INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1987, p. 198-210 Vol. 37, No. 3 0020-7713/87/030198-13$02 .OO/O Copyright 0 1987, International Union of Microbiological Societies

Oligella, a New Genus Including urethralis comb. nov. (Formerly Moraxella urethralis) and sp. nov. (Formerly CDC Group IVe): Relationship to Taylorella equigenitalis and Related Taxa R. ROSSAU,l K. KERSTERS,l E. FALSEN,* E. JANTZEN,3 P. SEGERS,l A. UNION,l L. NEHLS,2 AND J. DE LEY1* Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, B-9000, Ghent, Belgium’; Culture Collection, Department of Clinical Bacteriology, University of Goteborg, S-41346 Giiteborg, Sweden2; and Department of Methodology, National Institute of Public Health, Oslo, Norway3

The taxonomic relationships of Moraxella urethralis, the Centers for Disease Control (CDC) group IVe, Tayldrellu equigenitalis, and other grambnegative were studied by deoxyribonucleic acid (DNA)-DNA polyacrylamide gel electrophoresis of cellular proteins, and serological, biochemical, and auxanographic anrilyses. A high relationship was detected between M. urethralis and the CDC group IVe strains. However, no relationship of M. urethralis and CDC group IVe with genuine Moraxella species was obsek-ved. We describe a new genus, Oligella, containing two species: (to accommodate Moraxella urethralis) and Oligella ureolytica (to accommodate CDC group IVe strains). The type species is Oligella urethralis, with type strain ATCC 17960T. The type strain of Oligella ureolytica is CDC C379 (ATCC 43534, CCUG 1465, LMG 6519). Oligella is a member of rRNA superfamily 111, containing, e.g., the Pseudomonas acidovorans and Pseudomonas solandcearum complexes and Chromdbacterium, Janthisobacterium, and Neisseria species. The closest relatives of Oligella species are Taylorella equigenitalis and the family.

Moraxella urethralis strains are coccobacillary, aerobic, of M. urethralis and CDC group IVe was likewise observed gram-negative nonmotile bacteria, predominantly isolated by the systematic use of immunotyping during the last 15 from human urine. They are considered as commensals of years at the Culture Collection of the University of Goteborg the genitourethral tract, and in some rare cases they can be (CCUG), Goteborg, Sweden. involved in infections. Their role in pathogenicity is proba- In the present study, different methods were used to bly low. The species was first described by Lautrop et al. unravel the taxonomic relationships between the above- (28), who clearly stated that allocation in the genus Morax- mentioned taxa and other possibly related species. A new ella was only a matter of convenience. At present, the genus, Oligella, is proposed to accommodate Moraxella taxonomic status of M. urethralis is still doubtful, and in urethralis Lautrop, B@vre, and Frederiksen 1970 (28) as Bergey’s Manual of Systemic Bacteriology (2) this taxon is Oligella urethralis comb. nov., and CDC group IVe as included as a species incertae sedis in the genus Moraxella. Oligella ureolytica sp. nov. Taylorella equigenitalis and strains assigned by the Cen- ters for Disease Control (CDC), Atlanta, Ga., to group IVe MATERIALS AND METHODS equi- are also bacterial taxa of uncertain affiliation. T. Bacterial strains and media. The strains used are listed in genitalis, the causative agent of contagious equine metritis, HaemophiluS equigenitalis Table 1. They were routinely cultured on heart infusion agar was formerly known as Taylor, (Difco Laboratories, Detroit, Mich.), riutrient agar (Oxoid Rosenthal, Brown, Lapage, Hill, and Legros 1978 (37) and T. Taylorella Ltd., London, United Kingdom) or horse blood agar. rekently allocated to a new genus, Sugimato, equigenitalis was grown under microaerophilic conditions in Isayama, Sakazaki, arid Kuramochi 1983 (35). CDC group a jar on heart infusion agar or on chocolate agar in a CO;! IVe organisms are isolated from human urine with a much incubator at 37°C. Heat-killed cells (15 min at 70°C) of M. smaller frequency than is M. urethralis. Their pathogenicity urethralis NCTC 11008 and T. equigenitalis NCTC 11184T is not yet clear. Phenotypically, CDC group IVe strains Alcaligenes Bordetella were kindly provided by W. Mannheim (Institut fur resemble some species, some spe- Medizinische Mikrobiologie, Klinikum der Philipps- cies, and CDC group IVc-2 strains (33). However, prelimi- Universitat, Marburg, Federal Republic of Germany). Some nary deoxyribonucleic acid (DNA)-ribosomal ribonucleic additional reference strains were used in DNA-rRNA hy- acid (rRNA) hybridizations indicated that CDC group IVe bridizations (see Tables 4 and 5 and Fig. 3). Partly unpub- strains are not highly related to Alcaligenes or Bordetella lished hybridization data from the Ghent research group species (25). indicated that all these strains are genetic representatives of DNA-rRNA hybridizations also indicated that M. ,urethralis Moraxella the species and the rRNA branch to which they belong. does not belong to the genus (32), but SDS-PAGE of proteins. Cells were grown in Roux flasks at revealed a possible relationship with T. equigenitalis, CDC 28°C for 40 h on heart infusion agar, except for strains of T. group IVe, the family Alcaligenaceae (i.e., Alcaligenes and equigenitalis, which were incubated at 37°C. Whole-cell Bordetella species [9], and other species of rRNA superfam- protein extracts were prepared, and sodium dodecyl sulfate- illy I11 sensu De Ley (5). The taxonomically isolated position polyacrylamide gel electrophoresis (SDS-PAGE) was per- formed according to small modifications of the procedure of * Corresponding author. Laemmli (27) as described previously (26).

198 VOL. 37. 1987 OLIGELLA GEN. NOV. 199

TABLE 1. Strains used" Name as received Strain no.b Other strain designation Received fromh: Source (yr of isolation) Strains assigned to Oligella urethralis: M. urethralis ATCC 17960T' CCUG 13463T (=CMG 5303T), E. Juni and J. Shewan Ear LMG 1015T M. urethralis WM6 CCUG 994 (=LMG 5304), CIP K. Bgvre Cervix: Aarhus, Denmark (1961) 81.33 M. urethralis WM20 CCUG 995 (=LMG 5132), CIP K. Bgvre Urogenital tract; Aarhus, 81.34, CDC KC1290 Denmark (1962) M. urethralis CCUG 460 LMG 6232 Our isolate Ear; Goteborg, Sweden (1969) M. urethralis CCUG 1964 ATCC 43533, LMG 6231 Our isolate Urine; Goteborg, Sweden (1972) M. urethralis CCUG 3433 LMG 6227 Our isolate Urine; Goteborg, Sweden (1974) M. urethralis CCUG 4451 LMG 6835 Our isolate Pus; Goteborg, Sweden (1975) M. urethralis CCUG 5112 LMG 6837 Our isolate Urine; Goteborg, Sweden (1976) M. urethralis CCUG 7464 LMG 6836 Our isolate Urine; Goteborg, Sweden (1978) M. urethralis CCUG 7726 LMG 6838 Our isolate Urine; Goteborg, Sweden (1978) M. urethralis CCUG 10532 LMG 6229 Our isolate Urine; Goteborg, Sweden (1981) M. urethralis NCTC 11008 -J Pus from ear; United Kingdom (1971) M. urethralis CCUG 11920 LMG 6228 Our isolate Pus from foot wound; Goteborg, Sweden (1982) M. urethralis CCUG 12956 LMG 6230 Our isolate Urine; Goteborg; Sweden (1982) M. urethralis CDC C1098 CCUG 13464 (=LMG 6839) E. Juni Vaginal swab; Canada (1972)

