The Identification of Bacteroides Ureolyticus from Patients with Non-Gonococcal Urethritis by Conventional Biochemical Tests and by DNA and Protein Analyses

Home , Bacteroides ureolyticus

J. Med. MicrobioL-Vol. 21 (1986). 109-1 16 tr) 1986 The Pathological Society of Great Britain and Ireland The identification of Bacteroides ureolyticus from patients with non-gonococcal urethritis by conventional biochemical tests and by DNA and protein analyses

A. J. TAYLOR, CHRISTINE A. DAWSON and R. J. OWEN

National Collection of Type Cultures, Central Public Health Laboratory, London N W9 5HT

Summary. Twenty-seven strains of gram-negative anaerobic bacteria isolated from patients with non-gonococcal urethritis (NGU-associated strains) were compared with reference strains of Bacteroides ureolyticus and representatives of other species of Bacteroides and Fusobacterium. Conventional biochemical tests, electrophoretic (PAGE) protein profiles and DNA base compositions of 29-5+ 1-2 mol % G+C indicated striking similarities between the NGU-associated strains and B. ureolyticus. Three of the reference strains previously assigned to that species were atypical in some respects as was one of the strains from urethritis and one strain, NCTC 10939, was incorrectly classified as B. ureolyticus. Casein hydrolysis by B. ureolyticus was the principal test difference between the two sets of strains. DNA-DNA hybridisation results of 2 72% at optimum temperature conditions provided further evidence of the close relationship between B. ureolyticus and the NGU-associated strains and we conclude that these organisms should be identified as B. ureolyticus. There was insufficient evidence for recognising subspecies or biotypes within this revised concept of B. ureolyticus although several types (PAGE-type I, 17 strains; type II,6 strains; type III,3 strains) were detected which might provide the basis for future studies of hospital strains. The DNA base composition and hybridisation data indicated that B. ureolyticus was not related to other species of Bacteroides or Fusobacterium.

