International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2189–2195 Printed in Great Britain

Dysgonomonas gen. nov. to accommodate sp. nov., an organism isolated from a human gall bladder, and Dysgonomonas capnocytophagoides (formerly CDC group DF-3)

Tor Hofstad,1 Ingar Olsen,2 Emenike R. Eribe,2 Enevold Falsen,3 Matthew D. Collins4 and Paul A. Lawson4

Author for correspondence: Paul A. Lawson. Tel: j44 118 935 7224. Fax: j44 118 935 7222. e-mail: p.a.lawson!reading.ac.uk

1 Department of Results of a polyphasic taxonomic study on an unknown Gram-negative, Microbiology and facultatively anaerobic, coccobacillus-shaped organism isolated from an Immunology, University of Bergen, The Gade infected human gall bladder are presented. Phenotypic and molecular Institute, N-5021, Bergen, taxonomic studies revealed the organism to be close to, but distinct from, Norway organisms designated CDC (Centers for Disease Control and Prevention) group 2 Department of Oral DF-3. The unknown bacterium was readily distinguished from reference strains Biology, Dental Faculty, of Bacteroides, Prevotella, Porphyromonas and related taxa by 16S rRNA gene University of Oslo, N-0316 Oslo, Norway sequencing, biochemical tests, analysis of cellular long-chain fatty acids and electrophoretic analysis of whole-cell proteins. Based on the results of the 3 Culture Collection, Department of Clinical present study, it is proposed that the unknown bacterium be classified in a Bacteriology, University of new genus, Dysgonomonas,asDysgonomonas gadei sp. nov. (type strain CCUG Go$ teborg, Go$ teborg, 42882T l CIP 106420T). In addition, a new , Dysgonomonas S-413 46, Sweden capnocytophagoides sp. nov., is proposed to accommodate strains previously 4 Department of Food belonging to CDC group DF-3. The type species of the genus Dysgonomonas is Science and Technology, University of Reading, Dysgonomonas gadei. Whiteknights, PO Box 226, Reading RG6 6AP, UK Keywords: , phylogeny, Dysgonomonas, CDC group DF-3, 16S rRNA

INTRODUCTION indicated that CDC group DF-3 is phylogenetically related to, but nevertheless distinct from Bacteroides, CDC (Centers for Disease Control and Prevention) Porphyromonas, Prevotella and related species (Paster group DF-3 (dysgonic fermenter 3) is a group of et al., 1994; Vandamme et al., 1996). CDC DF-3 hitherto unclassified, fastidious, facultatively anaer- isolates are resistant to several antimicrobial agents, obic, Gram-negative coccobacilli (Wallace et al., including penicillins, cephalosporins, aminoglycosides 1989). CDC group DF-3 strains grow slowly on blood and ciprofloxacin. Most isolates are susceptible to agar medium, but not on MacConkey agar, ferment clindamycin, tetracycline, chloramphenicol, imipenem several carbohydrates, hydrolyse aesculin, do not and trimethoprim\sulfamethoxazole (Gill et al., 1991; reduce nitrate, and are catalase- and oxidase-negative. Blum et al., 1992). CDC group DF-3 organisms are These organisms have a distinct cellular fatty acid relatively rare isolates that have been recovered from profile characterized by large amounts of straight- stool samples, mainly in immunocompromised chain saturated, anteiso- and iso-methyl branched, patients and patients with severe underlying diseases and 3-hydroxy long-chain fatty acids (Wallace et al., (Blum et al., 1992; Heiner et al., 1992), but also from 1989). Comparative 16S rRNA sequence analysis has clinical materials such as blood, wounds and abscesses (Aronson & Zbick, 1988; Bangsborg et al., 1990). The ...... pathogenic potential of the organisms remains un- Abbreviation: CDC, Centers for Disease Control and Prevention. known. The isolation of organisms with biochemical The GenBank accession number for the 16S rRNA gene sequence of properties similar, but not identical, to those of CDC Dysgonomonas gadei strain CCUG 42882T is Y18530. group DF-3 (designated CDC group DF-3-like

