Phenotypic and genotypic analyses of clinical Fusobacterium nucleatum and Fusobacterium periodonticum isolates from the human gut.
ABSTRACT Fusobacterium nucleatum is a Gram-negative anaerobic rod that is part of the normal human microflora, and has also been associated with various infections. Bacterial strains belonging to the species are typically heterogeneous in both phenotype and genotype, which can hinder their identification in a clinical setting. The majority of F. nucleatum isolates originate from oral sites, however the species is also a resident of the human gastrointestinal tract. The aim of this study was to compare F. nucleatum isolates from human intestinal biopsy samples to try and determine whether isolates from this site are divergent from oral isolates. We used a variety of phenotypic and genotypic markers to compare 21 F. nucleatum and Fusobacterium periodonticum isolates from the GI tract to oral isolates and recognized type strains in order to study heterogeneity within this set. 16S rDNA and rpoB gene sequence analysis allowed us to build phylogenetic trees that consistently placed isolates into distinct clusters. 16S rDNA copy number analyses using Denaturing Gradient Gel Electrophoresis (DGGE) demonstrated potential for use as a method to examine clonality amongst species. Phenotypic analyses gave variable results that were generally unhelpful in distinguishing between phylogenetic clusters. Our results suggest that a) F. periodonticum isolates are not restricted to the oral niche; b) phenotypic classification is not sufficient to subspeciate isolates; c) heterogeneity within the species is extensive but constrained; and d) F. nucleatum isolates from the gut tend to identify with the animalis subspecies.
- SourceAvailable from: Richard Moore[Show abstract] [Hide abstract]
ABSTRACT: An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.Genome Research 02/2012; 22(2):299-306. DOI:10.1101/gr.126516.111 · 13.85 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase beta-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.Journal of clinical microbiology 12/2009; 48(2):545-53. DOI:10.1128/JCM.01631-09 · 4.23 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.Journal of clinical microbiology 04/2010; 48(4):1283-90. DOI:10.1128/JCM.01620-09 · 4.23 Impact Factor