). Probes specific for bacteria found in the human intestine have also been generated and characterized (Franks et al., 1998
; Langendijk et al., 1995
; McCartney et al., 1996
; Yamamoto et al., 1992
).
Although the population studies detailed above concentrated on the human intestinal microflora, many studies on the interaction between the intestinal microflora and the mucosal immune system are being done in mouse models (Cebra et al., 1999 ). The intestinal microflora of specific pathogen-free mice is still ill-defined. Many inbred strains of mice started out as germ-free strains that were colonized with a defined population of bacteria, the altered Schaedler flora (Dewhirst et al., 1999
) the 16S rRNA gene sequences of the eight bacterial strains of the altered Schaedler flora were published by Dewhirst et al. (1999)
. Under specific pathogen-free conditions, no monitoring of the population is performed, except for the exclusion of certain defined pathogenic organisms. A number of new bacterial strains have recently been identified in the murine intestinal tract. For example, a new species of Bacteroides, Bacteroides acidifaciens, isolated from the mouse caecum was described recently (Miyamoto & Itoh, 2000
). Also, a new unidentified Gram-positive rod (HCDA-1), involved in the metabolism of bile acids, was isolated from the rat intestinal microflora (Eyssen et al., 1999
).
Because the intestinal microflora of mice is still ill-defined, we were interested in evaluating the utility of existing probes used to study the human intestinal microflora to identify murine intestinal bacteria. We were also interested in the development of new 16S rRNA probes for the detection of murine intestinal bacterial species that did not hybridize to the existing probes. In this work, we examined the bacteria present in the murine small intestine, caecum, large intestine and faeces directly, and identified the bacterial species within these regions by their 16S rRNA gene sequences. Using the 16S rRNA gene sequences derived from these bacteria, we developed oligonucleotide probes and found them to be useful for the identification and quantification of bacterial populations in the mouse intestinal tract. These newly designed probes can be applied to study the role of intestinal bacteria in diverse mouse disease models.
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METHODS |
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Bacterial cultures and isolation of intestinal bacterial DNA.
Mice were killed using CO2, and total DNA was isolated from the large intestine, small intestine, caecum and faeces of the mice. Each specimen was homogenized in TE buffer. Lysozyme (5 mg ml-1) was added to the homogenate and the mixture was incubated at 37 °C for 1 h. Proteinase K (2 mg ml-1) was added to the homogenate, which was incubated for a further 1 h at 56 °C. SDS (1%, w/v) was then added to the homogenate, which was incubated at 37 °C for 30 min. The DNA was extracted from the homogenate with equal volumes of phenol/chloroform/isoamyl alcohol. The sample was centrifuged and the DNA (in the aqueous layer) was precipitated overnight with 3 M sodium acetate/ethanol. The sample containing the precipitate was centrifuged, the supernatant removed, and the resulting DNA pellet resuspended in TE buffer. A species belonging to the mouse intestinal bacteria (MIB) group was isolated from the caecum sample by plating dilution series of the untreated homogenate onto pre-reduced Brucella blood agar plates supplemented with 5% sheep blood (Summanen et al., 1993 ); the plates were incubated anaerobically for 48 h at 37 °C. After 48 h, random colonies were picked from the agar, and these were simultaneously subcultured and hybridized with the MIB661 probe. One MIB661-positive isolate was obtained. A partial DNA sequence was obtained from this isolate by direct sequencing of its 16S rDNA, after PCR amplification of the 16S rRNA gene.
PCR amplification of 16S rRNA gene sequences, cloning and sequencing.