Introduction ciated with the upper respiratory tract, were Eikenella corrodens Facultatively and obligately anaerobic gram- renamed (Jackson and Good- negative bacilli that produce a ‘pitting’ or ‘corrod- man, 1972). To avoid further confusion, they ing’ effect on solid agar media were described by subsequently re-classified the obligate anaerobes as B. ureolyticus 1978). Henriksen (1948) who reported the isolation of (Jackson and Goodman, three strains, one from the female genital tract, one Non-sporing gram-negative anaerobes comprise from a perineal abscess and one from a pulmonary a significant part of the flora of the female genital (1 abscess. Eiken (1958) proposed the name Bacter- tract in healthy individuals. Duerden 980) iso- Bacteroides oicies corrodens for 6 1 similar strains isolated mainly lated 113 strains of from 13 women from oral, dental and upper respiratory tract speci- attending a family planning clinic and found that B. melaninogenicuslB. oralis mens. It was subsequently established that these members of the group corroding strains were not obligate anaerobes, and were the commonest species. In a study of anaerobic Jackson et al. (1971) divided them into two groups: organisms isolated from the urogenital tract, Moss (1983) did not isolate B. ureolyticus strains from (a) facultative anaerobes, whose growth was female patients but reported one isolate of B. enhanced by COz, that did not produce urease and ureolyticus that had DNA base compositions of 57-58 mol% from a male patient with non-gonococ- guanine plus cytosine (G+C); and (b) obligate cal urethritis (NGU). However, strains originally identified as B. corrodens but now conforming to B. anaerobes, that produced urease and had DNA ureolyticus, base compositions of 27-28 molx G+C. The have been isolated from the female et al., facultative anaerobes, which are most often asso- genital tract by other investigators (Labbe 1977; Robinson and James, 1973). In a study of B. ureolyticus, Duerden et al. (1982) Received 12 Feb. 1985; accepted 7 May 1985. isolated 103 strains from patients with necrotic or 109 110 A. J. TAYLOR, CHRISTINE A. DAWSON AND R. J. OWEN gangrenous lesions. The highest prevalence (43 tation of carbohydrates. Arginine dihydrolase and lysine isolates) was in patients with perineal and perianal and ornithine decarboxylase production were tested by infections but strains were also found in ulcers and three methods: Maller (1955); NCTC micromethod lesions of the lower limbs of patients with peripheral (Cowan, 1974); and Minitek (BBL) system (Stargel et u/., vascular disease. B. ureolyticus was rarely isolated 1976). The results of the micromethod were recorded at 4 and 24 h, and those of the Minitek system at 48 h. in pure culture and most often was associated with Atmospheric requirements were tested by incubation for anaerobic gram-positive cocci. 7 days in anaerobic conditions, in 0. 5% and in air plus The association of B. ureolyticus-li ke organisms co*10%. with genital tract disease was first reported by Fontaine et ul. (1982), who investigated carriage of non-sporing anaerobes in men with urethritis and Polyacrylamide gel electrophoresis (PAGE) found one particular organism associated with Analysis of whole-cell protein extracts was performed patients with NGU. Subsequently these isolates by the method described previously (Taylor and Owen, were shown to be similar to B. ureolyticus (Fontaine 1984) with a buffer containing 0.25~Tris-HCI, 0.192~ et al., 1984 a and b). glycine, and sodium dodecyl sulphate (SDS) 0.19; at pH In this study, we examined the NGU-associated 8.5. The gels were dried then directly scanned (LKB 2202 strains of Fontaine and co-workers (1982, 1984 a Ultrascan Laser Densitometer) and data on peak areas, and h) and compared them in conventional bio- heights and retention times recorded (LKB 2220 Record- ing Integrator). chemical characteristics, polyacrylamide gel electrophoresis (PAGE) patterns of proteins, DNA base compositions and DNA-DNA hybridisation, DNA isofution B. ureolyticus, with type and reference strains of as Three-day-old cultures on BHIB were used to inoculate well as with low-G+C-content species of Bucter- 250 ml of modified Brain Heart Infusion Broth, contain- oicies and Fusohacterium. ing (/L), Brain Heart Infusion powder (Oxoid) 37g, Liver Digest (Oxoid) 5g, Yeast Extract (Oxoid) 5g, L-cysteine- HCI 0+5g,haemin 6 mg and Vitamin KI1 mg. Cultures Materials and methods were incubated on a rotary shaker for 5 days at 37°C with a replacement atmosphere of Hz 90%, COZ 10%. Cells Struins were harvested by centrifugation, washed, lysed with SDS Twenty-seven strains of gram-negative anaerobic bac- and the DNA extracted as described by Owen and Pitcher teria isolated from patients with non-gonococcal urethri- (1 985). Because of the small amounts of cellular material tis were studied. The strains, numbered A243/83-A247/ available, 4-ml volumes of solutions were used through- 83, A302/83, A303/83, A305/83-A307/83 and A 174184- out the procedure. The purity of all DNA samples was A190/84, were obtained from Mr E. A. Fontaine and Dr checked by gel filtration chromatography on Sepharose- S. P. Borriello, Clinical Research Centre, Northwick Park CL4B. If substantial quantities of residual RNA, protein Hospital, Middlesex. The following ten reference strains or polysaccharide were present, the sample was further were also studied: B. ureolyticus NCTC nos. 10939, purified on a preparative column of Sepharose-CL-4B. 10940, 10941, 10948 and 10949, B. praeacutus NCTC The DNA samples were considered sufficiently pure if 1 1 158, B. hypermegas NCTC 1057 I, B. termitidis NCTC their absorbance ratios (230nm/260nm and 280nm/ 11300, F. nucleaturn NCTC 10562 and F. necrophorum 260nm) were c. 0-450 and 0.515 respectively. The stock NCTC 10575. DNA solutions at a concentration of at least 50 pg/ml were stored in 0-1 or 0.3 x SSC (0-15 M NaCl buffered with 0.015 M trisodium citrate, pH 7.0) at - 30°C. Culture media All strains were grown on modified Brain Heart Estimation of DNA base composition Infusion Blood Agar (BHIB) as described by Taylor and Owen (1984). The DNA base composition (mol y” G+C) was estimated from the thermal denaturation temperature (Tm) as described previously (Owen et a/., 1978), except Conuentionul biochemical tests that the Tm was determined in either 0.33xSSC or 0.1 xSSC buffer. The base composition was calculated The following tests were performed on all strains by the and expressed relative to reference DNA from Esckwi- methods described by Taylor and Owen (1984): Gram’s chia coli NCTC 900 1 (51 -7 mol”:, G + C). stain, motility and nitrate reduction; production of oxidase, catalase, urease, lecithinase, lipase. H2S, DNAase and P-galactosidase (ONPG test); casein diges- DNA-D NA hybridisation tion; tyrosine, hippurate and starch hydrolysis; growth with and hydrolysis of Tween 20 and 80; and fermen- A membrane filter method was used based on that IDENTIFICATION OF BACTEROIDES UREOL YTICUS 111