Strains assigned to Oligella ureoly tica : CDC group IVe CDC C379T CCUG 1465T (=LMG 6519T), R. Weaver Urine; Louisiana (1971) ATCC 43534T CDC group IVe CDC B8375 CCUG 12412 (=LMG 3241) R. Weaver/(H. Urine; California (1971) Ericsson) CDC group IVe CDC FlOlO LMG 3242 (=CCUG 12688) R. Weaver Urine; Colorado (1981) CDC group IVe CDC F1147 LMG 3243 (=CCUG 12503) R. Weaver Urine; Alaska (1981) CDC group IVe CDC F3464 LMG 6614 (=CCUG 17692) R. Weaver Urine; California (1982) CDC group IVe CDC F4474 LMG 6613 (=CCUG 17691) R. Weaver Urine; Georgia (1983) CDC group IVe CDC F4479 LMG 6612 (=CCUG 17690) R. Weaver Urine; Tennessee (1983) CDC group IVe CDC F4585 LMG 6611 (=CCUG 17689) R. Weaver Urine; Pennsylvania (1983) CDC group IVe CDC F5560 LMG 6610 (=CCUG 17688) R. Weaver Urine; South Carolina (1984) CDC group IVe CDC F6167 LMG 6609 (=CCUG 176871, R. Weaver Catheterized urine; Kansas ATCC 35578, CDC F1582 (1981) CDC group IVe CCUG 17791 ATCC 43535, LMG 6773 Our isolate Contaminated cerebrospinal fluid; Bor&s, Sweden (1985)

Reference strains: T. equigenitalis NCTC 11184T CCUG 10786* (= LMG 6222T) NCTC" Equine; United Kingdom (1977?) T. equigenitalis CCUG 16464 LMG 6223 L. Mirtensson Equine; Uppsala, Sweden (1983) T. equigenitalis CCUG 16465 LMG 6224 L. Mirtensson Equine; Uppsala, Sweden (1983) T. eqyigenitalis CCUG 16466' LMG 6225 L. Mgrtensson Equine; Uppsala, Sweden (1982) T. equigenitalis CCUG 16467' LMG 6226 L. Mirtensson Equine; Uppsala, Sweden (1984) Bo rde te 11 a NCTC 452T CCUG 219T, LMG 1232T NCTC Lung of dog bronchiseptica Alcaligenes xylosoxidans ATCC 15173T LMG 1231T, NCTC 8582T ATCC and NCTC Soil subsp. denitr$cans (=CCUG 407T) Alcaligenes .faecalis NCIB 81.56' LMG 1229T, CCM 1052T NCIB and CCM Unknown (=CCUG 1814r) Alcaligenes eutrophus ATCC 17697T LMG 1199T, CCUG 1776T ATCC Soil; U.S.A. (1957) CDC group IVc-2 CDC E6793 LMG 3244 (=CCUG 12507) R. Weaver Human respiratory tract; Oregon (1979) CDC group IVc-2 CDC E8967 CCUG 12411, LMG 3245 R. Weaver/(H . Equine; Georgia (1980) (=CCUG 12505) Ericsson) M. lacunata CIP A182Tf CCUG 4441T, ATCC 17967T CIP Subacute conjunctivitis (=LMG 5301T) M. lacunata ATCC 17952 LMG 1009, NCTC 7911 J. Shewan and NCTC No information available (=CCUG 4862) M. osloensis ATCC 19976T CCUG 350T (;LMG 5131T), K. Bgvre Cerebrospinal fluid CDC A1920 M. osloensis CCUG 63 LMG 6916 Our isolate Urine; Goteborg, Sweden (1968) M. osloensis CCUG 11977 LMG 6917 Our isolate Blood; Goteborg, Sweden (1982) M. osloensis CCUG 13305 LMG 6918 Our isolate Pus; appendix, Goteborg, Sweden (1983) Coatinued 1200 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 1-Continued Name as received Strain no.b Other strain designation Received from:b Source (yr of isolation) M. osloensis CCUG 13369 LMG 6919 Our isolate Blood; Goteborg, Sweden (1983) M. osloensis CCUG 13705 LMG 6920 Our isolate Cerebrospinal fluid; Goteborg, Sweden (1983) M.phenylpyruvica ATCC 23333T CCUG 351T K. Bgvre Blood (=LMG 5372=)

a Other strains used for DNA-rRNA hybridizations are listed in Tables 4 and 5. ATCC, American Type Culture Collection, Rockville, Md.; CCM, Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia; CCUG, Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, University of Goteborg, Sweden; CDC, Centers for Disease Control, Atlanta, Ga.; CIP, Collection de I'Institut Pasteur, Paris, France; LMG, Culture Collection Laboratorium Microbiologie, Ghent, Belgium; NCIB, National Collection of [ndustrial Bacteria, Aberdeen, Scotland; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom; K. Bgvre, Kaptein W.Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Oslo, Norway; H. Ericsson, Department for Clinical Microbiology, Karolinska Sjukhuset, Stockholm, Sweden; E. Juni, Department of Microbiology and Immunology, University of Michigan, Ann Arbor; L. Mirtensson, The National Veterinary Institute, Uppsala, Sweden; J. Shewan, Torry Research Station, Aberdeen, United Kingdom; R. Weaver, Centers for Disease Control. Listed as Acinerobacrer Iwofii in the catalog of the American Type Culture Collection (1985), and received from J. Shewan in 1967 as "Mimu polymorpha var. oxiduns .'' Heat-killed cells of these strains were received from W. Mannheim, Institut fur Medizinische Mikrobiologie, Klinikum der Philipps-Universitat, Marburg, Federal Republic of Germany. Used solely for fatty acid analysis. Used solely for immunization and in DNA-rRNA hybridizations.

