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1 Department of Oral Biology, Royal Dental College, Faculty of Health Sciences, University of Aarhus, DK-8000 Aarhus C, Denmark
2 Institute of Medical Microbiology and Immunology, Faculty of Health Sciences, University of Aarhus, DK-8000 Aarhus C, Denmark
3 Department of Bacterial and Inflammatory Diseases, National Public Health Institute (KTL), FIN-00300 Helsinki, Finland
Correspondence
Ellen V. G. Frandsen
ef{at}microbiology.au.dk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the near-full-length 16S rRNA gene sequences reported in this paper are DQ009623 for strain AHN8855T, DQ009624 for strain A0404T and DQ009622 for strain AHN8471. The GenBank/EMBL/DDBJ accession numbers for the other 16S rRNA gene sequences determined in this study are DQ012296–DQ012382.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Alexandrakis et al., 2000; Martino et al., 2001; Okada et al., 2002; Bonatti et al., 2003; Sabbatani et al., 2004). They are fairly often associated with infections in children with neutropenia or leukaemia (Parenti & Snydman, 1985; Campbell & Edwards, 1991). The three original Capnocytophaga species, Capnocytophaga ochracea, Capnocytophaga sputigena and Capnocytophaga gingivalis, were initially isolated from periodontitis in adults, but subsequent studies demonstrated their presence also at periodontally healthy sites in both children and adults. Thus, their association with periodontal disease is a matter of controversy (Dzink et al., 1988; Moore & Moore, 1994; Colombo et al., 1998; Paster et al., 2001; Kumar et al., 2003). Early reports correlated the pathogenic potential of Capnocytophaga to an immunosuppressive effect on polymorphonuclear neutrophils, including suppression of their chemotaxis (Shurin et al., 1979; Van Dyke et al., 1982). Subsequently, sonic extracts of Capnocytophaga were reported to inhibit lymphocyte proliferation in response to mitogen (Ochiai et al., 1998). An uneven distribution of virulence factors among isolates suggests that pathogenic potential may be associated with some of the species, and possibly only with certain members of the species (Irving et al., 1978; Laughon et al., 1982a). Especially, C. gingivalis has stronger proteolytic activity than C. ochracea and C. sputigena (Gazi et al., 1997), and LPS from C. gingivalis has haemagglutinating activity, in contrast to LPS from C. ochracea (Okuda & Kato, 1987).
Studies of the pathogenic significance of individual Capnocytophaga species are hampered by ambiguous assignment of isolates to species level by conventional phenotypic tests (Laughon et al., 1982a; Kristiansen et al., 1984; Speck et al., 1987; Khwaja et al., 1990). The description of two novel oral species, Capnocytophaga haemolytica and Capnocytophaga granulosa, seems to have added to these difficulties (Yamamoto et al., 1994). Nevertheless, cluster analysis of RFLP patterns of 16S rRNA genes allowed differentiation between the three original species (Wilson et al., 1995), and multilocus enzyme electrophoresis (MLEE) proved to be a valuable method to distinguish between the five oral Capnocytophaga species (Frandsen & Wade, 1996). Subsequently, oligonucleotide probes specific for all recognized Capnocytophaga species were reported (Conrads et al., 1997).
