Detection,isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6–8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2 h exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, α-galactosidase, β-galactosidase, β-galactosidase-6-phosphate or α-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16:0 and C18:1ω9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (=DSM 101753(T) = LMG 29236(T)). We also demonstrated that Clostridium rectum and mortiferum Fusobacterium represent the same species, with nomenclatural priority for the latter.     

Phenotypic and genotypic analyses of clinical Fusobacterium nucleatum and Fusobacterium periodonticum isolates from the human gut

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.     

Glucose utilization and growth response to protein hydrolysates byFusobacterium species

Glucose uptake was measured in the supernatants of 18 strains ofFusobacterium species cultured in BM medium. Some species, such asF. nucleatum andF. necrophorum, used between 25% and 48% of the glucose in the medium, but the terminal pH remained near neutral. By contrast, strains ofF. mortiferum andF. necrogenes used on average over 90% of the available glucose in the medium and produced a predictably low acidic pH. Strains ofF. varium used between 86% and 91% of the glucose present but produced a near neutral pH of between 5.8 and 5.9. The metabolic fate of glucose inF. varium was, therefore, examined in more detail. Glucose stimulated the growth of this species, and [¹⁴C]glucose was incorporated into the metabolic end products and various cellular components. Protein hydrolysates, tested for their growth-promoting effects onFusobacterium species, produced two general growth response patterns. Most species grew prolifically on trypticase, proteose peptone, and yeast extract, but poorly in casamino acids and vitamin-free casamino acids. Growth in bactocasitone was poor, but for three species,F. necrophorum, F. varium, andF. nucleatum, there was an approximately linear growth response up to 0.5%. These results suggest a major role for nitrogen metabolism but do not preclude glucose as an energy source in at least some species ofFusobacterium.     

A selective medium for Fusobacterium spp.

J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Identification of Mycobacterium ulcerans in the Environment from Regions in Southeast Australia in Which It Is Endemic with Sequence Capture-PCR

We recently described the use of PCR to identify the environmental source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the M. ulcerans-specific insertion sequence, IS2404. In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two M. ulcerans sequences (IS2404 and IS2606) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of M. ulcerans infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for M. ulcerans. Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of M. ulcerans cells in positive samples from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.     

A selective medium for Fusobacterium spp

A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 microgram/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Fusobacterium nucleatum subsp. fusiforme Gharbia and Shah 1992 is a Later Synonym of Fusobacterium nucleatum subsp. vincentii Dzink et al. 1990

On the basis of the DNA–DNA hybridization patterns and phenotypic characteristics, Fusobacterium nucleatum was classified into five subspecies. Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme. The aim of this study was to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis using a single sequence (24,715 bp) concatenated 22 housekeeping genes of eight F. nucleatum strains including type strains of five F. nucleatum subspecies. The phylogenetic analysis indicated that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. These results suggested that F. nucleatum subsp. fusiforme and F. nucleatum subsp. vincentii can be classified into a single subspecies. F. nucleatum subsp. vincentii was early published name; therefore, F. nucleatum subsp. fusiforme Gharbia and Shah 1992 can be regarded as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.     

Characterization of aid1, a Novel Gene Involved in Fusobacterium nucleatum Interspecies Interactions

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.     

Long-Term Alteration of Intestinal Microbiota in Patients with Ulcerative Colitis by Antibiotic Combination Therapy

Previous work has demonstrated that intestinal bacteria, such as Fusobacterium varium (F. varium), contribute to the clinical activity in ulcerative colitis (UC); thus, an antibiotic combination therapy (amoxicillin, tetracycline, and metronidazole (ATM)) against F. varium can induce and maintain UC remission. Therefore, we investigated whether ATM therapy induces a long-term alteration of intestinal microbiota in patients with UC. Patients with UC were enrolled in a multicenter, randomized, double-blind, placebo-controlled study. Biopsy samples at the beginning of the trial and again at 3 months after treatment completion were randomly obtained from 20 patients. The terminal restriction fragment length polymorphism (T-RFLP) in mucosa-associated bacterial components was examined to assess the alteration of the intestinal microbiota. Profile changes of T-RFLP in mucosa-associated bacterial components were found in 10 of 12 patients in the treatment group and in none of 8 in the placebo group. Dice similarity coefficients using the unweighted pair group method with arithmetic averages (Dice-UPGMA) confirmed that the similarity of mucosal microbiota from the descending colon was significantly decreased after the ATM therapy, and this change was maintained for at least 3 months. Moreover, at 3 months after treatment completion, the F. varium/β-actin ratio, examined by real-time PCR using nested PCR products from biopsy samples, was reduced less than 40% in 8 of 12 treated patients, which was higher, but not significantly, than in 4 of 8 patients in the placebo group. Together, these results suggest that ATM therapy induces long-term alterations in the intestinal microbiota of patients with UC, which may be associated, at least in part, with clinical effects of the therapy.     