Morphological and biochemical features and carbon sub- acid methyl ester fractions were analyzed on a gas chro- strate assimilation tests. The techniques described by De Vos matograph equipped with a flame ionization detector and a et al. (13) were used. Nitrite reduction was carefully tested 25-m fused-silica capillary column coated with a nonpolar by incubation for 6 days in 0.05 and 0.001% nitrite as methyl silicone (BP-1; Scientific Glass Engineering Ltd., described by Falsen (16). API galleries (API 50CH, 50A0, Milton Keynes, United Kingdom). Additional analyses were and 50AA; API System, Montalieu-Vercieu, France) were performed on a 30-m polar (Carbowax), fused-silica capillary used for the carbon assimilation tests. column (Supelco Inc., Bellfonte, Pa,). The column temper- Numerical analysis of phenotypic features. The results of ature was usually programmed from 120 to 280°C at 8"C/min. the auxanographic tests were scored as follows: distinct Retention times and mass spectrometry were used to iden- growth after 1, 2, 4, and 7 days of incubation at 30°C were tify the eluting constituents. An electronic integrator calcu- coded as 7, 6, 5, and 4, respectively. Weak growth after 7 lated the amount of each fatty acid as a percentage of the days was coded as 3, and absence of growth after 7 days of total amount. incubation was coded as 1. An occasionally contaminated Taxonomic immunotyping. Antisera were prepared ac- cupule was coded as 0 (no comparison). Biochemical tests cording to previously reported procedures (13, 14) by immu- were coded as 5, 4, 3, 2, and 1 for, respectively, strong or nizing rabbits with bacterial extracts in Freund incomplete fast-positive, positive , weak or late-positive, questionable- adjuvant. The immunodiffusion technique used has been positive or probably negative, and negative. Nitrite reduc- described before (13, 14). tion was coded as follows: 5, total reduction of 0.05% nitrite; Transformation studies. Genetic transformation was car- 4, partial reduction of 0.05% nitrite; 3, 0.1% nitrate reduced ried out as described by Juni (23) with M. urethralis ATCC beyond nitrite; 2,0.001% nitrite reduced; 1, no reduction. Of 17960Tas the test strain. A cell paste of this strain was mixed the 235 characteristics tested, 126 were used in the numerical with sterile lysates of several other strains and incubated analysis. The API 20NE tests (21 features) were not included overnight on heart infusion plates at 33°C. A loopful of the in the computer analysis because they duplicated other tests, mixture was then transferred to a citrate-mineral medium and 88 other features were not used because all strains tested (23) and incubated at 33°C for 3 days. The test was consid- were either negative or displayed the same positive value. ered as positive when colonies were clearly visible to the The between-strain similarities were calculated by using the naked eye. Gower similarity coefficient (SG) (34). Cluster analysis was Isolation of DNA. Cells (2 to 7 g [wet weight]) were grown performed by the unweighted average pair group method in Roux flasks for 3 to 4 days. M. urethralis cells were first (34), using the Clustan 2.1 program of Wishart (38) on the treated with lysozyme (Boehringer Mannheim, Federal Re- Siemens 7551 (BS2000) computer of the Centraal Digitaal public of Germany) (2 mg/ml) for 10 min at 37°C in 0.033 M Rekencentrum, Ghent, Belgium. The reproducibility of clus- tris (hydroxymethyl) aminomethane (Tris) hydrochloride tering was estimated by duplicate tests of strains ATCC buffer containing 0.001 M ethylenediaminetetraacetate 17960T, WM20, CDC F1010, and ATCC 19976T. (EDTA) (final pH 8.0). After lysis in 1% SDS at 60"C, the Gas chromatography of cellular fatty acids. The following DNA was isolated by the method described by Marmur (29) strains were examined for cellular fatty acid composition: M. and further purified by CsCl gradient centrifugation. urethralis ATCC 17960T, WM6, CCUG 460, CCUG 1964, Filter fixation of DNA and determination of the amount of CCUG 3433, CCUG 10532, CCUG 11920, and CCUG 12956; DNA fixed. Thermally denatured DNA was fixed on nitro- CDC group IVe strains CDC B8375, CDC F1010, CDC cellulose filters (Sartorius, Gottingen, Federal Republic of F1147, CDC F3464, CDC F4474, CDC F4479, CDC F4585, Germany) as described previously (18). The amount of CDC F5560, CDC F6167, and CCUG 17791; and T. filter-fixed DNA was estimated chemically by the method of equigenitalis NCTC 11184T, CCUG 16464, CCUG 16465, Richards (30). CCUG 16466, and CCUG 16467. Strains were grown on DNA base composition. The average mole percent (mol%) standard heart infusion agar as described above and har- guanine-plus-cytosine (G + C) of the genomes was measured vested, freeze-dried, and methanolyzed (both alkaline and by the thermal denaturation method described by De Ley acidic) as described previously (20, 21), The resulting fatty and Van Muylem (11). VOL. 37, 1987 OLIGELLA GEN. NOV. 201

Preparation of 3H-labeled rRNA of M. urethralis WM6. patterns of any of the other taxa investigated (Fig. l), but After inoculation, approximately 2.5 mCi of [5,6-3H]uracil this in itself does not allow any conclusion on the degree of (New England Nuclear Corp., Boston, Mass.) was added to relatedness. The protein patterns of T. equigenitalis and 150 ml of heart infusion broth in a cotton-stoppered 1-liter CDC group IVc-2 differed from each other, and from the flask with a side tube. The incubation temperature was 33"C, patterns of the reference strains of Alcaligenes, Bordetella, and aeration was achieved by gentle magnetic stirring. At the and Moraxella species. end of the logarithmic phase, the cells were collected by Numerical analysis of phenotypic features. The strains centrifugation, and the labeled rRNA was isolated and listed in the Table 1, except M. urethralis NCTC 11008, the purified as described by De Ley and De Smedt (7). Because T. equigenitalis strains, and Moraxella phenylpyruvica of better growth behavior, strain WM6 was used instead of ATCC 23333T,were compared by numerical analysis of their the type strain of M. urethralis. Genetically and phenotypi- phenotypic features (Fig. 2). T. equigenitalis strains were cally this strain is as representative for the species as the not investigated in the auxanographic tests because sound type strain (see below). From 0.9 g [wet weight] of cells, 8.3 comparison of the results is not possible when different mg of rRNA was obtained with a specific activity of approx- conditions for incubation of the galleries are needed. In the imately 9 x lo4 dpdpg. experimental conditions used, M. phenylpyruvica ATCC DNA-rRNA hybridizations. The hybridizations were per- 23333T did not grow on any of the substrates and was formed as described by De Ley and De Smedt (7). The most therefore not included in the numerical analysis. We inves- important parameter is Tm(+ the temperature (in "C) at tigated six M. osloensis strains in search of new diagnostic which half of the DNA-rRNA duplex is denatured. Earlier features to differentiate this species from M. urethralis. The investigations (7, 10, 12) have shown that Tm(e)is a reliable following four phena were delineated above a Gower simi- measure of genetic relatedness, particularly useful for the larity coefficient (SG)of 85%: M. urethralis, CDC group IVe, detection of more remote relationships which cannot be M. osloensis, and CDC group IVc-2. Test reproducibility traced with most other methods. The higher the Tm(e)value, was at least 96% SG, and the cophenetic correlation coeffi- the higher the relatedness. A second parameter, of more cient was 0.95. The reference strains of Alcaligenes limited taxonomic value, is the percent rRNA binding, i.e., eutrophus, Alcaligenes faecalis, Alcaligenes xylosoxidans the amount of rRNA bound to 100 pg of DNA after ribonu- subsp. denitrgcans, Bordetella bronchiseptica, and Morax- clease treatment, ella lacunata occupied a separate position in the dendro- DNA-DNA hybridizations. The degree of binding (%D) was gram. estimated by the initial renaturation rate method (6). M. urethralis and CDC group IVe strains clustered at 84% Renaturations were performed in 2~ SSC with a DNA SG and shared 176 phenotypic features (all either positive or concentration of 42 pg/ml at 70"C, or in IXSSC with 53 pg negative; see below). of DNA per ml at 67°C. Hybridizations involving DNA of T. Comparison of gas chromatographic cellular fatty acid equigenitalis were carried out at 65°C in 2~ SSC with 42 pg patterns. As shown in Table 2 the intragroup fatty acid of DNA per ml. patterns of the three groups were homogeneous and compat- Determination of molecular complexity of the genome. The ible with the species definition. The intergroup pattern molecular complexity of bacterial genomes was calculated differences were also relatively small with the presence of from the initial renaturation rates obtained as described by lactobacillic acid (C19:cyc)in the CDC group IVe profiles as Gillis et al. (19). The complexity of the Escherichia coli B the single qualitative hallmark. Quantitatively T. equi- genome (2.71 x lo9 daltons) was used as a reference (17). genitalis showed small but distinct individual features by a relatively low content of myristic acid (C14:o) and 3- RESULTS hydroxypalmitic acid (3-OH-C16:o),and by its high level of 3-hydroxymyristic acid (3-OH-C14:o),stearic acid (C18:o),and Protein electropherograms. SDS-PAGE patterns of repre- the unidentified constituent XI. The patterns of M. urethralis sentative strains are shown in Fig. 1. The following protein and CDC group IVe (Table 2) were strikingly similar with electrophoretic groups of bacteria were recognized: M. C19:cycin the patterns of the CDC group IVe strains as the urethralis, CDC group IVe, T. equigenitalis, Moraxella single significant difference. osloensis, and CDC group IVc-2. The protein profiles of Immunotypical properties. Eight immunotypical groups seven M. urethralis strains (WM6, WM20, CCUG 445, were recognized (Table 3). Clear-cut serological cross- CCUG 7726, CCUG 10532, CCUG 11920, and CCUG 12956; reactions were observed between M. urethralis and CDC not all patterns are shown in Fig. 1) were nearly indistin- group IVe, whereas both groups displayed no or only very guishable from the pattern of the type strain ATCC 17960 weak cross-reactions with the following taxa: T. equigen- and showed reproducible differences from the patterns of the italis, B. bronchiseptica, A. faecalis (strain NCIB 813jT; other six M. urethralis strains for proteins in the zone with data not shown), A. eutrophus, CDC group IVc-2, M. relative mobilities of 0.3 to 0.4. Nevertheless, these latter six lacunata, and M. osloensis. strains are unmistakably part of the M. urethralis cluster. Genetic transformation. Transformation studies, with citr- Strains CCUG 1964, CCUG 7464, and CDC C1098 had ate as a marker, showed that all M. urethralis strains tested identical SDS-PAGE patterns, and strains CCUG 3433 and which grew on the citrate-mineral medium (strains WM6, CCUG 5112 were also indistinguishable from each other by WM20, CCUG 460, CCUG 1964, and CCUG 12956) readily electrophoresis. The pattern of strain CCUG 460 dif€ered transformed strain ATCC 17960T. However, all the investi- from the core of the M. urethralis cluster in a few minor gated CDC group IVe strains (CDC C379T, CDC F1010, bands only. Eleven CDC group IVe strains (including strains CDC B8375, and CDC F1147) and CDC group IVc-2 strains CDC F3464, CDC F4474, CDC 4479, and CCUG 17791; not (CDC E6793 and CDC E8967) failed to do so. Among the M. shown in Fig. 1) formed an extremely homogeneous electro- urethralis strains which did not grow on the citrate-mineral phoretic group, and so did three T. equigenitalis strains. medium (CCUG 3433, CCUG 10532, and CCUG 11920) only The SDS-PAGE patterns of M. urethralis and CDC group strain CCUG 10532 transformed strain ATCC 17960T to a IVe were on the whole more similar to each other than to the certain extent. The M. urethrazis strains CCUG 11920 and 2:02 ROSSAU ET AL. INT. J. SYST. BACTERIOL.