Despite improvements in potential means for identification, recent studies have revealed conflicting results regarding the Capnocytophaga species that colonize children. While a high prevalence of C. ochracea is a uniform finding, reports on the prevalence of C. gingivalis and C. sputigena vary among investigations (Conrads et al., 1996; Kamma et al., 2000a, b; Hayashi et al., 2001; Kimura et al., 2002; Ooshima et al., 2003). Moreover, a considerable diversity within the Capnocytophaga species has been demonstrated (Khwaja et al., 1990; Wilson et al., 1995; Frandsen & Wade, 1996). To elucidate the significance of Capnocytophaga species in disease in children and adults, it is mandatory to be able to identify clinical isolates unequivocally. The present study was undertaken to test the ability of cluster analysis based on MLEE results and 16S rRNA gene sequences to identify to species level the Capnocytophaga that colonize children, and to examine the diversity of members of the genus Capnocytophaga within individual subjects. As a result of the phylogenetic and phenotypic studies we describe a novel species and a novel genospecies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) on the basis that the cultured samples yielded both yellow–orange and beige Capnocytophaga suspect colonies (Leadbetter et al., 1979). All colonies with these characteristics were selected. This resulted in 62 isolates that fulfilled the characteristics of Capnocytophaga: aerotolerance (growth in air plus 5 % CO2 or air alone), negative catalase reaction and no indole production. A range of two to nine isolates per child were investigated, including two oxidase-positive strains with characteristics that otherwise were in accordance with the original description of Capnocytophaga (Socransky et al., 1979). The isolates were considered representative of the Capnocytophaga population in the children and were given a code consisting of the prefix AHN and a number. In addition, 40 type and reference strains of the five oral human Capnocytophaga species were included. These included strains whose species affiliation had been ascertained by DNA relatedness analysis (Speck et al., 1987), as well as strains that clustered together with these strains in a dendrogram based on MLEE (Frandsen & Wade, 1996). The species of animal origin (Capnocytophaga canimorsus and Capnocytophaga cynodegmi) were not included as reference strains because their key characteristics differ from those of the genus Capnocytophaga (Yamamoto et al., 1994). The reference strains used are shown in Table 1. All strains were cultivated on plaque agar (Jensen et al., 1968) at 37 °C in an atmosphere of air enriched with 5 % CO2. For inspection of colonial appearance, strains were grown on modified Columbia blood agar (Hunt et al., 1986). The modifications consisted of omission of antibiotics and of the use of freeze–thawed horse blood.
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, except that sonication was done intermittently for 5 min. The analyses were performed as described by Selander et al. (1986). Abbreviations of the enzyme names together with the buffer systems used according to Selander et al. (1986) are indicated in parentheses. The enzymes were: glutamate dehydrogenase (GDH, buffer system A), malate dehydrogenase (MDH, A), adenylate kinase (ADK, G), glyceraldehyde-3-phosphate dehydrogenase (GPI, G), leucyl-glycyl-glycyl peptidase (LGG, G), glutamic-oxaloacetic transaminase (GOT, G), hexokinase (HEX, H), mannose-phosphate isomerase (MPI, H), nucleoside phosphorylase (NSP, I), carbamate kinase (CDK, I), phosphoglucomutase (PGM, I) and phosphoglucose isomerase (PGI, I).
Determination of genetic diversity (h) at an enzyme locus among strains, i.e. the probability that two randomly chosen strains have different alleles of the locus, genetic distance between electrophoretic types (ETs), and construction of the dendrogram were performed using a matrix of pairwise genetic distances between ETs and the programs ETCLUS and ETMEGA of T. S. Whittam, Pennsylvania State University, PA, USA (http://foodsafe.msu.edu/Whittam/programs) and MEGA version 2.1 (Kumar et al., 2001).
16S rRNA gene sequencing.
Partial 16S rRNA gene fragments were amplified by PCR using forward primer 5'-AGAGTTTGATYMTGGCTCAG, positions 8–27 in the 16S rRNA gene of Escherichia coli, and reverse primer 5'-TATTACCGCGGCTGCTGGCA (positions 534–515). Cells from 7 ml liquid plaque medium (Jensen et al., 1968) were harvested, resuspended in 200 µl double-distilled water, boiled for 5 min and frozen. Bacterial suspension (1 µl) was used as template in the PCR with Ready to Go beads (Amersham) and 10 pmol of each primer in a 25 µl reaction volume. The samples were initially denatured at 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, annealing at 60 °C for 1 min, 72 °C for 2 min and then a final extension at 72 °C for 8 min in a Perkin-Elmer type 480 DNA thermal cycler. The PCR products were purified using Wizard Minicolumns (Promega). Sequencing in both directions was performed with the same primers using a Thermo Sequenase dye terminator cycle sequencing kit (Amersham) and analysed on an Applied Biosystems PRISM 377 automated sequencer. The sequence read was approximately 330 bp, corresponding to a stretch from position 97 to 425 in the C. ochracea 16S rRNA gene. Phylogenetic analysis was performed as described above using the MEGA program version 2.1. Distance options were the Kimura two-parameter model and calculation of bootstrap values was based on 500 replications.
