Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.     

Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation.     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii

Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29 NotI restriction fragments. The average resolution is 72.5 kb, or about 3. 5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative oriC has been located on a 27.5-kb NotI fragment. Gene organization upstream the oriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.     

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with BsaI and BbsI, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.     

PCR-ribotyping of Xenorhabdus and Photorhabdus isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.     

Evaluation of a new method for identification of bacteria based on sequence homology of 16S rRNA gene

A new identification method for bacteria based on partial sequences of divergent regions of the 16S rRNA gene was evaluated. The method involves PCR-based amplification of 16S rRNA gene fragments, followed by sequencing and comparison of sequences of about 300 nucleotides with those in the database of NCBI (National Center for Biotechnology Information) via the Internet. Most of the bacteria tested could be identified at the species level even if some unread nucleotides were present in the sequence. Although this method still requires improvement, it has the potential to be a highly reliable and practical identification method for bacteria.     

Molecular structure of a wheat chromosome end healed after gametocidal gene-induced breakage

In the progeny of the monosomic addition line of common wheat, Triticum aestivum, carrying the gametocidal chromosome of Aegilops cylindrica, deletion chromosomes carrying the break point within the nucleolar organizing region of chromosome 1B appeared. Attempts were made to amplify the break points by PCR using primers of telomere and rDNA (rRNA gene). In one deletion line, specific amplification of DNA fragments including the 18S rRNA gene, telomere repeats, and their junction occurred. At the junction of telomere and rRNA gene there was a 31-bp inverted duplication of the rRNA gene. Telomere sequences were initiated from the sequence TAG in the duplication. Between the duplications a small sequence was also inserted. This novel DNA sequence at the break point indicates that the breakage-fusion-bridge cycle(s) took place after the first chromatin breakage by the gametocidal gene.     

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.     

Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels

On the basis of DNA-DNA hybridization data, nine intestinal spirochete strains were grouped into five genospecies. Three of these genospecies were previously recognized Serpulina species, Serpulina hyodysenteriae (type strain, B78), Serpulina innocens (type strain, B256), and Serpulina pilosicoli (type strain, P43/6/78; previously "Anguillina coli"). The other two genospecies were found to be new Serpulina species, for which we propose the names Serpulina intermedia sp. nov. (with type strain PWS/A) and Serpulina murdochii sp. nov. (with type strain 56-150). S. intermedia and S. murdochii cells had a typical spirochete ultrastructure with 22 to 28 periplasmic flagella per cell. Various soluble sugars were growth substrates for S. intermedia and S. murdochii. During growth in basal heart infusion broth supplemented with fetal calf serum beneath an O2-N2 (1:99) atmosphere, cells of these new species consumed oxygen and glucose and produced H2, CO2, acetate, butyrate, and ethanol. The G + C content of the DNA of S. murdochii 56-150T was 27 mol%, and the G + C content of the DNA of S. intermedia PWS/AT was 25 mol%. In addition, a restriction fragment length polymorphism-PCR assay for the detection of intestinal spirochetes was developed. The assay was based on generation and restriction endonuclease analysis (with HinfI, TaqI, Sau3A, and MboII) of a 558-bp amplicon of ribosomal DNA (rDNA) encoding 16S rRNA. The PCR amplification was specific for Serpulina species and Brachyspira aalborgi. Four restriction digest patterns were found for the five Serpulina species. HinfI restriction differentiated S. murdochii and S. innocens from the other species. Sau3A and TaqI restrictions gave unique fragment patterns for S. murdochii and S. pilosicoli, respectively. S. hyodysenteriae and S. intermedia DNAs gave the same fragment pattern regardless of the enzyme tested. B. aalborgi was differentiated from the Serpulina species by MboII digestion of the 16S rDNA amplicon.     

IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF

The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages. Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-gamma. While C. burnetii multiplied in untreated monocytes, IFN-gamma reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h. IFN-gamma also affected the viability of infected cells. Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-gamma, it reached a maximum after 48 h and was followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-gamma. The role of cytokines was also investigated. IFN-gamma elicited a moderate release of IL-1beta in infected monocytes. Moreover, the IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1beta was not involved in the bacterial killing induced by IFN-gamma. TNF was involved in IFN-gamma-induced killing of C. burnetii and cell death. IFN-gamma induced mRNA expression and sustained secretion of TNF. Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-gamma-dependent killing of C. burnetii and cell death. These results suggest that IFN-gamma promotes the killing of C. burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.     

Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae

Representatives of the family Methanosarcinaceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Methanosarcinaceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16S rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 +/- 0.541 times more changes than the respective pairs of 16S rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16S rRNA gene. The qualitative similarity of the mcrI and 16S rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published mcrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinaceae do not have distinct mcrII genes.     

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.