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1 Research Service, VA Medical Center West Los Angeles, Building 304, Room E3-227, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA
2 Anaerobe Reference Laboratory, National Public Health Institute (KTL), Helsinki, Finland
3 Department of Medical Microbiology, University of Turku, Turku, Finland
4 Infectious Diseases Section, VA Medical Center West Los Angeles, Los Angeles, CA 90073, USA
5 Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA
6 Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA, USA
Correspondence
Yuli Song
yulis1{at}yahoo.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains WAL 8169T and WAL 8301T are AY974071 and AY974072, respectively.
The cellular fatty acid compositions of representative novel strains described in this work are presented in a supplementary table available in IJSEM Online.
Present address: Helsinki University Central Hospital Laboratory Services (HUSLAB), Helsinki, Finland. 
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MAIN TEXT |
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, 1989, 1990). Several other genera have subsequently been described for Bacteroides-like species that do not conform to these three major groups. The recent description of such a group, Alistipes, included two species, Bacteroides putredinis reclassified as Alistipes putredinis and Alistipes finegoldii as a novel species (Rautio et al., 2003). These species produce succinic acid as the principal metabolic end-product of glucose fermentation and iso-C15 : 0 as their major long-chain cellular fatty acid (CFA) (Rautio et al., 2003).
During studies on bile-resistant, pigment-producing strains from human intestinal sources, later leading to the description of the genus Alistipes and the species A. finegoldii (Rautio et al., 2003), another group of strains with similar phenotypic characteristics but a 16S rRNA gene sequence divergence of approximately 3 % was observed (Rautio et al., 1997a) but remained uncharacterized. In another project evaluating 16S rRNA gene sequencing for species identification of Bacteroides fragilis group isolates (Song et al., 2005), two groups of bile-resistant, pigment-producing organisms resembling A. finegoldii were found. Again, 16S rRNA gene sequencing revealed approximately 3 % sequence divergence between the unidentified bacteria and their phylogenetically closest species, A. finegoldii. A detailed comparison between these unknown groups of bile-resistant, pigment-producing organisms and A. finegoldii was performed. On the basis of the phenotypic and phylogenetic findings presented here, we propose two novel Alistipes species; in addition, we describe phenotypic tests useful in distinguishing between these novel organisms and related taxa.
Altogether, 32 isolates, including the type strains of A. putredinis and A. finegoldii, from human specimens were included in the present study. All of the strains were cultivated on Brucella blood agar supplemented with 5 % sheep blood, haemin and vitamin K1, and were incubated at 37 °C under anaerobic conditions. The strains were characterized biochemically by using a combination of conventional tests, as described in the Wadsworth–KTL Manual (Jousimies-Somer et al., 2002), plus the API ZYM and API Rapid ID 32A test kits (bioMérieux) and Rosco diagnostic tablets, according to the manufacturers' instructions; tests were performed in duplicate or in triplicate. Fermentation tests were performed using pre-reduced, anaerobically sterilized peptone/yeast extract/sugar broth tubes (Anaerobe Systems). The strains were grown in peptone/yeast extract and peptone/yeast extract/glucose broth for determination of metabolic end-products by GLC. The MICs of metronidazole, clindamycin, penicillin, cefotetan, ertapenem, ampicillin/sulbactam and vancomycin were determined for the seven WAL strains by the Clinical Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) reference agar dilution method (NCCLS, 2001). CFAs were detected with a gas chromatograph (Hewlett Packard) and Microbial Identification System software (MIDI). The isolates were grown on supplemented brain heart infusion agar with blood, and the bacterial mass was harvested directly from the plates because of poor growth in peptone/yeast extract liquid media. The corresponding library (ANAEROBE, version Moore 5.0) was used in successive analyses. The G+C content of the DNA (mol%) was determined by HPLC as described previously (Mesbah et al., 1989) except that the methanol content of the chromatographic buffer was reduced to 8 % and the temperature increased to 37 °C.
The 16S rRNA genes were amplified by PCRs using universal primers 8UA (positions 8–28, Escherichia coli numbering) and 1485B (positions 1485–1507) as described previously (Brosius et al., 1978). The amplified product was purified by using the QIAamp PCR purification kit (Qiagen) and directly sequenced with the Big Dye sequencing kit (Biotech Diagnostic) on an ABI 7700 sequencer (Applied Biosystems). The closest known relatives of the isolates were determined by performing database searches using BLAST software (Benson et al., 1997). Almost-complete 16S rRNA gene sequences (>1400 nt) of the isolates and of closely related bacteria were aligned using CLUSTAL W (http://genome.kribb.re.kr). A phylogenetic tree was constructed using the DNA analysis software PAUP*, version 4.0 (Sinauer Associates). The stability of the groupings was estimated by bootstrap analysis (1000 replications) using the same program. DNA–DNA reassociation experiments were carried out according to the spectrophotometric method of De Ley et al. (1970).