Near-full-length 16S rRNA gene sequences were determined for strains in clusters that were considered putative novel species according to the phylogenetic analyses of the MLEE and partial 16S rRNA gene data. The type strain of C. haemolytica, for which the 16S rRNA gene sequence was not available in databases at the time of investigation, was included. We used two primer pairs for amplification: the forward primer 5'-AGAGTTTGATYMTGGCTCAG, positions 8–27 in the E. coli 16S rRNA gene, combined with the reverse primer 5'-CCGGGAACGTATTCACCG, positions 1361–1344 in the C. ochracea 16S rRNA gene, and 5'-GCATGGTTGTCGTCAGCTC, positions 1027–1045 in the C. ochracea 16S rRNA gene, combined with the reverse primer 5'-CCGGGTTTCCCCATTCGG, position 130–113 in the 23S rRNA gene of E. coli. This resulted in two overlapping fragments which covered the 16S rRNA gene, except for the first 7 nt. For sequencing we used the same primers and six additional primers. The forward primers were 5'-ACGGGAGGCAGCAGT, positions 343–357 in the E. coli 16S rRNA gene, 5'-ATTAGATACCCTGGTAG (positions 785–802) and 5'-GGAATCGCTAGTAATCG (positions 1336–1352). The reverse primers were 5'-CTCACTGCTGCCTCCCGT, positions 348–330 in the C. ochracea 16S rRNA gene, 5'-CTACCAGGGTATCTAATC, positions 802–785 in the E. coli 16S rRNA gene, and 5'-GAGCTGACGACAACCATGC, positions 1045–1027 in the C. ochracea 16S rRNA gene. 16S rRNA gene sequences of the type strains of C. ochracea (GenBank accession no. U41350), C. sputigena (X67609), C. gingivalis (X67608), C. granulosa (U41348), C. canimorsus (L14637) and C. cynodegmi (L14638) were taken from GenBank. Alignments of the 16S rRNA gene sequences to those of the respective type strains and calculation of identity were performed with the BLAST program (http://www.ncbi.nlm.nih.gov) (Tatusova & Madden, 1999).
DNA–DNA relatedness and DNA G+C content assays.
DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite (Cashion et al., 1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) with modifications (Huß et al., 1983; Escara & Hutton, 1980), using a model 2600 spectrophotometer equipped with a model 2527-R thermoprogrammer and plotter (Gilford Instrument Laboratories). Renaturation rates were computed with the TRANSFER.BAS program of Jahnke (1992). The DNA G+C content was determined by the HPLC method described by Tamaoka & Komagata (1984) with the modifications described by Mesbah et al. (1989). The experiments were performed at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunsweig, Germany.
Biochemical tests.