FIG. 1. Normalized SDS-PAGE patterns of representative strains of Moraxella urethralis, CDC group IVe, Taylorella equigenitalis, Moraxella osloensis, CDC group IVc-2, and type strains of Alcaligenes xylosoxidans subsp. denitrijicans, Alcaligenes eutrophus, Alcaligenes faecalis, Bordetella bronchiseptica, Moraxella lacunata, and Moraxella phenylpyruvica.

CCUG 10532 were competent and could be transformed to a IVe and T. equigenitalis ATCC 11184T showed only a very citrate-positive phenotype by DNA from other M. urethralis low degree of DNA duplexing (14 to 26%). strains. M. urethralis CCUG 3433 was not transformable. DNA-rRNA hybridizations. 3H-labeled 23s or 16s rRNA G+C content, molecular complexity of the genomes, and from M. urethralis WM6 was hybridized with filter-fixed IDNA-DNA hybridizations. The average G+C values of the DNA from a variety of gram-negative bacteria (Table 4). The ]DNA of the investigated strains of M. urethralis and CDC similarity map (Fig. 3) was obtained by plotting Tm(e,values group IVe were strikingly similar, ranging from 46.0 to 47.5 versus the percentages of rRNA binding. The seven M. mol% (Table 4). The molecular complexities of their urethralis strains had a high degree of rRNA cistron similar- genomes were likewise in the same range of 1.2 x lo9 to 1.4 ity with the reference strain WM6 [T,,,, values ranging from :Y lo9 daltons. The type strain of T. equigenitalis had a much 78.9 to Sl.l°C]. With Tm(e,levels of approximately 77"C, lower G+C value of 37.6 mol%, and its genome complexity three representative strains of CDC group IVe clearly sepa- was lo9 daltons, this being only 37% of the complexity of the rated from M. urethralis but were still highly related with it genome of E. coli. (Table 4 and Fig. 3 and 4). The Tm,el values of the hybrids As expected from the similarities in the protein between rRNA of M. urethralis WM6 and DNAs from dectropherograms, M. urethralis ATCC 17960T showed 89 strains of B. bronchiseptica, Alcaligenes (members of rRNA to 100% DNA homology with M. urethralis WM6, CCUG superfamily 111), and T. equigenitalis were considerably 460, CCUG 1964, CCUG 3433, and NCTC 11008. Three lower and ranged from 72.4 to 74.2"C. Other taxa of rRNA strains of CDC group IVe (CDC C379T, CDC B8375, and superfamily I11 (5) such as Pseudomonas acidovorans, Pseu- (JDC F1147) had likewise a high degree of DNA relatedness domonas woodsii (a representative of the Pseudomonas (above 82%). However, strains of M. urethralis, CDC group solanacearum rRNA branch), Janthinobacterium lividum, VOL. 37, 1987 OLIGELLA GEN. NOV. 203

60 80 ‘losG 100 I 1 Phenon WM6 No. CCUG 7464 CCUG 4451 CCUG 5112 CCUG 7726 CDC C1098 CCUG 10532 CCUG 11920 1 M.URETHRALIS CCUG 3433 CCUG 1964 CCUG 12956, ATCC 17960 CCUG 460 WM20 CDC F1147 CDC 88375 CDC F4479 CDC C379T CDC F6167 CDC F4474 2 CDC GROUP Ee CCUG 17791 CDC F3464 CDC F4585 CDC FlOlO CDC F5560, r- r- ATCC 19976l I -r

I ATCC 17952 M.LACUNATA NCTC 452T 6. BRONCHISEPTICA ATCC 15173T A. XYLOSOXIDANS subsp. DENlTRlFlCANS NClB 8156T A. FAECALIS CDC €6793 b I

FIG. 2. Dendrogram showing the phenotypic relationships between the strains investigated, based on the Gower similarity coefficient (SG) and the unweighted average linkage cluster analysis.