described by Johnson (1981). A 2-ml solution of unla- Tween 20 and 80. They were non-saccharolytic, and belled DNA, containing DNA 150 pg in 0.1 x SSC, was gave negative results in tests for lipase, lecithinase denatured by heating then plunged into 8 ml of ice-cold and DNAase. They did not grow in air with or 7 x SSC. The solution was passed at a rate of about I ml/ without the addition of COz 10% (table I). The only min through a nitrocellulose membrane (Sartorius: 25- differences between the NGU-associated and refer- mm diameter, 0.2-pm pore size) presoaked in 6 x SSC for ence strains were as follows: The NGU-associated 30 min. The filter was washed thrice with 10-ml volumes strains utilised lysine, arginine and ornithine in of ice-cold 6xSSC. dried on filter paper and baked overnight at 60' C under vacuum. Membrane filters Mlallers medium, whereas the reference strains of B. containing about 5 DNA 50 pg cm2 were prepared for ureolyticus did not grow sufficiently and produced each test DNA and stored in a desiccator at 4°C. The equivocal results. In the Minitek system, only strain tritium labelled DNA probe was prepared by nick NCTC 10941 gave a positive result for arginine translation (Nick Translation Kit Code N 5000; Amer- hydrolysis. In the NCTC micromethod, all the sham International PLC) with c. 2 pm deoxy [ I ', 2', 5-3HI strains hydrolysed arginine but gave variable results cytidine triphosphate (spec. act. range 50-80 Ci/m mol) with lysine and ornithine. The type strain NCTC and stored in ethanol at -20°C (Leaper and Owen, 10941 gave positive results in tests with all three 1982). The DNA-DNA hybridisation was performed by amino acids. The NGU-associated isolates did not direct binding experiments. Small disks (0.5-cm diameter) B. ureolyticus were punched from each membrane and preincubated in hydrolyse casein whereas the refer- Denhardt's mixture (bovine serum albumin 0.02%, poly- ence strains did. vinylpyrolidone 0.02%, Ficoll400 0.02% in 2 x SSC) for 1 h at 45°C. Each set of DNA samples for hybridisation Electrophoretic protein patterns included duplicates of the homologous DNA and a blank filter. The radioactively labelled probe DNA resuspended The separation of whole-cell protein extracts by in 200 pl of 0.02 M NaCl was sonicated, heat denatured SDS-PAGE produced patterns containing at least and held on ice before hybridisation. The disks were 30 visually detectable bands (figs. 1 and 2). All the transferred to reaction vials each containing 97 pl of NGU-associated isolates except four had one of 3 x SSC, to which was added 28 pl of formamide (20% two PAGE profiles-type I (1 7 strains) and type I1 final concentration) and 15 pl of probe, to give a final (6 volume of 140 rnl and a concentration of 2.1 x SSC. The strains), which differed principally in the position vials were incubated at 45'C (optimum temperature of the major protein bands in the mo1.-wt region criterion in 20% formamide) with gentle shaking for 24h. (41-51) x lo3 (fig. 1). Two strains (A179/84 and The disks were removed to a perspex washing chamber A188/84) were slightly different (type 111) and a (Johnson, 1981) and washed in two 200-300-ml volumes single isolate (A 185/84) had a fourth unique, unas- of 2 x SSC at 45 C for a total of 10 min. They were then signed, profile (figs. 2 and 3). One of the NGU- dried on filter paper in an oven at 60 C for 5-10 min, associated strains (A307/84) had a profile identical transferred to scintillation vials and the radioactivity was with that of the reference cultures of B. ureolyticus measured. The percentage relative hybridisation (H) was (NCTC nos. 10940, 1094 1 and 10948). The remain- calculated from ing two B. ureolyticus strains (NCTC nos. 10939 Number of counts (cpm) bound and 10949) had different profiles from other DNA members of the species and from each other as H= by the heterologous filter Number of counts (cpm) bound shown in fig. 2. by the homologous DNA filter Fig. 3 shows the PAGE profiles of representatives of types I, I1 and 111, and the profile of the type strain of B. ureolyticus (NCTC 10941). The densito- Results metric traces of selected strains are illustrated in fig. 4 for comparison. Conimtional biochemical characteristics B. ureolyti- The NGU-associated strains and the DNA base compositions cus reference strains had the following characteristics: they were all gram-negative rod-shaped bac- DNA was extracted from 12 NGU-associated teria measuring 2 pm x 0-4pm, non-flagellate, non- strains and from three reference strains and one motile, oxidase-positive and catalase-negative. NGU-isolate of B. ureolyticus. Their guanine plus They grew anaerobically and in an atmosphere cytosine contents (mol % G+C), which are sum- containing 025% and at temperatures of 37°C and marised in table IT were 2631 mol %. The means 42 C. All gave positive results in tests for nitrate and standard deviations of the base compositions of reduction, and urease and HZS production. They the NGU-associated strains and B. ureolyticus were did not hydrolyse hippurate, tyrosine, starch or identical-29.5 f 1'2% and 29.3 f0.7mol% G + C 112 A. J. TAYLOR, CHRISTINE A. DAWSON AND R. J. OWEN