All strains and isolates were tested for haemolysis on horse blood agar and for oxidase activity. Fermentation of carbohydrates was evaluated both as a colour change induced in phenol red broth base and as a pH drop measured in trypticase broth (Socransky et al., 1979), each supplemented with 1 % of the following sugar substrates: amygdalin, arabinose, cellobiose, fructose, galactose, glucose, lactose, raffinose, sucrose and xylose. The pH measurements were evaluated as indicated by Socransky et al. (1979): a pH drop 
1.0 units below the pH of both the uninoculated carbohydrate broth and the inoculated carbohydrate-free broth controls was recorded as a positive fermentation. A pH drop between 0.5 and 0.99 units was recorded as a weak fermentation, and a pH change of less than 0.5 units was considered negative. Hydrolysis of aesculin and starch was tested according to Cowan (1974). Dextran hydrolysis was measured by the method of Staat et al. (1973). The ability to produce β-galactosidase (ONPG) and N-acetyl-β-glucosaminidase and to degrade N-benzoyl-DL-arginine-2-naphthylamide was detected as described previously (Kilian, 1978; Laughon et al., 1982b). API ZYM enzyme-substrate tests were performed as recommended by the manufacturer (bioMérieux). A score of 3–5, corresponding to 20 nmol of substrate hydrolysed, was recorded as a positive reaction and a score of 1–2, corresponding to 5–20 nmol, was recorded as weakly positive. The following tests were performed on selected strains: hydrolysis of gelatin [incubation for 7 and 14 days at 37 °C, followed by refrigeration as described by Cowan (1974)] and urea (Kilian, 1985), reduction of nitrate using freshly prepared indole-nitrite medium (BBL) supplemented with haemin and menadione as described by Holdeman et al. (1977) and fermentation of glycogen, inulin, melibiose and starch (Socransky et al., 1979). To test for human IgA1 cleavage, human dimeric IgA1 from a patient (Kah) with multiple myeloma, purified as described previously (Frandsen, 1994), was used. Bacterial colonies were suspended in 40 µl of 1 mg IgA1 ml–1 solution and subjected to immunoelectrophoresis after incubation for 24 h at 36 °C (Kilian, 1981).
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RESULTS |
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). Treating the absence of enzyme activity for these two enzymes as either missing data or as a signifying ‘zero allele’ resulted in identical clustering of strains in the MLEE dendrograms.
Fig. 1 presents the UPGMA tree based on MLEE data. The reference strains of the five recognized human Capnocytophaga species clustered in the same subdivisions as observed previously (Frandsen & Wade, 1996), supporting the usefulness of MLEE for species differentiation in this genus. The MLEE dendrogram revealed two major lineages, termed divisions MLEE-A and MLEE-B, each of which showed several subdivisions (marked in Fig. 1). A negative reaction for the enzyme HEX and a positive reaction for the enzyme GOT were characteristic of strains in MLEE-B1 (C. granulosa cluster) and MLEE-B2 (C. gingivalis cluster). All other isolates and strains were positive for HEX and negative for GOT, except for AHN9647 and AHN10017 from division A, which were positive for GOT.
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) and nine isolates (plus four duplicates) belonged to C. sputigena (MLEE-A5 in Fig. 1). Three child isolates clustered with C. granulosa, whereas no C. gingivalis or C. haemolytica isolates were found. A total of 14 isolates clustered independently of the clusters that represented recognized species. These were located in the following clusters: MLEE-A1 (five isolates), MLEE-A2 (three isolates), MLEE-A3 (one isolate), MLEE-A4 (four isolates) and MLEE-A7 (one isolate) (Fig. 1). Despite the limited number of isolates from each child, intra-subject diversity was observed (Table 2).
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Fig. 2 presents the neighbour-joining tree based on partial (approx. 330 bp) 16S rRNA gene sequences (SEQ-tree). The maximum-parsimony tree showed essentially the same topology (data not shown). The SEQ dendrogram also consisted of two divisions, SEQ-A and SEQ-B, each of which showed a number of subdivisions. The clustering profile of the SEQ dendrogram had an overall similarity to the profile of the MLEE dendrogram. The differences observed did not represent stable clusters according to bootstrap values (Fig. 2).
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Based on the phylogenetic analyses of data from MLEE and partial 16S rRNA gene sequencing, three evolutionary lineages could represent putative novel species, as none of them included recognized type strains and they separated from other clusters at the same level as recognized species. SEQ-B1 contained only one isolate; it was therefore not investigated further. SEQ-A3 contained strains AHN8471, AHN9528, AHN9576, AHN9607 and AHN9798, which clustered in MLEE-A3+A4, plus AHN8751 from MLEE-A6. SEQ-A5 contained strains AHN8855, AHN8708, AHN8725, AHN8730 and AHN8996, which clustered in MLEE-A1, plus AHN9708 from MLEE-A2.