01532 # 2000 IUMS 2189 T. Hofstad and others organisms) has also been reported (Daneshvar et al., MIC of metronidazole was determined after 2 d anaerobic 1991). incubation (Mart Anoxomat system; Mart Microbiology Automation). FAA, supplemented with 5% human blood, In this study, the cultural and biochemical properties, was used as growth medium instead of PDM Antibiotic cellular fatty acid composition and SDS-PAGE pro- Sensitivity medium because of poor growth on the latter, tein profile of an isolate, which resembles CDC group even when supplemented with 5% horse blood. DF-3 organisms, recovered from an infected gall SDS-PAGE of whole-cell proteins. To assess the overall bladder are reported. In addition, results of 16S rRNA phenotypic resemblance of the new isolate and reference gene sequencing indicate that the organism represents species, a comparative analysis of whole-cell protein profiles a new species that clusters together with CDC group by SDS-PAGE was performed. PAGE analysis of whole-cell DF-3 organisms. It is therefore proposed that a new proteins was performed as described by Pot et al. (1994) genus, Dysgonomonas, be created to accommodate the and Vandamme et al. (1998). For densitometric analysis, new organism isolated from an infected gall bladder normalization and interpretation of protein patterns the classified as Dysgonomonas gadei sp. nov. (type strain GCW 3.0 software package (Applied Maths) was used. The T T CCUG 42882 l CIP 106420 ) and organisms pre- similarity between all pairs of traces was expressed by the viously designated CDC group DF-3 be classified as Pearson product–moment correlation coefficient, converted for convenience to a percentage similarity. Dysgonomonas capnocytophagoides sp. nov. The type species of the genus Dysgonomonas is Dysgonomonas Long-chain cellular fatty acid analysis. Cells were cultured on gadei. chocolate agar using Columbia agar base and incubated for 48 h at 37 mC and centrifuged. Saponification, methanolysis, extraction and identification of the fatty acid methyl esters METHODS were made using the Microbial Identification system (Mi- crobial ID) as described previously (Moore et al., 1994; Source of the organism. Strain CCUG 42882T was originally Debelian et al., 1997). isolated from a 68-year-old male with known non-insulin- dependent diabetes mellitus and essential hypertension. The Determination of 16S rRNA gene sequences and phylo- patient was admitted to hospital because of fractures of the genetic analysis. Phylogenetic determination was performed left tibia and the left third, fourth and fifth metatarsal bones by comparative 16S rRNA gene sequence analyses. A large following a car accident. Treatment was conservative. Nine fragment of the 16S rRNA gene (corresponding to positions 30–1521 of the E. coli 16S rRNA gene) was amplified by days later, the patient was suddenly ill with fever, chills and PCR using conserved primers close to the 3 and 5 ends of vascular collapse. Escherichia coli was recovered in blood h h the gene. The PCR products were directly sequenced using a cultures. Ultrasound scanning of the abdomen, performed a Taq dye-deoxy terminator cycle sequencing kit (Applied few days later, showed gallstones and a distended gall Biosystems) and an automatic DNA sequencer (model bladder. Aerobic and anaerobic cultivation of pus (which 373A; Applied Biosystems). The closest known relatives of contained no trace of bile) aspirated from the gall bladder the new isolate were determined by performing a database yielded growth of E. coli,aKlebsiella species and enterococci. search using the program  of the Genetics Computer In addition, a slow-growing, Gram-negative coccobacillus Group package (Pearson & Lipman, 1985). These sequences was recovered by anaerobic cultivation on kanamycin\ and those of other known related strains were retrieved from vancomycin laked blood agar. The patient was treated with the EMBL or Ribosomal Database Project databases and cefuroxime and metronidazole for 2 weeks and recovered aligned with the newly determined sequences using the uneventfully. program  (Devereux et al., 1984). The resulting Cultural and biochemical characterization. The unknown multiple sequence alignment was corrected manually and Gram-negative coccobacillus from the infected gall bladder approximately 100 bases at the 5h end of the rRNA were was cultured aerobically at 35 mC in a thermostat supple- omitted from further analyses because of alignment mented with 7n5% CO# on Fastidious Anaerobic Agar ambiguities. Pairwise evolutionary distances were then (FAA; Lab M) and Columbia agar base (Difco), both of computed from a continuous stretch of 1320 bases using the which were supplemented with 5% human blood. Growth correction of Jukes & Cantor (1969). A phylogenetic tree requirements for X and V factors were examined using discs was constructed according to the neighbour-joining method impregnated with X factor, V factor or both, and nutrient with the program  (Felsenstein, 1989). The stability agar as basal medium. Bile sensitivity was determined by the of the groupings was estimated by bootstrap analysis (500 Oxgall test using Diatabs diagnostic tablets (Rosco) as replications) using the programs , , described by Weinberg et al. (1983). The strain was bio-  and  (Felsenstein, 1989). chemically characterized by using a combination of con- ventional tests and the API ID32A, API ID32E and API ZYM systems according to the manufacturer’s instructions RESULTS (API bioMe! rieux). Cultural and biochemical properties Susceptibility to antimicrobial agents. MICs of a range of The isolate originating from the infected gall bladder antibacterial agents, including penicillins, cephalosporins, glycopeptide antibiotics, aminoglycosides, fluoroquino- consisted of non-motile, Gram-negative coccobacilli lones, macrolides, quinolones, chloramphenicol, doxy- that grew relatively slowly on blood agar. The or- cycline, metronidazole, sulphadiazine and trimethoprim\ ganism grew with or without the addition of 7n5% sulphamethoxazole, were examined using the E-test (AB CO#, in addition to growing anaerobically. After 48 h Biodisk). The tests were performed as recommended by the incubation aerobically at 35 mCinaCO#-enriched manufacturers and read after incubation aerobically for 2 d. atmosphere (7n5%), the colonies were 1–2 mm in