Table I. Biochemical characteristics of NGU-associated strains and B. ureolyticus references strains

All strains positive in tests for: All strains negative in tests for: * Oxidase production Motility Nitrate reductase production Catalase production * Urease production Lipase production H2S production Lecithinase production t Lysine decarboxylase production DNAase production t Ornithine decarboxylase production Tyrosine hydrolysis Arginine dihydrolase production Tween 20 and 80 hydrolysis Growth at 42°C Saccharolytic activity Growth in 025% Growth in air+COz 10% Hippurate hydrolysis Starch hydrolysis $. Casein hydrolysis

* B. ureolyficus NCTC 10939 gave negative results in tests for oxidase and urease. t Variable results given by B. ureolyficus reference strains. $ B. ureolyficus reference strains gave positive results. respectively, with a value of 29.5 f 1-2mol % G + C DNA-DNA hybridisation for all strains. The base compositions of NGU- associated strains with different PAGE profiles were Table I1 shows the results of DNA-DNA not different. Reference strains of B. praeacutus, B. hybridisations between 3H-labelled DNA from termitidis and B. hypermegas had base compositions of 30.9, 33.3 and 29.4 mol % G+C respectively. F. 12345678 necrophorum and F. nucleatum reference strains had base compositions of 33.5 and 24.2 mol G+C respectively.

1 2 3 4 5 6 7 8 9 1011 76-78 66

Fig. 2. Gel of protein extracts of B. ureolyticus reference strains in tracks I4(NCTC nos. 10939, 10941, 10948 and 10949) and representative NGU-associated strains of PAGE-type I (track 5, Fig. 1. Gel of protein extracts of NSU-associated strains. strain A182/84), PAGE-type 11 (track 6, strain A176/84) and an PAGE-type I, tracks 1-6 (strains A243/83, A183/84, A245/83, unassigned strain (track 7, strain A185/84). Track 8 contains the A 18 1/84, A 182/84 and A 175/84). PAGE-type 11, tracks 7-10 protein markers ovo-transferrin, albumin, ovalbumin, and (strains A174/84, A244/83, A276/83 and A176/84). PAGE-tyPe chemotrypsinogen A (BDH) of mol. wts (lo3) 76-78, 66.25, 45 111, track I I (strain A188/84). and 25.7 respectively. IDENTIFICATION OF BACTEROIDES UREOL YTICUS 113