Fig. 3 presents the neighbour-joining tree based on nearly full-length 16S rRNA gene sequencing of the above-mentioned strains together with the type strain of C. haemolytica (A0404T) (Fig. 3). The 16S rRNA gene sequence of all strains included 1484–1491 bp, except for strain AHN8730 which, due to ambiguous sequences, was not readable beyond position 1331. The strains within each of the two novel taxa showed 99 % mutual similarity (no gaps). The closest relatives to both taxa were C. ochracea and C. sputigena (95 % similarity and 8–10 gaps). Lower sequence similarities (
92 %) were found to all other described species of the genus Capnocytophaga, including the animal species C. cynodegmi and C. canimorsus, and to the strains in the other novel taxon. Strain AHN9708 from MLEE-A2 and SEQ-A5 was lost before near-full-length sequencing and DNA relatedness analyses. Strains AHN10044 and AHN9756 from MLEE-A2 had only 96 % similarity (0–1 gap) to the strains in SEQ-A5.
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). AHN9528 approached this threshold (68.6 %) and had a significantly lower relatedness to the type strains of both C. ochracea (33.6 %) and C. sputigena (51.8 %), for this reason it was considered part of the new taxon. The taxonomic position of the remaining isolate, AHN9576, was uncertain due to a combination of low DNA relatedness and high 16S rRNA gene sequence similarity to AHN8471. Consequently, it was considered aberrant. In addition to strain AHN8471, taxon AHN8471 consisted of AHN9528, AHN9607, AHN9798 and AHN8751. The four isolates clustering with AHN8855T in both the MLEE and the SEQ dendrograms showed DNA relatedness from 72.0 to 75.6 %. AHN9708 from MLEE-A2 was lost before these experiments and was thus unavailable for analyses and AHN10044 and AHN9756, also from MLEE-A2, had low values of DNA relatedness (<42 %). In addition to AHN8855T, taxon AHN8855 consisted of AHN8708, AHN8725, AHN8730 and AHN8996. The DNA G+C content of AHN8471 was 38.8 mol% and that of AHN8855T was 41.3 mol%.
Biochemical characterization
With the purpose of identifying phenotypic tests suitable for species differentiation, all 102 isolates and reference strains of human oral Capnocytophaga species were subjected to a battery of 37 biochemical tests, the results of which are presented in Table 3. The isolates and strains representing the individual species are those that clustered unequivocally with the type and reference strains in both the MLEE and the SEQ dendrograms. The results for C. haemolytica are based on the type strain only. In agreement with the original description of the species, this strain had lost its β-haemolytic activity (Yamamoto et al., 1994). The species C. cynodegmi and C. canimorsus were not included in Table 3 because these are animal species and are reported to be distinct from human Capnocytophaga species by positive catalase and oxidase reactions (Brenner et al., 1989). Strains and isolates in the present study were negative in catalase and oxidase reactions, except for WW331 and AHN9647, which were positive for oxidase. Twenty of the tests showed variations among isolates (Table 3). Of these, a minor proportion was suitable for species differentiation: acid production from cellobiose, amygdalin and sucrose, dextran hydrolysis and tests for trypsin-like (hydrolysis of N-benzoyl-DL-arginine-2-naphthylamide) and β-glucosaminidase activities when performed in the API ZYM kit. The two latter tests had a much higher sensitivity, leading to more positive reactions when they were performed with alternative chromogenic substrates (data not shown). This was also the case for β-galactosidase (ONPG) activity, where all strains were positive when performed with alternative chromogenic substrates.