Chromobacterium violaceum, several Neisseria species, and a CDC group IVc-2 strain were located at Tmfe)values of 65.3 to 71.6”C (Table 4 and Fig. 3). Genuine moraxellae (M. lacunata and M. osloensis, members of rRNA superfamily TABLE 2. Cellular fatty acid composition of M. urethralis, CDC 11) and a Rhizobium meliloti strain (belonging to rRNA group IVe, and T. equigenitalis superfamily IV) had much lower Tm(e,values (Table 4 and Fatty acid Fig. 3). These data were confirmed by DNA-rRNA hybrid- (C atoms:double M. urethralis (8)” T’ equigenitalis bonds) “pv”, :zyp (5) izations with reference rRNAs from several other bacterial taxa (Table 5). A dendrogram based on the Tmfe,values of the 14:O 4.4-5.3 5.0-8.0 0.7-1.4 DNA-rRNA hybrids is shown in Fig. 4. 15:O 0.2-2 .o 0.2-1.2 0.0-0.3 16:1 2.2-3.6 1.2-3.4 1.0-1.8 16:O 25.7-30.6 19.5-30.5 22.1-29.6 DISCUSSION 17:O 0.1-1.3 0.1-0.5 0.3-0.7 18:2 tr”-0.5 0.1-0.4 0.5-0.9 When Lautrop et al. (28) proposed M. urethralis as a new A9-18: Id 0.0-0.5 tr-0.2 0.1-0.8 species of the genus Moraxella, they emphasized that their All-18~1~ 45.3-49.6 37.8-47.9 42.3-5 1.6 results neither proved nor contradicted relationships to true 18:O 0.9-1.4 0.5-1.8 3.1-7.2 Moraxella species. The separate taxonomic position of M. XIf 0.8-2.5 0.7-1.7 4.4-7.6 urethralis was further substantiated by chemotaxonomic x,f 0.6-1.0 0.5-0.7 0.3-0.9 studies (4, 20, 21). Resemblance to the overall fatty acid xd 0.3-0.4 0.2-0.7 0.4-0.8 composition of the Neisseriaceae such as the absence of - - 19:cyc 2.7-10.8 branched-chain fatty acids, led Bgivre and Hagen (3) and 3-OH-14 :0 4.7-7.3 4.6-7.3 9.2-1 1.5 M. urethralis 3-OH-16:O 4 -6-6.4 3.7-5.1 1.0-1.5 Bgvre (2) to conclude that might constitute a new genus within the family Neisseriaceae. M. urethralis a Total number of strains investigated. was considered to be a species incertae sedis of the genus Range expressed as percentage of the total amount. Moraxella (2). Our serological data (Table 3) and DNA- Less than 0.1%. Oleic acid. rRNA hybridizations results with labeled rRNA of M. cis-Vaccenic acid. urethralis clearly show (Tables 4 and 5; Fig. 3 and 4) that this Unidentified constituent. taxon is not related to any of the species of the Neis- 204 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 3. Immunodiffusion analyses Antiserum against: M. urethralis group Antigen from: CDC T. B' A. eutrophus CDC group M*lacu- M. osloensis ATCC CCUG IVe equigenitalis septica IVC-2 CDC nUkI CIP ATCC 19976T 17960T 1964 CDC C379T NCTC 11184T NCTC 452T 17697T E8967 A182T

M. urethralis ATCC 17960T 6" 8 4 0 1 0 0 1 0 IM. urethralis WM6 4 7 4 0 1 0 0 0 0 111. urethralis WM20 4 7 4 0 0 0 0 0 0 M. urethralis CCUG 460 5 6 4 0 1 0 0 1 0 111. urethralis CCUG 1964 5 7 4 0 0 0 0 0 0 M. urethralis CCUG 3433 6 8 4 0 0 0 0 0 0 M. urethralis CCUG 4451 6 8 4 0 0 0 0 0 0 .W. urethralis CCUG 5112 6 8 4 0 0 0 0 0 0 ,W. urethralis CCUG 7464 6 8 4 0 0 0 0 0 0 M. urethralis CCUG 7726 6 8 4 0 1 0 0 0 0 ,V. urethralis CCUG 10532 5 8 4 0 1 0 0 0 0 ,V. urethralis CCUG 11920 5 8 4 0 1 0 0 0 0 ,W. urethralis CCUG 12956 5 8 4 0 1 0 0 0 0 CDC group IVe CDC C379T 4 5 7 0 0 0 0 0 0 CDC group IVe CDC B8375 3 6 7 0 0 0 0 0 0 CDC group IVe CDC FlOlO 3 5 6 1 0 0 0 0 0 CDC group IVe CDC F1147 4 5 7 1 0 0 0 0 0 {CDCgroup IVe CDC F3464 3 6 7 1 0 0 0 0 0 CDC group IVe CDC F4474 3 6 7 0 0 0 0 0 0 CDC group IVe CDC F4479 3 6 7 0 0 0 0 0 0 CDC group IVe CDC F4585 4 6 8 0 0 0 0 0 0 CDC group IVe CDC F5560 3 4 7 0 0 0 0 0 0 lCDC group IVe CDC F6167 4 5 7 1 0 0 0 0 0 [CDC group IVe CCUG 17791 3 6 8 0 0 0 0 0 0 T. equigenitalis NCTC 11184T 1 1 0 6 0 0 0 0 1 T. equigenitalis CCUG 16464 1 1 0 6 0 0 0 0 1 T. equigenitalis CCUG 16465 1 1 0 7 1 0 0 0 1 B. bronchiseptica NCTC 452T 0 2 1 0 6 1 1 0 0 .4. xylosoxidans subsp. 0 1 1 0 4 1 1 0 0 denitrijicans ATCC 15173T .4lcaligenesfuecalis NCIB 8156T 0 2 2 0 3 1 1 0 0 .4lcaligenes eutrophus ATCC 0 1 0 0 0 7 5 0 0 17697= CDC group IVc-2 CDC E6793 0 1 0 1 (CDC group IVc-2 CDC E8967 0 1 0 0 M. lacunata ATCC 17952 0 1 7 2 M. osloensis ATCC 19976T 0 0 0 4 44. osloensis CCUG 63 0 0 0 5 M. osloensis CCUG 11977 0 0 1 5 44. osloensis CCUG 13305 0 0 1 5 -44. osloensis CCUG 13705 00 2 6

~ ~ ~~ 0, No precipitate; 1 or 2, weak reaction with uncertain interpretation; 3, weak reaction usually revealing relatedness when serum specificity was high; 4 or 5, moderate reaction revealing relatedness or identity with unsatisfactory serum; 6,7 or 8, strong reaction observed only with closely related strains or with identical ,strains when the serum specificity was high. Each value is the average of at least two immunodiffusion analyses.

seriaceae. M. urethralis is a member of rRNA superfamily a limited number of tests (Tables 6 and 7), and possess some 111, and a high relationship was found with CDC group IVe phenotypic traits in common with Bordetella and Alcal- strains and representative strains of the genera Alcaligenes, igenes. However, M. urethralis and CDC group IVe strains i!?ordetella, and Taylorella (Tables 4 and 5; Fig. 3 and 4). resemble also some Moraxella species and differentiation ,4lthough phenotypical similarities between the CDC group among M. urethralis, M. osloensis, and M. phenylpyruvica We, Bordetella and Alcaligenes species have been observed might be difficult (31). (.33), these bacteria have never been thought to be related to Although previously published taxonomic data cannot M. urethralis. Yet, the detected rRNA relatedness between support our results, none really disagrees with the newly A4. urethralis and the CDC group IVe was further evidenced established suprageneric relationships of M. urethralis. by phenotypical data (Fig. 2), G+C content (Table 4), DNA-DNA hybridizations failed to reveal relatedness of M. molecular complexity of the genomes, protein electrophero- urethralis to Acinetobacter, Bordetella, Brucella, Hae- grams (Fig. l),and fatty acid composition (Table 2). How- mophilus, Kingella, Legionella, Moraxella, and Taylorella ever, interrelatedness between both groups could be detected (22, 35). M. urethralis was neither transformed to strepto- neither by genetic transformation, using citrate as the marker, mycin resistance (28) nor to prototrophy (23) by DNA from nor by DNA-DNA hybridizations (see above). other species. The high phenotypic relationship between M. urethralis Our polyphasic approach indicates that M. urethralis and the CDC group IVe is particularly obvious in the should be removed from Moraxella and placed in a new j>henogram(Fig. 2). Both taxa can only be differentiated by genus in which the CDC group IVe strains should also be VOL. 37, 1987 OLIGELLA GEN. NOV. 205