2190 International Journal of Systematic and Evolutionary Microbiology 50 Dysgonomonas gen. nov.

...... Fig. 1. Similarity dendrogram based on whole-cell protein patterns of Dysgonomonas gen. nov., sp. nov. and related species. Levels of correlation are expressed as percentages of similarity for convenience.

diameter, non-adherent, entire, grey-white, smooth different API systems employed. Using the API ID32A and non-haemolytic, and had a slightly aromatic and rapid ID32E systems, β-glucuronidase was odour. Following incubation for a few more days, the detected, but with the API ZYM kit this enzyme was colonies were somewhat coalesced, butyrous and α- not produced. The API rapid ID32A profile for the haemolytic. Growth was obtained at 25 mC, but not at unknown bacterium was 4577640222 when compared 43 mC. Growth was not observed on MacConkey agar. to 4736440202 obtained for DF-3 strains CCUG The coccobacillus grew on nutrient agar around X and 17996T and CCUG 42515. XV discs, suggesting a growth factor dependency for haem. The organism grew under microaerophilic and strictly anaerobic conditions. It was catalase-positive SDS-PAGE of whole-cell proteins and oxidase-negative, it failed to reduce nitrate, and it A numerical analysis of the whole-cell protein patterns did not produce hydrogen sulphide or acetoin, but did of the unknown bacterium and two CDC group DF-3 produce indole. Aesculin was hydrolysed, but gelatin strains (CCUG 17996, CCUG 42515) together with and urea were not. Growth and precipitation were reference strains of Bacteroides, Capnocytophaga and observed around the Oxgall tablets demonstrating that Prevotella is shown in Fig. 1. The unknown gall the organism was resistant to ox bile. Glucose was bladder isolate was shown to be separate from the two fermented with production of acid but no gas. Acid DF-3 strains and all reference strains used. The nearest was produced from -arabinose, cellobiose, fructose, correlation was with Bacteroides uniformis at a level of lactose, -mannose, raffinose, -rhamnose, -ribose approximately 58%. In addition, the two CDC DF-3 (weak reaction), salicin, starch, sucrose and trehalose. strains clustered together with a correlation level of Adonitol, dulcitol, erythritol, glycogen, inositol, about 75%. -mannitol and -sorbitol were not acidified. Posi- tive reactions were obtained for N-acetyl-β-glucos- aminidase, acid phosphatase, alanine arylamidase, Susceptibility to antimicrobial agents alkaline phosphatase, α-arabinosidase, ester lipase C8 (weak reaction), α-galactosidase, β-galactosidase, α- The unknown clinical isolate was sensitive to " glucosidase, β-glucosidase, β-glucuronidase, glutamyl metronidazole (MIC 1 5 µgmlV ), clindamycin (MIC " n " glutamic acid arylamidase, α-mannosidase (weak re- 0 25 µgmlV ), doxycycline (MIC 0 19 µgmlV ), n " n action), α-fucosidase, chymotrypsin, alanine aryl- imipenem (MIC 0 5 µgmlV ), meropenem (MIC " n amidase, leucyl glycine arylamidase, phosphoamidase 0 064 µgmlV ) and trimethoprim\sulphamethoxazole n " and trypsin. Arginine dihydrolase, arginine aryl- (MIC 0 125 µgmlV ). The organism was resistant to n " amidase, cystine arylamidase, esterase C-4, β- cefoxitin (MIC 24 µgmlV ) and the other cephalo- galactosidase 6-phosphate, glutamic acid decarboxyl- sporins tested [cefotaxime, cefpirome, ceftazidime, ase, glycine arylamidase, histidine arylamidase, lipase ceftriaxone, cefuroxime and cephalothin (MIC " C14, leucine arylamidase, phenylalanine arylamidase, 256 µgmlV )]. In addition, the isolate was resistant to " proline arylamidase, pyroglutamic acid arylamidase, the aminoglycosides [gentamicin (MIC 256 µgmlV ), " serine arylamidase, tyrosine arylamidase, urease and netilmicin (MIC 256 µgmlV ) and sulphadiazine (MIC " valine arylamidase were not detected. Results for the 256 µgmlV )], fluoroquinolones [ciprofloxacin (MIC " " production of β-glucuronidase differed between the 32 µgmlV ) and oxafloxacin (MIC 8 µgmlV )] and the

International Journal of Systematic and Evolutionary Microbiology 50 2191 T. Hofstad and others

Table 1. Composition (%) of cellular fatty acids in strain CCUG 42882T and CDC group DF-3 strains CCUG 17996T and 42515 ...... , Not detected.

Fatty acid Strain

DF-3 CCUG 17996 T DF-3 CCUG 42515 CCUG 42882 T

iso-C"#:!  0n31n9 C"$:!  0n9  iso-C"$:! 9n50n50n9 anteiso-C"$:!  0n73n5 C"%:! 2n41n91n6 iso-C"%:! 19n812n312n9 C"&:! 11n314n22n9 C"&:" 3n42n7  anteiso-C"&:! 19n619n423n9 iso-C"&:! 2n72n24n0 iso-3OH C"&:!  0n8  3OH C"&:!  3n8  3OH C"':! 4n65n37n9 iso-3OH C"(:! 3n33n5  C"':! 3n43n615n2 iso-C"':!   1n3 iso-3OH C"':! 12n39n310n5 3OH C"':! 4n65n37n9 C"':! cis* 4n73n91n2 iso-3OH C"(:! 3n33n5  3OH C"(:!  1n60n5 anteiso-C"(:" 3n2   2OH C"(:!   1n8 C"):! cis*  1n41n1