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. Additional tests selected from the proposed minimal standard tests for describing new taxa in the genus Capnocytophaga (Bernardet et al., 2002) were performed on the strains from taxon AHN8471 and two reference strains each of C. ochracea (CCUG 9716T and CCUG 15407) and C. sputigena (CCUG 9714T and CCUG 9277). None of the strains hydrolysed gelatin or urea, reduced nitrate or fermented melibiose. All strains fermented glycogen and starch, and all strains, except AHN8471, fermented inulin. Thus, inclusion of these additional tests did not result in differential tests for taxon AHN8471. Strains from taxon AHN8855 reduced nitrate, and tests for hydrolysis of gelatin and urea and fermentation of melibiose, glycogen, starch and inulin were negative, except for AHN8730, which fermented starch and inulin.
Upon primary isolation on Brucella blood agar, colonies of the strains belonging to taxon AHN8855 were beige (Könönen et al., 1994). We found that this feature was variable and depended on the substrate. On plaque agar plates (Jensen et al., 1968) containing haemolysed blood, the colonial appearance varied from beige to yellow–orange and pink. On Columbia blood agar containing defibrinated freeze–thawed blood (modified from Hunt et al., 1986), the colonial appearance varied from beige to yellow–orange, green or pink. Strains belonging to taxon AHN8471 produced typical yellow–orange or green colonies on fresh plates of plaque agar and Columbia blood agar, whereas use of 10-day-old plates for inoculation resulted in beige colonies for some strains.
Of the 102 isolates and strains tested, 93 (91 %) cleaved human IgA1 in a pattern typical of IgA1 protease activity, as revealed by immunoelectrophoresis (Table 3).
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DISCUSSION |
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) because oral sample cultures yielded growth of Capnocytophaga ‘suspect’ colonies of both yellow–orange and beige appearance. Despite the inclusion of both colony types, a limited number of Capnocytophaga isolates (mean 5.5, range 2–9) were recovered from each child. This supports the findings of others that, although Capnocytophaga bacteria are found in a large proportion of young children, they are not prominent members of the oral flora (Könönen et al., 1994; Conrads et al., 1996; Kamma et al., 2000a, b; Hayashi et al., 2001; Kimura et al., 2002; Ooshima et al., 2003).
Lack of oxidase activity was originally described as a defining feature of the genus Capnocytophaga (Socransky et al., 1979). Brenner et al. (1989) reported animal strains that produced oxidase but otherwise resembled Capnocytophaga species and, on this basis, described them as two Capnocytophaga species. The unequivocal affiliation to the genus of two oxidase-positive strains (AHN9647 and W331) in this study on the basis of partial 16S rRNA gene sequences (98 % identity to other Capnocytophaga 16S rRNA gene sequences) confirms that the oxidase test is not reliable for discrimination of the genus from related genera.
The usefulness of MLEE for differentiation between Capnocytophaga species was confirmed in the present study and extended to include the two new taxa, taxon AHN8471 and taxon AHN8855. Moreover, presence of the enzyme GOT and absence of the enzyme HEX discriminated C. granulosa and C. gingivalis from the other Capnocytophaga species.
Interpretation of MLEE results is based on the assumption that differences in mobility of individual enzymes reflect differences in protein sequence and thus a genetic difference. This is less precise than nucleotide sequencing and, as a consequence of sequencing becoming faster and less expensive, MLEE is losing its significance. In this respect, however, it is noteworthy that MLEE proved much better at identifying C. ochracea (Fig. 1) than partial sequencing of the 16S rRNA gene (Fig. 2).
The existence of three new taxa was indicated by the MLEE and the SEQ dendrograms. We refrained from investigating further the MLEE-A7/SEQ-B1 subdivision because it contained only a single isolate (AHN9647) and as such it did not merit an elevation to species rank. Taxon AHN8471 and taxon AHN8855 consisted of several isolates from four and two children, respectively, and thus warranted further investigation. The near-full-length 16S rRNA gene sequences (Fig. 3) and DNA–DNA relatedness studies confirmed that the two taxa represent novel species.