TABLE 4. Parameters of hybrids between ['HIrRNA of M. urethralis WM6 and DNAs of selected strains and G+C contents

Sequence" DNA from strainb: 7ac M. URETHRALIS 1 M. urethralis WM6 81.1 0.17 47.2 5 2 M. urethralis ATCC 17960T 80.6 0.13 47.5 3 M. urethralis WM20 79.3 0.16 47.2 ,:t=-g CDC GROUP He 4 M. urethralis NCTC 11008 80.4 0.16 47.4 5 M. urethralis CCUG 3433 78.9 0.13 46.7 6 M. urethralis CCUG 1964 79.9 0.13 46.1 7 M. urethralis CCUG 460 79.2 0.09 46.3 ,&*?L IGENACEAE TAYLORELCA? 8 CDC group IVe CDC C379T 77.4 0.11 46.0 9 CDC group IVe CDC F1147 77.2 0.14 46.4 I? SOLANACEARUM 10 CDC group IVe CDC B8375 77.0 0.11 46.6 r RNA branch 11 T. equigenitalis NCTC 11184T 73.0 0.18 37.6 JANTHIN8BACTERIUM 12 B. bronchiseptica NCTC 8761 74.2 0.08 69.5 0 2! 13 A. faecalis Lautrop AB220 72.4 0.09 57.5 -2'1 CHROMOBACTERIUM 14 A. xylosoxidans subsp. deni- 73.7 0.06 68.6 I? ACI DOVOR- r RNA branch triJicans NCIB 11015 25 NElSSERlA 19-18 22 15 CDC group IVc-2 CDC E8967 71.6 0.08 65.9 24 16 P. woodsii NCPPB 2441 70.3 0.07 17 P. caryophylli NCPPB 349 68.1 0.06 18 P. acidovorans ATCC 15668T 65.8 0.06 66.6 19 A. paradoxus ATCC 17713T 65.6 0.03 67.0 20 J. lividum NCTC 9796T 68.6 0.14 65.5 21 C. viofaceum NCTC 9757T 68.7 0.21 67.2 6 MORAXELLA 22 N. rneningitidis NCTC 10025T 65.5 0.15 52.8 RHlZOBlUM 23 N. subflava ATCC 10555 67.3 0.12 p 28 &27 24 N. denitrificans ATCC 14686= 65.3 0.06 56.2 I I I I N. lactarnica NCTC 10616 66.4 53.8 0.05 0.10 0.15 0.20 25 0.14 */. rRNA binding 26 M. lacunata ATCC 17967T 58.9 0.07 43.9 27 M. osloensis ATCC 19976T 58.7 0.08 FIG. 3. rRNA similarity map of DNA-rRNA hybrids between 28 M. caviae ATCC 14659T 58.1 0.06 46.9 3H-labeled rRNA of Moraxella urethralis WM6 and DNAs from a 29 R. mefiloti NZP 4009 57.7 0.04 variety of bacteria, including seven M. urethralis strains, three CDC group IVe strains, and Taylorella equigenitalis NCTC 1118AT. " Sequence numbers (see Fig. 4) are not strain numbers. Strains (represented by sequence numbers [Table 41) are joined by Abbreviations: Lautrap, H. Lautrop, Statens Serum Institutet, Copenha- gen, Denmark; NZP, Applied Biochemistry Division, Department of Scien- lines when they belong phenotypically or genetically to the same tific and Industrial Research, Palmerston North, New Zealand (also see Table taxon. P., Pseudomonas. 1). ' Some GiC values were previously published by the Ghent research fermented nor oxidized. Oxidase positive and usually group. catalase positive. Indole and hydrogen sulfide are not formed, and gelatin is not hydrolyzed. included as a separate species. We propose the name The major cellular fatty acids are cis-vaccenic acid (C18:1) Oligella gen. nov. with two species: Oligella urethralis (Lautrop, Bgvre, and Frederiksen 1970) comb. nov., replac- M. urethralis 1970 (28), ALCALIGENACEAE ing Lautrop, Bqjvre, and Frederiksen ' v, '3 and Oligella ureolytica sp. nov., for the CDC group IVe [L strains. For the moment, Oligella does not seem to fit in one of the known bacterial families. Characteristics for differen- tiating Oligella urethralis and Oligella ureolytica from each other and from some other taxa are summarized in Table 7. Description of Oligella gen. nov. Oligella (0.lig.el'la. Gr. adj. oligos, little; scanty; M.L. dim. ending -ella;M. L. fem. n. Oligella, referring to small bacterium with limited nutri- MORAXELLA tional properties). BRANHAMELLA falqe neisseriae Gram-negative, noncapsulated, nonsporeforming small 70 JANTHINOBACTERIUM rods, mostly not exceeding 1 pm and often occurring in IrRNA Superfamily II pairs; they lack the typical plumpness of moraxellae. Mostly I rRNA branch CHROMOBACTERIUM rRNA Superfamily I nonmotile but some strains of 0. ureolytica are peritri- I I chously flagellated (Fig. 5). They are moderately fastidious, 6ot I aerobic chemoorganotrophs growing on nutrient agar, but I rRNA Superfamily Ip growth is enhanced by the addition of, e.g., yeast autolysate, FIG. 4. rRNA cistron similarity dendrogram of Moraxella serum, or blood. Colonies on blood agar develop rather urethralis and related taxa. The Tm(e)values were taken from the slowly and are more overtly white than those of all recog- present study, De Ley et al. (9), and Rossau et al. (32) and were nized species of Moraxella. No pigments and no odor are clustered by the unweighted average linkage method. The black produced. Nonhemolytic. Biochemically they are rather areas at the top of most branches represent the ranges of Tm(e)values inert, and only a few organic acids and amino acids are of the reference taxa. Broken lines show branches from which no utilized as the sole carbon source. Carbohydrates are neither labeled rRNA was included. 206 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 5. T,,,(,, values of DNA-rRNA hybrids between DNA from M. urethrali~,CDC group We, T. equigenitcilis, and CDC group IVc- 2, and labeled rRNA from 14 reference strains T,,,c,,("C) M. urcthrcilis CDC group IVe CDC group IVc-2 Labeled rRNA from: T. equigenitcilis ATCC WM20 NCTC CCUG CDC CDC NCTC 11184' CDC CDC 17960T WM6 11008 1946 F1147 B8375 E8967 E6793 Bordetella bronchiseptica 71.1 70.6 70.9 71.0 70.5 70.6 71.1 NCTC 452T Alcaligenes xylosoxidans subsp. 72.6 72.0 71.7 71.2 72.1 72.3 denitrifcans ATCC 15173T Alcaligenesfaecalis NCIB 8156T 70.9 71.1 70.8 71.2 70.5 Pseudornonas solanacearum 70.0 70.2 68.7 70.4 70.1 76.1 76.8 NCPPB 325T Alcaligenes eutrophus ATCC 70.4 70.8 78.2 78.5 17697T Janthinobacteriurn lividurn 70.3 70.0 67.6 69.5 68.2 70.1 69.7 NCTC 9796= Pseudornonas acidovorans 67.8 68.0 65.5 ATCC 1566gT Alcaligenes paradoxus ATCC 67.6 67.4 59.2 17713T Chrornobacteriurn violaceurn 68.8 68.3 NCTC 9757T Neisseria flavescens ATCC 66.1 66.2 65.3 67.6 13120T Moraxella lacunata ATCC 61.4 60.7 59.2 17952 Acinetobacter calcoaceticus 61.5 59.2 ATCC 23055T Ha ern ophilus injuenza e NCTC 59.2 8143T Ha ern ophilus uph rop hilus 60.6 NCTC 5906T and palmitic acid (C16:o). Two 3-hydroxylated acids (3-OH- butyfate, 2-naphthyl caprylate, and L-leucyl-2-naphthylamide. C14:oand 3-OH-CI6:,) are present, whereas branched-chain The following substrates are pot hydrolyzed (using API ZYM acids are absent. galleries): 2-naphthyl mydstate, ~-valyl-2-napbthylamide, L- Farameters of DNA-rRNA hybrids reveal that Oligella is cystyl-2-naphthylamide, N-benzoyl-~~-arginine-2-naph- related at the suprageneric level to Taylorella, Bordetella, thylamide, jV-glutarylphenylalanine-2-naphthylamide,2- and Alcaligenes. naphtbylphosphate (at pH 5.4), 6-bromo-2-naphthyl-a-~- Mainly isolated from the genitourinary tract of humans. galactopyranoside, 2-naphthyl-P-~-galactopyranoside,naph- Their pathogenicity is unknown but most probably low. thol-AS-BI-P-D-glucuronate, 2-naphthyl-a-~-glucopyranoside, The G+C content of DNA is 47 2 1 mol% (T, method). 6-bromo-2-naphthyl-~-~-glucopyranoside,1-naphthyl-N- Type species is Oligellu urethralis (Lautrop, Bqbre, and acetyl-P-D-glucosaminide, 6-bromo-2-naphthyl-a-p-manno- Frederiksen 1970) comb. nov. pyranoside, and 2-naphthyl-q-~-fucopyranoside.The following Description of Oligella urethralis. Oligella urethralis substrates are utilized as the sole carbon source: acetate, (Lautrop, Bovre and Frederiksen 1970) comb nov. (Morax- succinate, fumarate, DL-lactate, ~~-3-hydroxybutyrate,L- ella urethralis Lautrop, BBvre and Frederiksen 1970) malate, pyruvate, 2-ketoglutarate, bengoate, and L-glutamate.