Non-motile, Gram-negative, rod-shaped bacteria were recovered from specimens of human intestinal origin (Table 1), together with other strict anaerobes (e.g. species of the B. fragilis group) and/or aerobes, and often showed heavy growth upon primary isolation. The isolates grew anaerobically on agar media well but very poorly in liquid media. No growth occurred in subcultures when exposed to oxygen. Typically, the isolates produced pigment on laked rabbit blood agar and grew on Bacteroides/bile/aesculin agar, showing tolerance to 20 % bile. All isolates were resistant to special-potency antimicrobial discs containing vancomycin (5 µg), kanamycin (1000 µg) and colistin (10 µg), produced indole and hydrolysed gelatin. The results for lipase and urease production, for arginine hydrolysis and for nitrate reduction were negative. Acid was produced from glucose. Because of the scanty growth in liquid pre-reduced, anaerobically sterilized biochemicals, we failed to demonstrate the fermentation of other carbohydrates reliably. Mannose and raffinose were fermented according to the API Rapid ID 32A system (bioMérieux). Positive enzyme reactions were obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, 
-galactosidase, -glucosidase and N-acetyl--glucosaminidase with the API ZYM kit, and for -galactosidase, -galactosidase, -glucosidase, N-acetyl--glucosaminidase, leucyl glycine arylamidase, alanine arylamidase and glutamyl glutamate arylamidase with the API Rapid ID 32A kit. The major metabolic end-product was succinic acid; minor amounts of acetic and propionic acids were also produced. The principal long-chain CFA was iso-C15 : 0 (20–53 % of the total CFAs). For the seven WAL strains tested, the MICs with respect to metronidazole, clindamycin, ampicillin/sulbactam and ertapenem were 
0.5 µg ml–1, and those with respect to penicillin G were 1.0 µg ml–1; however, resistance to cefotetan (MICs 
16 µg ml–1) and vancomycin (MICs >32 µg ml–1) was shown. Notably, penicillin resistance due to -lactamase production may be found (Rautio et al., 1997b).
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) was confirmed, but one isolate (AHN 19871) did not cluster with A. finegoldii or with any of the isolates examined in the present study; the remaining two isolates (AHN 2398 and AHN 19499) clustered with four previously unknown isolates (WAL 8301T, WAL 11404, WAL 11550 and WAL 12401) (Table 1
). Pairwise analysis showed that these six isolates formed a novel group and 15 previously unknown isolates another group where the isolates within each group were phylogenetically closely related to each other (>99 % sequence similarity). Sequence searches of the GenBank and Ribosomal Database Project libraries revealed that these groups represent two hitherto unknown sublines within the genus Alistipes, being members of the phylum Bacteroidetes. A tree constructed by the maximum-parsimony method (Fig. 1), depicting the phylogenetic affinity of the two novel bacterial groups, as exemplified by strains WAL 8169T (=CCUG 48946T) and WAL 8301T (=CCUG 48947T), confirmed the placement of the unknown bacteria in the genus Alistipes. It is evident from the branching pattern in the tree that the two novel species have a significantly close relationship with A. finegoldii. Pairwise comparison based on almost-complete (>1400 nt) 16S rRNA gene sequences revealed approximately 4 % sequence divergence between these novel taxa and approximately 3 % sequence divergence between them and the type strain of their most closely related species with a validly published name, A. finegoldii. Furthermore, DNA–DNA reassociation values of 43.5, 49.6 and 33.6 % were observed between AHN 2357 (=CCUG 46019) and CCUG 46020T (the type strain of A. finegoldii), between WAL 8169T (=CCUG 48946T) and CCUG 46020T and between WAL 8301T (=CCUG 48947T) and CCUG 46020T, respectively, thereby confirming that these unidentified bacterial strains represent novel species.
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). Biochemical characteristics useful for distinguishing A. finegoldii and the two novel groups within the genus from each other are based on reactions with the API ZYM kit (Table 2). In contrast to the corresponding API ZYM reactions, the reactions obtained with individual Rosco diagnostic tablets were positive for -glucosidase and -fucosidase for most strains. Table 3 presents a list of distinguishing characteristics for the genus Alistipes and related genera. The pigment-producing A. finegoldii and two novel groups within the genus Alistipes can be distinguished readily from pigment-producing species of the genera Porphyromonas and Prevotella by their resistance to 20 % bile. In addition, the novel isolates are fermentative, unlike the pigment-producing species within the genus Porphyromonas. Our data, based on phylogenetic and phenotypic analyses, show that both novel groups represent hitherto unknown sublines within the genus Alistipes and can be separated from each other and from other Alistipes species.