Based on a combination of clustering in the MLEE and SEQ-based dendrograms, a threshold of 97 % 16S rRNA gene similarity and a threshold of 70 % DNA–DNA relatedness for species identity (Stackebrandt & Goebel, 1994; Wayne et al., 1987), each of the two taxa contained five strains. AHN9528 showed only 68.6 % DNA–DNA relatedness to strain AHN8471, but belonged to taxon AHN8471 by all other analyses and was consequently retained in the taxon. Strain AHN9576 was more distantly related to AHN8471 (55.1 % DNA–DNA relatedness), for which reason it was not included in the taxon. The taxonomic status of this strain remains a puzzle because it appeared to be closely related to the other strains in this cluster according to both dendrograms. Collectively, these results emphasize that the species affiliation of an isolate should be based on several different analyses.
The phenotypic examination of the strains in our collection confirmed the difficulties in distinguishing between Capnocytophaga species (Laughon et al., 1982a; Kristiansen et al., 1984; Khwaja et al., 1990). Especially, distinction between C. ochracea and C. sputigena is difficult, prompting others to stop using phenotypic tests for species identification (Ciantar et al., 2005). The limited usefulness of phenotypic tests for these two species was underscored by the finding of variations in test results in duplicate isolates from the same individual that otherwise presented with identical ET and partial 16S rRNA gene sequences. Taxon AHN8471 increased the problems of species differentiation. Additional tests selected from the proposed minimal standard tests for describing new taxa in the genus Capnocytophaga (Bernardet et al., 2002) were included in order to search for tests that allow differentiation of taxon AHN8471 from C. ochracea and C. sputigena. For the majority of these tests, variations among strains belonging to the same species were previously reported (Socransky et al., 1979; Kristiansen et al., 1984; Speck et al., 1987; Yamamoto et al., 1994). This clearly illustrates the difficulties in species differentiation within the genus Capnocytophaga by traditional phenotypic analysis.
The heterogeneity in the genus Capnocytophaga has puzzled researchers for a long time and is probably a major reason why the association between members of this genus and periodontal disease remains an open question. Application of genetic analyses in future studies may facilitate clarification of the role of these bacteria in disease (Gevers et al., 2005).
We found by far the highest prevalence of C. ochracea and C. sputigena in young Finnish children. C. granulosa and C. haemolytica were originally isolated from supragingival plaque in adults (Yamamoto et al., 1994) and later recovered from subgingival plaque from adults as well (Ciantar et al., 2001). For C. granulosa, this may now be extended to include the oral cavity of children. In addition, taxon AHN8471 and taxon AHN8855 were isolated, but at low frequencies. C. gingivalis was not found. The latter observation is in contrast to other studies. In Greek children with primary dentition, C. ochracea was found in 40 %, C. gingivalis in 55.8 % and C. sputigena in 2.5 % of the samples, according to speciation by conventional phenotypic tests (Kamma et al., 2000b). Based on PCR with species-specific primers, the prevalence of the three species in periodontally healthy Japanese children was 100 % for C. ochracea, 96 % for C. gingivalis and 48 % for C. sputigena (Hayashi et al., 2001). Kimura et al. (2002) using the same technique found C. ochracea and C. sputigena in approximately 50 % of Japanese children with primary dentition. This study did not include C. gingivalis in the analysis. In addition to the employment of different identification criteria, the differences between the studies, especially with regard to C. gingivalis, may be due to racial and/or geographical differences.
Search for rRNA gene sequences from non-cultivated bacteria (http://rdp.cme.msu.edu/index.jsp) that matched the partial rRNA gene sequences of the strains belonging to the two novel species revealed a 100 % match for the sequences of three of the strains of C. leadbetteri to sequence AY807298 and a 99.1 % match for two of the Capnocytophaga genospecies AHN8471 strains to sequence AY806644. Both of these sequences originated from an airway sample of a child with cystic fibrosis (GenBank data). The partial sequences of three of the C. leadbetteri strains showed a 97.7–99.7 % match to sequence AY005075, and three of the Capnocytophaga genospecies AHN8471 strains revealed a 96.6 % match to sequence AY005076. Both of these sequences originated from a human subgingival plaque sample (Paster et al., 2001).