(u.re.thra'1i.s. Gr. n. ourethra, urethra; M.L. gen. n. The fdlowing compounds are not utilized as I carbon source: urethralis, of the urethra). N-acetyl-glucosamhe, amygdalin, D-arabinose, L-arabinose, Description is as for the genus (see above). Nonmotile. arbutin, cellobiose, esculin, D-fructose, D-fucose, L-fucose, Growth at 42T, urease negative (Christensen's), no growth D-galactose, gentiobiose, D-gluconate, D-glucosamine, D- on p-hydroxybenzoate. Nitrate is not reduced. Susceptible glucose, glycogen, inulin, 2-ketogluconate, 5-ketogluconate, to penicillin. Features in which one or more of the strains lactose, a-lyxose, maltose, D-mannose, rnelezitose, melibiose, differed are summarized in Table 6. methyl-a-D-glucoside, methyl-a-D-mannoside, methyl-P-D- Other characteristics common to 0. urethralis and 0. xyloside, raffmose, L-rhamnose, D-ribose, salicin, L-sorbose, ureolytica are listed below. Growth at 30 apd 37°C. Reduc- starch, sucrose, D-tagatose, trehalose, turanose, D-XylOSe, tion of nitrite. Growth in the presence of up to 3% NaCl, and L-xylose, adonitol, D-arabitol, L-arabitol; dulcitol, meso- on Drigalski agar. The following physiological features are erythritol, glycerol, meso-inositol, D-mannitol, sorbitol, meso- negative: acid production ip oxidative-fermentative pedia xylitol, caprate, caprylate, heptanoate, pelargonate, maleate, containing D-glucose, D-fructose, D-xylose, maltose, ado- malonate, oxalate, glycolate, adipate, azelate, pimelate, sebac- nitol; formation of fluorescent pigment on King B medium; ate, suberate, DL-glycerate, D-tartrate, meso-tartrate, levulin- growth on cetrimide; hydrolysis of acetamide, DNA, ate, aconitate, citraconate, D-mandelate, phthalate, iso- esculin, gelatin, starch, and Tween 80; lysine and ornithine phthalate, terephthalate, p-alanine, L-arginine, L-citrulline, L- decarboxylase; arginine dihydrolase. The following sub- cysteine, glycine, L-histidine, DL-kynurehine, L-lysine, L- strates are hydrolyzed (using API ZYM galleries): 2-naphthyl methionine, L-ornithine, L-phenylalanine, L-serine, VOL. 37, 1987 OLIGELLA GEN. NOV. 207

TABLE 6. Characteristics in which the strains of M. urethrulis and CDC group 1Ve differ M. ur-etkrrdis CDC group We No. of Result of Feature strains ‘Ype Strains that gave the less ::ainof Of Strains that gave the less positive common result positive type strain common result (n = 14) (*TCC (n = 11) (CDC C379) 17960) Motility 0 CDC B8375, CDC F1147, CDC F3464, CDC F4585, CDC F4479 Growth in the pres- 8 WM6, CCUG 1964, CCUG 3433, ence of 4.5% NaCl CCUG 5112, CCUG 7464, CCUG 12956 Reduction of nitrate to 0 CDC F4474. CCUG 17791 nitrite Denitrification 12 CCUG 1964, CCUG 3433 CDC B8375, CDC F1147 Urease (Christensen) 0 Hydrolysis of b: 2-Naphthyl phos- 2 WM20, CCUG 7464 CDC F1010, CDC F4474(w)‘, phate at pH 8.5 CDC F4479(w), CDC F4585, CCUG 17791 Naphthol-AS-BI- 1 CDC C1098(w) CDC F1010, CDC F3463, phosphodiamide CDC F4474, CDC F4585, CDC F6167 Susceptibility to peni- 14 CDC B8375, CDC F4474, cillin (10 pg) CCUG 17791 Growth at 42°C 14 Growth ond: Benzoate 13 CCUG 13464 Butyrate 14 CDC B8375, CDC F3464 n-Capraate 4 CCUG 4451, CCUG 5112, CCUG CDC F5560 7726, CCUG 13464 Citrate 9 CCUG 3433, CCUG 4451, CCUG 10532, CCUG 11920, ATCC 17960T Glutarate 14 CDC B8375 o-H y droxybenzoate 0 CDC FlOlO vn-Hydroxybenzoate 1 CCUG 1964 CDC B8375, CDC F4474, CDC F4585, CDC F5560 p-Hydroxybenzoate 0 lsobutyrate 13 WM6 CDC C379T, CDC B8375, CDC F4479, CDC F5560, CDC F6167 Isovalerate 12 CCUG 3433, CCUG 10532 CDC F1147 Itaconate 14 CDC F4479 D-Malate 13 CCUG 460 CDC B8375, CDC F3464, CDC F4585 CDC F5560 L-Mandelate 1 CCUG 460 Mesaconate 12 CCUG 460, ATCC 17960T CDC F4479 Phenylacetate 0 CDC B8375, CDC F1010, CDC F1147, CDC F4479, CDC F5560 Propionate 14 CDC F1147, CDC F6167 L-Tartrate 2 WM6, CCUG 460 CDC C379T, CDC F4585 n-Valerate 13 CCUG 1964 CDC F3464 D-a- Alanine 13 CCUG 3433 CDC F3464 L-a- Alanine 10 WM6, CCUG 460, CCUG 11920, CDC F1010, CDC F1147 CCUG 3433 DL-2-Aminobut yrate 0 CDC F4479 DL-4-Aminobut yrate 0 CDC FlOlO ~~-5-Aminovalerate 13 WM20 CDC FlOlO L-Aspartate 0 CDC F5560 L-Isoleucine 6 WM6, CCUG 4451, CCUG 5112, CDC FlOlO CCUG 7726(w), CCUG 13464(w), ATCC 17960T L-Leucine 8 +(w) WM20, CCUG 460, CCUG 1964, 2(w) CDC F1010, CDC F4585 CCUG 3433, CCUG 10532, CCUG 11920 L-Norleucine 10 +(w) CCUG 460, CCUG 1964, CCUG 5 CDC F1010(w), CDC 3433, CCUG 11920 F4479(w), CDC F4585(w), CDC F5560, CCUG 17791 Continued 208 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE &Continued M. urethrulis CDC group IVe No. of Result of Feature strains Strains that gave the less %aii: Of Strains that gave the less positive common result positive type strain common result (ATCC (n = 11) (CDC C379) (n = 14) 17960)