1234567

76 -78 66

Fig. 3. Gel of protein extracts of NGU-associated strains representing PAGE-type I, tracks 1 and 2 (strains A234/83 and A24b/83), PAGE-type 11, tracks 3 and 4 (strains A174/84 and A247/83), PAGE type 111, track 5 (A I88/84) and B. ureolyricus track 6 (NCTC 10941). Track 7 contains the protein mo1.-wt markers.

NGU-associated strain A245/83 and filter-fixed DNA from other NGU-associated strains, from B. ureolyticus and from various reference strains of Bacteroides and Fusohacterium with DNA base compositions in the 24-34 mol % G + C range. The data show that 10 of the NGU-associated strains and three of the B. ureolyticus strains, including the type strain (NCTC 10941) had a high degree of similarity (72% at optimum temperature criteria) with the reference NGU-associated strain. The main exceptions were NGU-associated strain A185/84 and B. ureolyticus NCTC 10948, which were only partly related to strain A245/83 with relative hybridisation values of 33 and 2 1% respectively. The other reference strains of Bacteroides and Fusohacterium were unrelated to strain A245/83. The strains are listed in Table I1 according to their protein profile and the results show that the NGU-associated strains Of PAGE-type were Fig. 4. Densitometric traces of gels of protein extracts of B. ( 920/,) with a mean Of ureolytjcus (a+-) and NGU-associated strains (d-f): (a) NCTC 96+4%. The four PAGE-type 11 strains were less 10939, (b) NCTC 10948, (c) NCTC 10941, (d) A182/84, PAGE similar to the type-I reference strains with a mean Type I, (e) A176/84, PAGE type 11, (f) A188/84, PAGE type 111. 114 A. J. TAYLOR, CHRISTINE A. DAWSON AND R. J. OWEN

Table 11. DNA base compositions and DNA-DNA hybridisation levels with 3H- labelled DNA from NGU-associated strain A245/83

Source of unlabelled DNA Relative DNA sequence G + C content homology with Species Strain no. PAGE Type (mol%) strain A2451831 - NGU-associated A245183 I 30.lt 100 (homologous) A243183 I 3 I *2t 100 A305183 I 29.4 95 A 182184 I 28-3 92 A306183 I 31.2 ... A 188184 111 29.9 81 A 179184 111 29.0 78 A 176184 I1 26.5 82 A246183 I1 29.0f 78 A244183 I1 29.9t 73 A247183 I1 30.8t 72 A 185184 not 29.1 33 designated B. ureolyticus NCTC 10940 - 29.0 86 NCTC 10941 * - 29.0 80 A3071838 - 29.0 76 NCTC 10948 - 30.3 21 NCTC 10939 - ... <1 B. praeaculus NCTC I I158* - 30.9 8 B. termitidis NCTC 11300* - 33-3 <1 B. hypermegas NCTC 10571 - 29.4