The results of this study facilitate more unequivocal assignment of clinical isolates of the genus Capnocytophaga to species level and create a platform for future studies of the clinical significance of the individual species and of potential differences in pathogenic potential between and within the species.
Description of Capnocytophaga genospecies AHN8471
Due to lack of an easily performed phenotypic test, we describe taxon AHN8471 as Capnocytophaga genospecies AHN8471. Cells are slender (0.35–0.45x3–5 µm), Gram-negative rods, motile by gliding. Yellow–orange or green colonies, 1–2 mm in diameter, appear 2–3 days after anaerobic incubation on fresh plaque agar and Columbia blood agar plates. Pellets of cells grown anaerobically in liquid culture are orange. Grows in air enriched with 5 % CO2, but not in air alone. Tests for catalase, indole, oxidase, β-haemolysis on blood agar, hydrolysis of gelatin and urea, reduction of nitrate and fermentation of melibiose are negative. All strains ferment glycogen and starch, and all strains except AHN8471 ferment inulin. Other phenotypic characteristics are presented in Table 3. The genospecies is not distinguishable from C. ochracea and C. sputigena by means of traditional phenotypic tests. However, MLEE and partial 16S rRNA gene sequencing are useful for differentiation. All strains cleave human IgA1 in the hinge region.
The reference strain is AHN8471 (=CCUG 51856=NCTC 13374) and has a G+C content of 38.8 mol%. Other strains belonging to the genospecies are AHN8751, AHN9528, AHN9607 and AHN9798. All strains have been isolated from the oral cavity of children.
Description of Capnocytophaga leadbetteri sp. nov.
Capnocytophaga leadbetteri [lead.bet'te.ri. N.L. gen. masc. n. leadbetteri of Leadbetter, in honour of E. R. Leadbetter, the American microbiologist who (as first author) described and proposed the genus Capnocytophaga].
Cells are slender (0.35–0.45x3–5 µm), Gram-negative rods motile by gliding. Colonies 1–2 mm in diameter and ranging from beige to yellow–orange or pink appear 2–3 days after anaerobic incubation on plaque agar. On Columbia blood agar, the colonial appearance varies from beige to yellow–orange, green or pink. Pellets of cells grown anaerobically in liquid culture are beige. Grows in air enriched with 5 % CO2, but not in air alone. Tests for catalase, indole, oxidase, β-haemolysis on blood agar, hydrolysis of gelatin and urea and fermentation of melibiose, glycogen, starch and inulin are negative, except for AHN8730, which ferments starch and inulin. All strains reduce nitrate. Other phenotypic characteristics, including the specific reactions of the type strain, are presented in Table 3. The species consists of strains with weak saccharolytic activity. Lack of fermentation of sucrose has the highest discriminatory power from other species. MLEE and partial 16S rRNA gene sequencing are also useful for species differentiation. All strains cleave human IgA1 in the hinge region.
The type strain is AHN8855T (=CCUG 51857T=NCTC 13375T) and has a DNA G+C content of 41.3 mol%. Other strains belonging to the species are AHN8708, AHN8725, AHN8730 and AHN8996. All strains have been isolated from the oral cavity of children.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-glucosidase. All strains were negative for haemolysis on horse blood agar, fermentation of xylose, fermentation of arabinose (except AHN9798, which was weakly positive) and oxidase activity (except AHN9647, which showed variable results, and W331, which was repeatedly positive) and were negative for the following API ZYM tests: β-glucuronidase (except for the weakly positive reaction of AHN8730, AHN9708 and B0611),