16S rRNA genes were specifically amplified by PCR using the conserved 16S-rRNA-specific primer pairs 8FE (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGMTACCTTGTTACGACTT-3'), and 519FB (5'-ATTGGATCCCAGCMGCCGGGGTAA-3') and 1392RS (5'-TGAGTCGACACGGGCGGTGTGTRC-3'). The PCR conditions used were 30 cycles at 96 °C for 1 min, 48 °C for 2 min and 72 °C for 2 min, followed by a final elongation at 72 °C for 5 min. PCR products were purified by using preparative low-melting-point-agarose gel electrophoresis to separate the products. The band corresponding to 1500 bp was excised from the gel. The excised piece of gel was heat-sealed in a plastic pouch (Kapak) and frozen at -20 °C; it was then squeezed to remove the DNA. The DNA was precipitated from the recovered liquid by using 3 M sodium acetate/ethanol. The sample containing the PCR product was centrifuged, the supernatant discarded, and the resulting pellet was dissolved in TE buffer. The PCR product was then introduced into pPUC18 by using the TA Cloning Kit (Invitrogen). pPUC18 containing the PCR product was used to transform Escherichia coli DH5; plasmids of the resulting clones were isolated for sequencing or slot-blot hybridization using the Wizard Mini-Prep Kit (Promega) and extraction with phenol/chloroform. The clone sequences were obtained by using vector-specific primers and an ALFwin automated sequencing device (Amersham Pharmacia).
Analysis of the 16S rRNA gene sequences and the design of new probes.
The clone sequences were compared with sequences of reference organisms from the Ribosomal Database Project (release 8.0) (Maidak et al., 2001 ), which contains about 16000 sequences, including sequences of the altered Schaedler flora (Dewhirst et al., 1999
). The clone sequences and the 16S rDNA sequences of their nearest relatives were aligned; the ARB package (Ludwig et al., 1998
) was used to produce the multiple-sequence alignment. A phylogenetic tree, generated by using the neighbour-joining method with JukesCantor 2-correction parameter, was produced to illustrate the clustering of the different clone groups with their nearest relatives. The distance matrix (generated from the multiple-sequence alignment) used in the neighbour-joining analysis included stretches of sequence corresponding to E. coli positions 411464 and only used positions with more than 50% invariability, as implemented in the ARB software. Hence, the phylogenetic analysis was based on 1192 nt. The topology of the tree was analysed by bootstrap analysis (1000 replications). New probes were designed from the sequences of the clones. Existing probes were tested to identify the target groups of bacteria that were grouped on the basis of the obtained sequences, by screening for group-specific target sequences using the ARB software (Ludwig et al., 1998
). Specificity of the probes was tested by slot-blot hybridization of clones with known sequences using increasing stringency, i.e. increasing the temperature and the formamide concentration, as described previously (Franks et al., 1998
).
Slot blots and hybridization with 16s rRNA oligonucleotide probes.
A series of slot blots were generated for each set of clones obtained from the different intestinal sites. 32P-labelled 16S rRNA oligonucleotides were used to probe the slot blots, as described previously (Franks et al., 1998 ). Hybridizations were done overnight at 50 °C in 30% formamide. The samples were then washed at room temperature for 1 h, followed by washing at 37°C in 2xSSC and 0·1% SDS for 1 h.
Sample preparation and enumeration of intestinal bacteria.
The complete contents of the caeca and the large intestines of five 5-week-old FvB mice were collected and frozen at -20 °C. After thawing the samples on ice, PBS (137 mM NaCl, 2·7 mM KCl, 5·4 mM Na2HPO4, 1·8 mM KH2PO4, pH 7·4) was added to them to give 1 g sample (10 ml PBS)-1. For each sample, the suspension was homogenized by using a Polytron homogenizer for 5 s and centrifuged at 35 g for 15 min. The supernatant was mixed with 3 vols of freshly prepared 4% paraformaldehyde in PBS. The bacteria were fixed overnight at 4°C. An aliquot (2 ml) of the fixed bacteria was centrifuged at 12000 g for 10 min and washed twice with PBS. The pellet was taken up in 400 µl of 50% ethanol in PBS, which was kept at -20 °C. This procedure typically yielded 1x109 bacteria (ml ethanol-fixed stock)-1 (as counted with a PetroffHausser counting chamber; Hausser Scientific).
FISH.
From the ethanol-fixed stock, 6x106 cells were added to 300 µl of 50 °C hybridization buffer [0·9 M NaCl, 20 mM Tris/HCl (pH 7·2), 0·1% SDS] containing 10 ng oligonucleotide probe µl-1 (oligonucleotide probes with FITC incorporated into their 5' and 3' ends were synthesized by Operon Technologies). The bacteria/hybridization buffer/probe mixture was incubated overnight at 50 °C, to allow hybridization. The mixture was then centrifuged at 12000 g for 5 min. The resulting pellet was washed once with a 50 °C washing solution (hybridization buffer without SDS) and once with PBS (pH 8·5); it was then taken up in 500 µl PBS (pH 8·5).