* Type strain. t Value previously published (Taylor and Owen, 1984). Other literature values for the strains given below are from Holdeman et al. (I 984): NCTC 10941 28-30 mol%; NCTC 1 I300 34-36 mol %; NCTC 11 158 28 mol%. 1 Repeats of at least two determinations. Pooled standard deviation for all determinations was 6%. 5 Strain included in B. ureolylicus on the basis of its PAGE protein profile. The PAGE types of the remaining strains not included in the DNA analyses were: PAGE-type I; strains A302/83, A303183, A1 75/84, A1 77/84, A 178184, A 180184, A1 8 1/84, A 183184, A 184184, A186/84, A189184 and A190184; and PAGE-type 11; strains A174184 and A187/84. value of 76 & 5%, whereas the two PAGE-type 111 pattern (figs. 2 and 4) was quite distinct from those strains were slightly more similar to the type-I of the other strains and from those of the NGU- reference strain with a mean value of 79 f 2%. Apart associated group. The DNA hybridisation data also from the atypical strains NCTC 10939 and NCTC provided evidence strongly suggesting that the 10948, B. ureofyticus strains showed 7686% simi- strain should be excluded from B. ureolyticus. The larity (mean 81 5%) with the NGU-associated second atypical strain of B. ureofyticuswas NCTC reference strain. 10948. The conventional biochemical characteristics and PAGE protein profile (position of major Discussion peaks) conformed to those of the type strain of B. ureofyticus but it could be inferred from the DNA- This study showed that three of the five reference DNA hybridisation data (2 1% relatedness with strains of B. ureofyticus differed from the type strain strain A245/83) that the strain was partly but not (NCTC 10941) in certain respects. NCTC strain closely related to typical members of the species. 10939 ( = VPI 7902) appeared microscopically as The DNA differences correlated with differences in gram-negative coccal forms, not observed in typical the fine structure of the protein profile (fig. 4). The strains of B. ureolyticus. Furthermore it did not third atypical strain of B. ureolyticus was NCTC produce urease and oxidase, and its PAGE protein 10949, which differed from the other strains in its IDENTIFICATION OF BACTEROIDES UREOL YTICUS 115

PAGE profile (fig. 4). No DNA data are available polar pili, agar pitting, /3-haemolysis, oxygen toler- on this strain because all attempts to isolate high- ance, H2S production, protease activity, decarboxy- mo1.-wt DNA were unsuccessful. The NGU-asso- lation of amino acids and types of metabolic end ciated strain A185/84 also differed both in its products (acetate and lactate). In the case of casein protein profile and in DNA-DNA hybridisation digestion, we agree with Fontaine et al. (1 984 6) that data from the other NGU-associated strains. there are some differences between the two sets of The results of this study of NGU-associated strains, but as discussed above, we question the strains are consistent with a preliminary investiga- differential value of most of these characteristics tion on these micro-organisms (Taylor and Owen, apart from the metabolic end products and p- 1984), and support most of the observations of haemolysis, which have not been examined in our Fontaine et al. (1 984 b).There are certain discrepan- study. cies in the conventional biochemical tests. Pitting of Fontaine et al. (1984 b) suggested that the NGU- agar is a feature attributed to B. ureolyticus (Jack- associated strains may be biotypes or variants of B. son and Goodman, 1978) but was evident in only ureolyticus. However our results, in particular the 50% of the NGU-associated strains when first DNA-DNA hybridisations, showed that both sets received in our study. On repeated subculture, the of strains with the exceptions already discussed, characteristic was lost in all but three strains. comprised a homogeneous (highly related) group. Fontaine el al. (1 984 b) reported that strains never The level of DNA-DNA homology used to define a pitted agar on primary isolation but observed that species is arbitrary but it is generally accepted that 25% did so on subculture, so it does not appear to be strains with homologies in the 70-100% range are a reliable characteristic for use in identification. members of the same species (Johnson, 