glycopeptide antibiotics [vancomycin (MIC Phylogenetic analysis " " 48 µgmlV ) and teicoplanin (MIC 256 µgmlV )]. The MICs of other antibiotics tested were: ampicillin, To investigate the genealogical affinity between the " " 6 µgmlV ; amoxicillin, 6 µgmlV ; amoxicillin\ unknown bacterium and its relationship with other " clavulanic acid, l µgmlV ; benzylpenicillin, Gram-negative taxa, comparative 16S rRNA gene " " 32 µgmlV ; erythromycin, 16 µgmlV ; azithro- sequence analysis was performed. The almost complete " " " mycin, 16 µgmlV ; clarithromycin, 16 µgmlV ; and gene sequence (" 1400 nt) of the unknown isolate was " chloramphenicol, 4 µgmlV . determined. Sequence searches of GenBank and RDP databases revealed that the unknown isolate was phylogenetically most closely associated with members Cellular fatty acid composition of the genera Bacteroides, Prevotella and Porphyro- monas, which belong to the Bacteroides subgroup of The percentage fatty acid compositions of strain the flavobacterium–bacteroides subphylum. A tree CCUG 42882T and CDC group DF-3 strains (CCUG constructed by the neighbour-joining method 17996 and CCUG 42515) are given in Table 1. The depicting the phylogenetic affinity of the unknown fatty acids of the unknown isolate CCUG 42882T were coccobacillus is shown in Fig. 2. The unknown found to be iso-C"%:!, anteiso-C"&:!,C"':! and iso-3OH bacterium was phylogenetically placed on the per- C"':!. This fatty acid pattern was similar to those of the iphery of members of the genus Porphyromonas two reference CDC group DF-3 strains examined. clustering with CDC group DF-3 strain CCUG 17996.

2192 International Journal of Systematic and Evolutionary Microbiology 50 Dysgonomonas gen. nov.

...... Fig. 2. Unrooted tree showing the phylogenetic relationships of Dysgonomonas species and some related Gram-negative . The tree constructed using the neighbour-joining method was based on a comparison of approx. 1327 nt. Bootstrap values, expressed as a percentage of 500 replications, are given at branching points. Scale bar, 1% sequence divergence.

A second strain belonging to CDC group DF-3 similarity), Bacteroides forsythus (89% sequence simi- (CCUG 42515) was found to be identical to CCUG larity), Bacteroides merdae (89% sequence similarity) 17996 (100% sequence similarity in 1420 bases com- and Bacteroides splanchnicus (83% sequence simi- pared). The sequence divergence between strain larity) (Fig. 2). Based on tree topology considerations CCUG 42882T and CDC group DF-3 strains was 7%. and sequence divergence values, it is clear that the unknown bacterium cannot be assigned to any of the DISCUSSION currently described genera. The association between the unknown bacterium and the CDC group DF-3 It is evident from the findings of this investigation strains is, however, very significant and the depth of that the bacterium originating from a human gall the cluster formed by the two taxa is indicative of a bladder infection represents a hitherto unrecognized single genus. Chemotaxonomic findings (analysis of species within the Bacteroides subgroup of the whole-cell proteins and long-chain fatty acids) and its flavobacterium–bacteroides subphylum. Phylogen- facultative nature also demonstrate the separateness of etically, the coccobacillus-shaped bacterium clus- the unknown clinical isolate from currently named tered together with strain CDC group DF-3 (93% members of the Bacteroides subgroup and strongly sequence similarity) and bootstrap resampling showed support its affinity with the CDC group DF-3 strains, this relationship to be statistically significant (100% which also exhibit a facultative mode of respiration. recovery in 500 resamplings). Other taxa displayed Until a more complete inventory of the organisms significantly lower levels of sequence similarity in- present within this supercluster of bacteria is estab- cluding Bacteroides (85–87%), Porphyromonas (84– lished, it is not possible to draw conclusions on the 88%) and Prevotella (79–85%) and the misclassified significance of the association of facultative organisms strict anaerobes Bacteroides distasonis (86% sequence with strict anaerobes. The recovery of the newly