Fluorescence microscopy.
To 200 µl of the hybridized bacteria, 10 µl of 4',6-diamidino-2-phenylindole (DAPI; 15 µg ml-1) was added. The bacteria were suspended in 3 ml PBS and then collected on a 0·2 µm filter (Millipore). The filter was mounted onto a slide using Vectashield fluorescence mounting medium (Vector Laboratories). Slides were viewed under oil immersion, using a Nikon microphot FXA epifluorescence microscope equipped with DAPI and FITC filter cubes and an MTI 3CCD camera. DAPI and FITC images were captured and analysed using COUNT-PRO PLUS software (Media Cybernetics). Percentages of bacterial populations of which the ribosomes hybridized to specific 16S fluorescently labelled oligonucleotide probes were counted in a total of 1000 DAPI-positive bacteria.
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RESULTS |
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Along with three known Bacteroides spp., a large group of our sequences (n=16) belonged to a separate, so far unrecognized branch of the Bacteroides group of bacteria. This new operational taxonomic unit (OTU) is designated here as MIB. Unique sequences in this OTU were obtained from samples from the small intestine (n=2), the large intestine (n=3) and the faeces (n=6) of two different mouse strains (FvB and C57BL/6) that were housed in two independent laboratories. To confirm the presence of these species within the mouse intestinal microflora, we isolated a bacterial species from the mouse caecum by plating serial dilutions of a caecal sample onto Brucella blood agar medium supplemented with 5% sheep blood. Random colonies from the plates were screened with a probe specific for the MIB OTU (MIB661; see below). Analysis of the 16S rRNA gene sequence of this isolate (MIB-CB3), obtained by direct sequencing of the PCR-amplified 16S rRNA gene (accession no. AJ418059), confirmed that this isolate belonged to the MIB OTU.
Eight of our library sequences clustered within the Eubacterium rectaleClostridium coccoides group. Two sequences, L10-4 and L10-21, were more distantly related to the E. rectaleC. coccoides group; these sequences had two and four mismatches, respectively, with the Erec482 probe. One sequence (L10-6), which occurred four times, was related (although distantly) to Verrucomicrobium spinosum and was grouped within the Verrucomicrobium group (Suau et al., 1999 ; Wilson & Blitchington, 1996
). Another sequence, also occurring four times, was distantly related to Bacillus mycoides and was referred to as belonging to the Bacillus mycoides group. Nine sequences represented by only one clone did not cluster together and were designated as miscellaneous (Table 1
). Four of these sequences (F13, L10-14, L10-21 and L11-2) belonged to the heterogeneous Clostridium leptum group, which includes the fusiform bacteria Clostridium sp. ASF356 and Eubacterium sp. ASF500. This number of clones was surprisingly low, since fusiform bacteria are morphologically the most dominant group of organisms present in mouse faecal samples.
Design of new probes and testing of existing probes
To obtain probes that could be used for the quantitative analysis of mouse intestinal and faecal bacterial populations we tested existing probes to see whether they could be used to identify the different groups of bacteria within these environments. For the E. rectaleC. coccoides group (Franks et al., 1998 ), the Bacteroides group (Manz et al., 1996
), the SFB (Snel et al., 1995
) and the Lactobacillus group (Harmsen et al., 1999
), the published probes were appropriate for identifying the different target groups (Table 2
). The Erec482 probe could target all of the new clones that belonged to the E. rectaleC. coccoides group. All of the sequences belonging to the new MIB OTU had one or more mismatches with Bac303. Therefore, we designed two new oligonucleotide probes, MIB661 and MIB724, which could identify members of this new OTU (Fig. 2
). MIB661 revealed all new members of the MIB OTU, except for clone F1, which had one mismatch with the probe. MIB724 could target most (9/11) members of the new OTU, but it had one mismatch with two sequences, S30-4 and S30-5. Furthermore, probes were designed that could bind to the Verrucomicrobium group (Ver620) and the Bacillus mycoides group (Bmy843) of bacteria (Table 2
; Fig. 2
). The specificity of the newly designed probes was tested using slot-blot hybridization, with the target clones as reference. Hybridizations were found to be optimal at 50 °C in a buffer containing 30% formamide. Increasing the formamide concentration to 50% or the hybridization temperature to 65 °C resulted in the loss of stable hybridization. Decreasing the formamide concentration did not significantly decrease the specificity of hybridization, unless it was accompanied by a decrease in the hybridization temperature.