1984). There Spreading growth from the edge of the colonies was is insufficient genomic variation in our view to a more consistent feature in all the strains examined justify placing the NGU-associated strains in a and was the feature that originally led to their different subspecies to separate them from the other isolation (Fontaine et al., 1984 a).In contrast to the B. ureolyticus strains. Likewise, there are insuffi- observations of Fontaine et al. (1984 b), we found cient consistent test differences between the two sets that all NGU isolates produce H2S strongly after of strains, apart from casein digestion, to provide incubation for 18-48 h. The difference was attri- the basis for a biotyping scheme. We found that the buted to the greater sensitivity of the lead acetate most reliable method for subgrouping the strains paper strip method employed in our study. The was PAGE of proteins, and the DNA homology NGU-associated and B. ureolyticus strains were results confirmed a small but significant genome non-motile in hanging-drop preparations. It is difference between PAGE-types I and 11. As PAGE important to ensure that Brownian motion of the of proteins can be applied to a large number of cells in such preparations is not confused with the strains, we suggest it might provide the best “twitching” motility attributed to B. ureolyticus by approach if more detailed investigations of clinical Jackson and Goodman (1 978), which is a phenome- isolates are required to identify types or variants non associated with growth on a solid medium within B. ureolyticus. (Henrichsen, 1983). Our results of conventional biochemical tests Jackson and Goodman (1978) stated that B. (with the exception of casein digestion and decar- ureolyticus tolerated 021 % but not 5%, whereas our boxylation of amino acids), DNA base composi- findings were that all the NGU-associated strains tion, DNA-DNA homologies and protein electro- and B. ureolyticus grew well on BHIB in 025% after phoretic patterns confirmed a high degree of 3 days. These differences may be an effect of the similarity between the NGU-associated isolates and medium; for instance, there could be a difference reference strains of B. ureolyticus. We conclude between BHIB and the casein-cystine-haemin agar from these results that the NGU-associated strains used by Jackson and Goodman (1978) in the should be classified as B. ureolyticus as represented availability of suitable electron acceptors. by the type strains NCTC 10941 and NCTC 10940. Fontaine et al. (1984 b) reviewed the properties of We would include NCTC nos. 10948 and 10949 and B. ureolyticus reported by various authors, and strain A185/84 as atypical members of the species, those of the NGU-associated strains. Although but the identity of NCTC 10939 is uncertain. they noted that the relationship between the two The results of DNA-DNA hybridisations pre- groups was striking (i.e., similarities in G+C sented in this study show that B. ureolyticus is content, cell-wall structure and common biochemi- unrelated to B. praeacutus, B. hypermegas and to B. cal features), they drew attention to variations termiditis, which are the only other species in the between the two groups, notably the presence of genus Bacteroides with unusually low DNA base 116 A. J. TAYLOR, CHRISTINE A. DAWSON AND R. J. OWEN compositions of <33 mol% G+C. Likewise no ureolyticus, the atypical strains and allied taxa of similarities with F. necrophorum and F. nucleaturn low G + C content is consequently uncertain as they were detected. As the genus Bacteroides is heteroge- would be excluded from Bacteroides according to neous, Shah and Collins (1983) proposed that the criteria proposed by Shah and Collins (1983). Bacteroides sensu strictu should be restricted on the Further studies with techniques such as DNA- basis of a number of chemical criteria, and in the ribosomal RNA hybridisation are needed to clarify case of DNA base composition, to taxa with values their generic and suprageneric relationships. between 40 and 52 mol % G+C; that rearrange- ment would include the type species, B. fragiZis (41- We thank Mr. E. Fontaine and Dr. S. P. Borriello for 44 mol % G+C). The taxonomic position of B. supplying the clinical isolates used in this study.