International Journal of Systematic and Evolutionary Microbiology 50 2193 T. Hofstad and others described organism from the diseased gall bladder may somewhat coalesced, butyrous and α-haemolytic. be indicative that it was present as an opportunistic Growth is obtained at 25 mC but not at 43 mC. Grows pathogen. Moore et al. (1994) found that anteiso- under microaerophilic and strictly anaerobic con- C"&:!, iso-C"&:!, iso-3OH C"(:! and C"':! are the major ditions. The organism does not grow on Mac- cellular fatty acids in Bacteroides and Prevotella. These Conkey agar but grows on nutrient agar around X findings were consistent with earlier studies (Miyagawa and XV discs, suggesting a growth factor dependency et al., 1979; Shah & Collins 1980; Mayberry et al., for haem. The organism is catalase-positive and 1982). There is a general consensus that iso-C"&:! is the oxidase-negative. It does not reduce nitrate or produce major fatty acid in Porphyromonas strains, accounting hydrogen sulphide or acetoin. Aesculin is hydrolysed for 33–58% of the fatty acids present (Moore et al., but gelatin and urea are not. Indole is produced. 1994). Therefore, the fatty acid profile of strain CCUG Resistant to ox bile. Glucose is fermented with 42882T is incompatible with the genus Porphyromonas. production of acid but no gas. Acid is produced from The fatty acids of the aforementioned strain, however, -arabinose, cellobiose, fructose, lactose, -mannose, closely resemble those reported for CDC group DF-3 melezitose, melibiose, raffinose, -rhamnose, -ribose strains (Daneshvar et al., 1991; Wallace et al., 1989; (weak reaction), salicin, starch, sucrose, trehalose and Table 1). Therefore, based on both phenotypic and xylose. Adonitol, dulcitol, erythritol, glycogen, in- phylogenetic findings of this and previous studies, it is ositol, -mannitol and -sorbitol are not acidified. proposed that the unknown Gram-negative cocco- Positive reactions for N-acetyl-β-glucosaminidase, bacillus and organisms previously designated CDC acid phosphatase, alanine arylamidase, alkaline phos- group DF-3 merit classification in a new genus, phatase, α-arabinosidase, ester lipase C8 (weak Dysgonomonas gen. nov., as Dysgonomonas gadei sp. reaction), α-galactosidase, β-galactosidase (weak reac- nov. and Dysgonomonas capnocytophagoides sp. nov., tion), α-glucosidase, β-glucosidase, glutamyl glutamic respectively. acid arylamidase, α-mannosidase (weak reaction), α- fucosidase, chymotrypsin, alanine arylamidase, leucyl Description of Dysgonomonas gen. nov. glycine arylamidase, phosphoamidase and trypsin. Arginine dihydrolase, arginine arylamidase, cystine Dysgonomonas (Dys.go.no.mohnas. N.L. n. Dysgonic a arylamidase, esterase C-4, β-galactosidase 6- type of fermenter; Gr. fem. n. monas a monad, unit; phosphate, glutamic acid decarboxylase, glycine aryl- N.L. fem. n. Dysgonomonas