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Sequence analysis of three of the clones that hybridized with Bact338 but not with any of the other specific probes confirmed that these clones lacked the target sequences for the probes used. The three clones were found to represent a Helicobacter species (L10-17) or additional, unrecognized species (L10-4 and L10-21).
Quantification of the bacterial species found within the murine caecum and large intestine by in situ hybridization
For a more accurate determination of the size of the bacterial population within the mouse intestinal tract, we isolated intestinal bacteria and quantified populations by in situ hybridization and fluorescence microscopy, using both the established and the newly designed fluorescently labelled 16S-rRNA-targeted oligonucleotide probes. Quantification of the bacterial populations was performed on specimens from the caeca and large intestines of 5-week-old FvB mice. The Bact338 probe was able to account for approximately 95% of all of the DAPI-stained bacteria in the large intestine and caecum. Since MIB661 and MIB724 targeted strongly overlapping sequences and revealed almost similar counts in the initial counts, only MIB661 was used for quantification of the tested samples. In the large intestine samples, 27±7·1% of the bacteria were labelled with Erec482, 0% were labelled with SFB1008, 10±3·6% were labelled with Lab158, 1% were labelled Ver620, 11±2·2% were labelled with Bac303 and 31±4·6% were labelled with MIB661; these values accounted for 80% of all of the bacteria present within these samples. In the caecal samples, 33±7·1% of the bacteria were labelled with Erec482, 1% were labelled with SFB1008, 11±2·5% were labelled with Lab158, 5±0·5% were labelled with Bac303, 19±4·0% were labelled with MIB661 and 2% were labelled with Ver620; these values accounted for 71% of the bacteria present within the caecal samples.
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DISCUSSION |
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Of the 16S rRNA gene clones analysed, 57% contained unique sequences. Thirty of these sequences were represented only once, whereas 10 sequences were present in multiple clones. About 25% of our 16S rRNA gene sequences differed only slightly (<2% nucleotide differences) from those of known bacterial species. This percentage includes non-cultivable members of the gut microflora, such as SFB, which dominate the small intestine of the mouse, especially around weaning; the numbers of SFB present in the small intestine decline after weaning, although they remain present in low numbers within the caecum (Snel et al., 1998 ). We identified five clones from the small intestine that belonged to the SFB; however, our in situ hybridization studies revealed that only 1% of the caecal bacteria belonged to this group. Our method for the isolation of intestinal bacteria may favour the retrieval of lumenal bacteria over that of attached bacteria, which may result in an underestimation of numbers for this bacterial group.
Four members of the altered Schaedler flora (ASF360 and ASF361, Lactobacillus sp.; ASF502, C. coccoides; ASF519, Bacteroides sp.) were identified in our clone libraries. Surprisingly, no clones were found in our libraries that corresponded to the fusiform bacteria of the altered Schaedler flora (ASF356, ASF492 and ASF500), even though fusiform bacteria are morphologically very dominant in faecal samples. These fusiform bacteria may be represented by the sequences belonging to the E. rectaleC. coccoides group or by the four sequences belonging to the C. leptum group. No new probe was developed to target these sequences belonging to the C. leptum group, since only four clones were found and they did not cluster in a clear manner. However, in clone libraries generated from human faecal samples, this group of bacteria is strongly represented (Suau et al., 1999 ; Wilson & Blitchington, 1996
). In future studies on the mouse gut microflora, it will be important to test new probes designed based on the sequences from the human faecal bacteria libraries. The fusiform bacteria are of particular interest, as a Gram-positive rod that belongs to this group was isolated from the rat intestine and has been found to be important in bile-acid metabolism (Eyssen et al., 1999
).