REFERENCES “Bucteroides corrodens, anaerobic”. International Journal of Systematic Bacteriology 28: 197-200. Cowan S T 1974 Cowan and Steel’s Manual for the identification Johnson J L 1981 Genetic characterization. In: Gerhardt P (ed) of medical bacteria, 2nd Edn. Cambridge University Press. Manual of methods for general bacteriology, American Duerden B I 1980 The isolation and identification of Bacreroides Society for Microbiology, Washington D.C. pp 450472. spp from the normal human vaginal flora. Journal of Johnson J L 1984 Bacterial classification 111: Nucleic acids in Medical Microbiology 13: 79-87. bacterial classification. In: Krieg N R (ed) Bergey’s Manual Duerden B I, Bennet K W, Faulkner J 1982 Isolation of of systematic bacteriology vol. 1. Williams and Wilkins, Bucteroides ureolyticus (8.corrodens) from clinical infec- Baltimore pp 8-1 1. tions. Journal of Clinical Pathology 35: 309-3 12. Labbe M, Hansen W, Schoutens E, Yourassowsky E 1977 Eiken M 1958 Studies on an anaerobic, rod-shaped, Gram- Isolation of Bacieriodes corrodens and Eikenella corrodens negative micro-organism: Bacteroides corrodens N. sp. Acta from human clinical specimens. Comparative study of Puthologica el Microbiologicu Scundinuuica 43; 404-4 16. incidence and methods of identification. Infection 5: 159- Fontaine E A. Taylor-Robinson D, Hanna N F, Coufalik E D 162. 1982 Anaerobes in men with urethritis. British Journcrl qf Leaper S, Owen R J 1982 Differentiation between Campylobtrcter Venereal Diseuse 58:321-326. jejuni and allied thermophilic campylobacters by hybridiza- Fontaine E A, Borriello S P, Taylor-Robinson D, Davies H A tion of deoxyribonucleic acids. FEMS Microbiology Letters 1984a Characteristics of a Gram-negative anaerobe isolated 15 :203-208. from men with non-gonococcal urethritis. Journal uf Medi- Msller V 1955 Simplified tests for some amino acid decarboxy- rul Microbiology 17: 129-140. lases and for the arginine dihydrolase system. Acta Putholo- Fontaine E A, Borriello S P, Taylor-Robinson D, Davies H A gica et Microbiologica Scundinavica 36: 158- 172. 1984b Differential characteristics of a small Gram-negative Moss S 1983 Isolation and identification of anaerobic organisms anaerobe associated with non-gonococcal uret hritis which from the male and female urogenital tracts. Brirish Journal morphologically resembles Bacteroides ureolytirus. In: of Venereal Disease 59: 182-185. Mhrdh P-A, Taylor-Robinson D (eds) Bacterial vaginosis. Owen R J, Legros R M, Lapage S P 1978 Base composition. size Almqvist and Wiksell International, Stockholm, pp 157- and sequence similarities of genome deoxyribonucleic acids 165. from clinical isolates of Pseudoomonas putrefaciens. Journul Henrichsen J 1983 Twitching motility. Annual Reziiew qf Micro- of General Microbiology 104: 127-1 38. biology 37:81-93. Owen R J, Pitcher D 1985 Current methods for estimating DNA Henriksen S D I948 Studies in Gram-negative anaerobes: Gram- base composition and levels of DNA-DNA hybridization. negative anaerobic rods with spreading colonies. Acta In: Goodfellow M, Minnikin D E (eds) Chemical methods Pathologica et Microbiologica Scandinauica 25 :368 -375. in bacterial systematics, Academic Press, Orlando, F A, pp Holdeman L V, Kelley R W, Moore W E C 1984 Anaerobic gram 67-93. negative straight, curved and helical rods. Genus 1. Bacter- Robinson J V A, James A L 1973 Some serological studies on oides Castellani and Chalmers 1919, 959a I. In: Krieg NR Bacteroides corrodens. Journal of‘ Generul Microbiologv 78: (ed) Bergey’s Manual of systematic bacteriology vol 1. 193- 197. Williams and Wilkins, Baltimore, pp 604631. Shah H N, Collins M D 1983 Genus Bacteroides: A chemotaxo- Jackson F L, Goodman Y E, Be1 F R, Wong P C. Whitehouse nomical perspective. Journal of Applird Buctrriology 55: RLS 1971 Taxonomic status of facultative and strictly 403-4 16. anaerobic “corroding bacilli” that have been classified as Stargel M D, Thompson F S, Phillips S E. Lombard G L, Dowell Bacteroides corrodens. Journal of Medical Microbiology V R 1976 Modification of the Minitek miniaturizcd diHir- 4: 171-184. entiation system for the characterization of anaerobic Jackson F L, Goodman Y E 1972 Transfer of the facultatively bacteria. Journal of Clinical Microbiology 3:29 1-30 1. anaerobic organism Bacteroides corrodens Eiken to a new Taylor A J, Owen R J 1984 Morphological and chemical genus Eikenella. International Journal of Systematic Bacter- characteristics of anaerobic curved rod-shaped bacteria iology 22:73-77. from the female genital tract. In: MArdh P-A and Taylor- Jackson F L, Goodman Y E 1978 Bacteroides ureolyticus, a new Robinson D (eds) Bacterial vaginosis. Almqvist and Wiksell species to accommodate strains previously identified as International, Stockholm, pp 97-106.