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Cells are slender rods with rounded ends and are 0.2–0.5x0.5–3 µm in size. After incubation for 48 h anaerobically at 37 °C on supplemented Brucella blood agar, colonies are circular, entire, convex, grey, opaque, weakly -haemolytic and 0.5–0.8 mm in diameter. After 4 days incubation on laked rabbit blood agar, colonies appear black. Under UV light (365 nm), colonies are brown but no fluorescence is observed. Strictly anaerobic. Resistant to 20 % bile. Indole-positive and catalase-negative. Nitrate is not reduced to nitrite. Aesculin hydrolysis is variable. When tested with the API Rapid ID 32A system, mannose and raffinose are fermented, and positive reactions are obtained for -galactosidase, -galactosidase, -glucosidase, N-acetyl--glucosaminidase, indole, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, -fucosidase and glutamyl glutamate arylamidase. Variable results for -glucosidase, -arabinosidase, glutamate decarboxylase and -fucosidase (when positive, the reaction is weak). Other reactions are negative. With the API ZYM kit, positive enzyme reactions are obtained for alkaline phosphatase, esterase (weak), esterase lipase (weak), acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucosidase and N-acetyl--glucosaminidase. Variable reaction for -chymotrypsin, but, when positive, the reaction is weak. Negative for lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -glucuronidase and -mannosidase. The principal long-chain fatty acid of the six isolates tested (iso-C15 : 0) varies in content from 23 to 27 % (of total CFAs).
The type strain is WAL 8169T (=CCUG 48946T=ATCC BAA-1178T). Fifteen strains were isolated from human specimens: 14 were of intestinal origin and one was from urine. The habitat is probably the human gut. The DNA G+C content of the type strain is 58 mol%.
Description of Alistipes shahii sp. nov.
Alistipes shahii (sha'hi.i. N.L. gen. n. shahii of Shah, to honour Haroun N. Shah, a contemporary microbiologist, for his contribution to anaerobic bacteriology).
Cells are slender rods, 0.1–0.2 µm by 0.6–4 µm. After incubation for 48 h anaerobically at 37 °C on supplemented Brucella blood agar, colonies are circular, entire, convex, grey, opaque, weakly -haemolytic and 0.5–1 mm in diameter. After 4 days incubation on laked rabbit blood agar, colonies appear black. Under UV light (365 nm), colonies are brown but no fluorescence is observed. Strictly anaerobic. Resistant to 20 % bile. Indole-positive and catalase-negative. Nitrate is not reduced to nitrite. Aesculin is hydrolysed. When tested with the API Rapid ID 32A system, mannose and raffinose are fermented, and positive reactions are obtained for -galactosidase, -galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, indole, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, -fucosidase and glutamyl glutamate arylamidase. All other reactions are negative. With the API ZYM system, positive enzyme reactions are obtained for alkaline phosphatase, esterase (weak), esterase lipase (weak), acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucosidase and N-acetyl--glucosaminidase. Two strains have very weak reactions to -chymotrypsin. Negative for lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -glucuronidase and -mannosidase. The content of the principal long-chain fatty acid (iso-C15 : 0) of six tested strains varies from 36 to 43 % (of total CFAs).
The type strain is WAL 8301T (=CCUG 48947T=ATCC BAA-1179T). The six strains were from human specimens of intestinal origin. The habitat is probably the human gut. The DNA G+C content of the type strain is 56 mol%.
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REFERENCES |
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Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A 75, 4801–4805.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Jousimies-Somer, H., Summanen, P., Citron, D. M., Baron, E. J., Wexler, H. M. & Finegold, S. M. (2002). Wadsworth – KTL Anaerobic Bacteriology Manual, 6th edn. Belmont, CA: Star Publishing.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
NCCLS (2001). Methods for antimicrobial susceptibility testing of anaerobic bacteria, 5th edn. Approved standard M11-A5. Wayne, PA: National Committee for Clinical Laboratory Standards.
Rautio, M., Eerola, E., Jalava, J. & Jousimies-Somer, H. (1997a). Phylogenetic description of a bile-resistant anaerobic pigmented Gram-negative bacillus, probably conforming to a new genus and species. Rev Med Microbiol 8 (Suppl. 1), S103.
Rautio, M., Lönnroth, M., Saxen, H., Nikku, R., Väisänen, M.-L., Finegold, S. M. & Jousimies-Somer, H. (1997b). Characteristics of an unusual anaerobic pigmented Gram-negative rod isolated from normal and inflamed appendices. Clin Infect Dis 25 (Suppl. 2), S107–S110.
Rautio, M., Eerola, E., Väisänen-Tunkelrott, M.-L., Molitoris, D., Lawson, P., Collins, M. D. & Jousimies-Somer, H. (2003). Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources. Syst Appl Microbiol 26, 182–188.[CrossRef][Medline]
Shah, H. N. & Collins, M. D. (1988). Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 38, 128–131.
Shah, H. N. & Collins, M. D. (1989). Proposal to restrict the genus Bacteroides (Castellani and Chalmers) to Bacteroides fragilis and closely related species. Int J Syst Bacteriol 39, 85–87.
Shah, H. N. & Collins, M. D. (1990). Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 40, 205–208.
Song, Y., Liu, C., Bolaños, M., Lee, J., McTeague, M. & Finegold, S. M. (2005). Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species. J Clin Microbiol 43, 1531–1537.
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