Of the unique sequences in our clone libraries, eight belonged to the E. rectaleC. coccoides group; these sequences could all be targeted with the existing Erec428 probe (Franks et al., 1998 ). The majority of these eight clones were derived from the large intestine, where bacteria of this group are commonly found.
One clone (L10-17) corresponded to a Helicobacter sp. (MIT 97-6810) that was recently isolated from the caecum of IL-10-deficient mice and was shown to be associated with the occurrence of colitis in these mice (Fox et al., 1999 ). As we have been able to isolate the 16S rRNA gene from the same Helicobacter sp. from immunocompetent mice, we have shown that this species can belong to the resident murine gut microflora without inducing pathology. Therefore, under conditions of a compromised immune system, this bacterium may opportunistically challenge the host.
We identified several Bacteroides spp., related or identical to known species, within our clone libraries that could be targeted with the existing Bac303 probe. A very surprising and important finding of this study was that the majority of these new sequences (n=11) formed a novel OTU within the CytophagaFlavobacterBacteroides phylum; we designated this OTU MIB. The phylogenetic position of this OTU was within the highly diverse Bacteroides group, although members of the OTU clustered tightly as a new lineage. This clustering was similar to that of the genera Prevotella and Porphyromonas, which form separate, distinct lineages within the Bacteroides group. The presence of members of the MIB OTU within the mouse intestinal tract was confirmed by isolation of a bacterial species belonging to this OTU from the mouse caecum. Analysis of all known Bacteroides sequences in the database showed no overlap between species containing the Bac303 sequence and our sequences that contained the new MIB-probe sequences. In the dot-blot assay, we found one clone that reacted with both of the MIB-probes and with the Bac303 probe; this result could represent some false hybridization by a clone that only had one mismatch with Bac303.
Clones of members of the MIB group were isolated from all parts of the mouse gastrointestinal tract, including the small and large intestine and the faeces. The new MIB probes, MIB661 and MIB724, identified a large population of small, rounded, rod-like bacteria that have not been detected previously with existing probes (Fig. 2). This suggests the usefulness of the MIB probes in quantification studies on intestinal populations.
Additional unique sequences from the libraries, while less abundant, were distantly related to the bacteria V. spinosum and Bacillus mycoides. Members of the Verrucomicrobium group of bacteria have also been identified in low numbers in human-derived 16S rRNA gene libraries (Suau et al., 1999 ; Wilson & Blitchington, 1996
) and in clone libraries of environmental samples, such as soil and water, but members of this group have not yet been identified in cultured bacteria. A new probe, Ver620, targeting the Verrucomicrobium group of bacteria has been designed; this probe detected low numbers (12%) of small, round bacteria in both the caeca and the large intestines of the mice studied (Fig. 2
). This probe may be more useful in defining bacterial populations within the small intestine, as it was identified from clones derived from that segment of the gastrointestinal tract. The Bacillus mycoides-related species identified in this study has not previously been described and was only represented by one sequence in our libraries, although this sequence did occur four times. The newly designed probe Bmy843, which was targeted at this species, did not detect Bacillus mycoides-related bacteria in the caeca or the large intestines of the mice studied here.
The application of 16S rRNA gene sequence analysis to studying the bacterial populations within the mouse gastrointestinal tract has uncovered large populations of previously unidentified bacterial species that contribute significantly to the composition of the intestinal commensal microflora. Using a combination of four pre-existing probes and our four newly designed probes we were able to identify the majority of bacteria within the murine caecum and large intestine. By using the newly designed and existing probes in FISH studies, we have established baseline information on the composition of the gut microflora from unmanipulated strains of inbred laboratory mice kept under specific pathogen-free conditions. Ultimately, similar approaches to the ones used here should allow a rapid assessment of commensal bacterial populations in a variety of mouse model systems looking at responses to diet, stress and disease.
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ACKNOWLEDGEMENTS |
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Received 25 February 2002;
revised 11 June 2002;
accepted 8 July 2002.