Development of species-specific polymerase chain reaction primers for detection of Fusobacterium periodonticum

The purpose of this study was to develop species-specific PCR primers for detection of Fusobacterium periodonticum. The specificity data showed that two sets of PCR primers, Fp-F3/Fp-R2 and Fp-F1/Fp-R2 PCR, produced amplicons from all the F. periodonticum, but not from the other species tested, which included 12 Fusobacterium species or subspecies and representative oral bacteria. The sensitivity of the primer sets was 4 or 40 pg of the chromosomal DNA from F. periodonticum ATCC 33693(T) . These results suggest that these two sets of PCR primers are quite sensitive in detection of F. periodonticum in molecular epidemiological studies of periodontitis.     

A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model

We have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the form of the PCR product(s), while the length of the PCR product makes it possible to differentiate between bacterial strains. If there is a single copy of the target sequence within genomic DNA, one specific PCR product is created (simplex ddLMS PCR), whereas for multiple copies of the gene the fingerprinting patterns can be obtained (multiplex ddLMS PCR). The described ddLMS PCR method is designed for rapid and specific strain differentiation in medical and microbiological studies. In comparison to other LM PCR it has substantial advantages: enables specific species'' DNA-typing without the need for pure bacterial culture selection, is not sensitive to contamination with other cells or genomic DNA, and gives univocal “band-based” results, which are easy to interpret. The utility of ddLMS PCR was shown for Acinetobacter calcoaceticus-baumannii (Acb) complex, the genetically closely related and phenotypically similar species and also important nosocomial pathogens, for which currently, there are no recommended methods for screening, typing and identification. In this article two models are proposed: 3′ recA-ddLMS PCR-MaeII/RsaI for Acb complex interspecific typing and 5′ rrn-ddLMS PCR-HindIII/ApaI for Acinetobacter baumannii intraspecific typing. ddLMS PCR allows not only for DNA-typing but also for confirmation of species in one reaction. Also, practical guidelines for designing a diagnostic test based on ddLMS PCR for genotyping different species of bacteria are provided.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Identification by quantitative gas chromatography of common Fusobacterium species isolated from clinical specimens

Two unusual hydroxy acids have recently been described in broth cultures of certain anaerobes. Based on these and other known metabolic end-products, an identification scheme from quantitative gas chromatography for common Fusobacterium species was established. After examining 137 isolates and eight type strains the scheme agrees with conventional procedures. This was assisted by only three simple tests: gas, indole and lipase production. No other subculture was needed.     

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

Identification and Characterization of Fusolisin,the Fusobacterium nucleatum Autotransporter Serine Protease

Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61–65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55–101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96–113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55–65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.     

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.     

Heterogeneity among Mycobacterium ulcerans from French Guiana Revealed by Multilocus Variable Number Tandem Repeat Analysis (MLVA)

Buruli ulcer is an emerging and neglected tropical disease caused by Mycobacterium ulcerans. Few cases have been reported so far in the Americas. With 250 cases reported since 1969, French Guiana is the only Buruli ulcer endemic area in the continent. Thus far, no genetic diversity studies of strains of M. ulcerans from French Guiana have been reported. Our goal in the present study was to examine the genetic diversity of M. ulcerans strains in this region by using the Multilocus Variable Number Tandem Repeat Analysis (MLVA) approach. A total of 23 DNA samples were purified from ulcer biopsies or derived from pure cultures. MVLA was used in the study of six previously-described Variable Number of Tandem Repeat (VNTR) markers. A total of three allelic combinations were characterized in our study: genotype I which has been described previously, genotype III which is very similar to genotype I, and genotype II which has distinctly different characteristics in comparison with the other two genotypes. This high degree of genetic diversity appears to be uncommon for M. ulcerans. Further research based on complete genome sequencing of strains belonging to genotypes I and II is in progress and should lead soon to a better understanding of genetic specificities of M. ulcerans strains from French Guiana.     

Phylogenetic Positions and Assignment of Swine and Ovine Corynebacterial Isolates Based on the 16S rDNA Sequence

The nucleotide sequences of the 16S ribosomal RNA gene (rDNA) of swine and ovine corynebacterial strains were determined. The sequences of the strains that identified as Corynebacterium pseudotuberculosis by their biochemical characteristics were homologous with each other. The phylogenetic position of C. pseudotuberculosis strains was closest to C. ulcerans and next closest to C. diphtheriae. The nucleotide sequence of another swine isolate, SC8, was similar to that of a recently proposed species, C. seminale, and a non-validated species, “C. glucuronolyticum,” with about 0.01 to 0.02 evolutionary distances. Analysis of the predicted secondary structure of the 16S rRNA molecule agreed with the close phylogenetic relationships between C. pseudotuberculosis and C. ulcerans and between C. seminale and strain SC8.