a monad from a Dysgonic amidase, histidine arylamidase, lipase C14, leucine fermenter). arylamidase, phenylalanine arylamidase, proline aryl- amidase, pyroglutamic acid arylamidase, serine aryl- Cells consist of non-motile, Gram-negative cocco- amidase, tyrosine arylamidase, urease and valine bacilli to short rods. Growth is not observed on arylamidase are not detected. The type strain of MacConkey agar. Requires X growth factor. Dysgonomonas gadei is CCUG 42882T ( CIP Facultatively anaerobic. May be catalase-positive or l 106420T). The type strain was recovered from a human -negative. Oxidase-negative. Glucose is fermented infected gall bladder. Habitat is not known. producing acid but no gas. Alkaline phosphatase is produced but not arginine dihydrolase. Does not reduce nitrate. Hydrogen sulphide and acetoin are not Description of Dysgonomonas capnocytophagoides produced. Aesculin may or may not be hydrolysed but sp. nov. gelatin and urea are not. Indole may or may not be produced. The long-chain cellular fatty acids are of the Dysgonomonas capnocytophagoides (cap.no.cy.to.pha. straight-chain saturated, anteiso- and iso-methyl goihdes. Gr. n. kapnos smoke; Gr. n. kytos hollow branched and 3-hydroxy types. The GjC content of vessel; Gr. v. phagein to eat; Gr. adj. suffix -oides DNA is 38 mol% (Vandamme et al., 1996). Isolated alike; N.L. adj. capnocytophagoides capnocytophaga- from human clinical specimens and stool samples. like, referring to some properties shared between Habitat is not known. The type species is Dysgono- these organisms). monas gadei. The description is based on results obtained in this study and those obtained by Wallace et al. (1989). Description of Dysgonomonas gadei sp. nov. Cells consist of non-motile, Gram-negative cocco- bacilli to short rods. After 48 h incubation aerobically Dysgonomonas gadei (ga de.i. N.L. gen. masc. n. gadei h at 35 CinaCO-enriched atmosphere (7 5%), the of Gade Institute, Bergen, Norway where the organism m # n colonies are 1–2 mm in diameter, non-adherent, entire, was first isolated). grey-white, smooth and non-haemolytic and have a Cells consist of non-motile, Gram-negative cocco- slight aromatic odour. The organism does not grow on bacilli that grow relatively slowly on blood agar. After MacConkey agar. Facultatively anaerobic. The or- 48 h incubation aerobically at 35 mCinaCO#-enriched ganism is catalase- and oxidase-negative. It does not atmosphere, the colonies are 1–2 mm in diameter, non- produce hydrogen sulphide or acetoin. Aesculin may adherent, entire, grey-white, smooth and non-hae- or may not be hydrolysed but gelatin and urea are not. molytic, and have a slightly aromatic odour. Following Indole may or may not be produced. Resistant to ox incubation for a few more days, the colonies become bile. Nitrate is not reduced. Major products of glucose

2194 International Journal of Systematic and Evolutionary Microbiology 50 Dysgonomonas gen. nov. fermentation are propionic, lactic and succinic acids. Felsenstein, J. (1989).  – phylogeny inference package Glucose is fermented with production of acid but no (version 3.2). Cladistics 5, 164–166. gas. Acid is produced from -arabinose, lactose, Gill, V. E., Travis, B. & Williams, D. Y. (1991). Clinical and maltose, -mannose, melibiose, raffinose, sucrose and microbiological observations on CDC group DF-3, a Gram -xylose. Adonitol, -arabitol, -arabitol, dulcitol, negative coccobacillus. J Clin Microbiol 29, 1589–1592. inositol, -mannitol, -sorbitol and trehalose are not Heiner, A. M., Disario, J. A., Carroll, K., Cohen, S., Evans, T. G. & acidified. Positive reactions are obtained for acid Shigeoka, A. O. (1992). Dysgonic fermenter-3 – a bacterium phosphatase, alanine arylamidase, alkaline phos- associated with diarrhoea in immunocompromised hosts. Am J phatase, α-arabinosidase, α-galactosidase, β- Gastroenterol 87, 1629–1630. galactosidase, β-galactosidase 6-phosphate, α-gluco- Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein sidase, β-glucosidase, glutamyl glutamic acid aryl- molecules. In Mammalian Protein Metabolism, pp. 21–132. amidase, leucyl glycine arylamidase and phospho- Edited by N. H. Munro. New York: Academic Press. amidase. Arginine arylamidase, arginine dihydro- Mayberry, W. R., Lambe, D. W., Jr & Ferguson, K. P. (1982). lase, N-acetyl-β-glucosaminidase, α-fucosidase, Identification of Bacteroides species by fatty acid profiles. Int J β-glucuronidase, glutamic acid decarboxylase, glycine Syst Bacteriol 32, 21–27. arylamidase, histidine arylamidase, lysine decarboxyl- Miyagawa, E., Azuma, R. & Suto, T. (1979). Distribution of ase, leucine arylamidase, α-mannosidase, ornithine sphingolipids in Bacteroides species. 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