DL-Norvaline 3 - CCUG 4451, CCUG 5112(w), 0 - CCUG 12956(w) L-Proline 14 + 8 + CDC F3464, CDC F4479, CDC F5560 Trigonellin 0 - 1 - CDC F5560 L-Valine 3(w) +(w) CCUG 4451, CCUG 12956, ATCC 0 - 17960T ' +, Positive; -, negative. Determined by using API ZYM strips (API System, France). w, Weak positive reaction. When supplied as sole source of carbon and energy, using API auxanographic galleries.

TABLE 7. Differentiation among 0. uyethralis, 0. ureolytica, T. equigenitalis, and several species of the genera Alcaligenes, Bordetella, Maraxella, and CDC group IVc-2"

'- B. ~.fae-A. xylo- A. eu- LuL . uvium culis soxidansb trophus groupIVC-2

Motility/peritrichous - c' d - + + + + + flagella Growth at 42"Cd + - + d - + d + + d d d Growth in presence + + - + + + d + + + d - of 3% NaCld Urease (Christensen) + Nitrate reductiond d Nitrite reduc tied + Denitrificatiod +f - - - Hydrolysis of Tween d + d 80d Acid phosphataseg + - + + + + + + + Carbon sources for growth? Acetate + d + Adipate d + Benzoate - d D-Fructose - D-Gluconate + D-XylOSe - p-H ydrox yben- + zoate L-Malate + + + + + + + + + Pimelate ------+ + + Differentiating cellu- lar fatty acids': 12:o + tr' tr tr tr tr 2-OH-12:O - + tr + + + 3-OH-1210 + tr tr tr tr tr - - 3-OH-16:O - tr tr - - - tr tr l7:cyc - + + + + + + + 19:cyc - + - - - - + tr + + tr tr + G+C content of 46-47 46-87 38 43-46 42.5-43.5 40-45.5 68-69.5 68-69 62-63 56-59 64-70 66-67.5 66-67 DNA (mol%)

a Data from references 2, 24, 25, and 33; from E. Falsen and K. Kersters (unpublished data); and from the present paper. Including Alculigenes xylosoxidans subsp. xylosoxiduns and subsp. denitrijcuns (26). t,more than 90% of the strains positive; -, less than 10% of the strains positive; d, 11 to 89% of the strains positive. Data from E. Falsen (unpublished data), and the present paper: some of these results may differ from published data. ' Exceptionally fast positive reaction. Demonstration of gas may be difficult. Tested with API ZYM galleries. Tested with auxanographic API galleries. ' Data from Jantzen et al. (211, E. Jantzen and E. Falsen (unpublished data), and the present paper; tr, less than 1%. VOL. 37, 1987 OLIGELLA GEN. NOV. 209

except for the following features: some strains are motile by means of long peritrichous flagella (Fig. 5); the urease test (Christensen's medium) is usually positive in 4 h; does not grow at 42"C, grows on p-hydroxybenzoate as a carbon source; most strains reduce nitrate and are resistant to penicillin. Lactobacillic acid (C19:cyc)is present in moderate amounts. Features in which one or more strains differed are listed in Table 6. The following API 20NE profiles (API System, France) were obtained: 0200045, 1200044, 1200045, and 1200047 (after 2 days of incubation). Mainly isolated from human urine. Pathogenicity unknown. The G+C content of DNA is 46-47 mol% (T, method). Type strain is ATCC 43534 (= CDC C379, CCUG 1465, LMG 6519). Isolated from human urine in California. Char- acteristics of the type strain are given in Table 6. It was the only strain with predominantly rod-shaped cells (Fig. 5). Taxonomic positions of Taylorella equigenitalis and CDC group IVc-2. Haernophilus equigenitalis was thoroughly studied by Taylor et al. (37) and Sugimoto et al. (35, 36). It was concluded that H. equigenitalis could not belong to Huernophilus and a new genus Taylorella was proposed (35). However, the (supra)generic relationships of this genus have not been elucidated up to now. Our genetic results support the conclusions of Sugimoto et al. (35) concerning the separate generic rank of these organisms, because a high rRNA cistron similarity could not be found with any of our reference rRNAs. The highest relationship was observed with Oligella spp. and the Alcaligenaceae. Both taxa are equidistant from Taylorella. The 7'm(e,values (Tables 4 and 5) and serological data (Table 3) exclude the incorporation of Taylorella as a true member of any of the above mentioned groups. With the inclusion of TaylorelZa in rRNA superfam- ily 111, the G+C span of this superfamily now ranges from 38 to 70 mol%. CDC group IVe (0. ureolytica) is usually mentioned in connection with the CDC group IVc-2 (33). It has been shown previously (8,15) that CDC group IVc-2 strains differ phenotypically, serologically, and genetically from 0. FIG. 5. Electron micrograph of a cell of the type strain CDC ureolytica and are closely related to Alcaligenes eutrophus C379 of Oligella ureolytica (CDC group IVe) showing long, slender, (see also Fig. 2; Tables 3 and 5). peritrichous flagella. Bar, 1 pm. ACKNOWLEDGMENTS We thank R. E. Weaver, CDC, Atlanta, Ga., for supplying L-threonine, D-tryptophan, L-tryptophan, L-tyrosine, ace- strains; and API System, La Balme-les-Grottes, France, for the API tamide, 2-aminobenzoate, 3-aminobenzoate, 4-aminobenzoate, 50CH, API 50A0, and API 50AA strips. E.F. is grateful to M. ~~-3-aminobutyrate,amylamine, benzylamine, betaine, Blomqvist for phenotyping and to A. Borjesson for immunotyping. butylamine, creatine, diaminobutane, ethanolamine, Electron microscopy was kindly carried out by P. Conway and L. ethylamine, histamine, sarcosine, spermine, tryptamine, and Toyra. E.F. is grateful to many scientists for strains, particularly to urea. L. Martensson for the strains of T. equigenitalis. The following API 20NE profiles (API System, France) J.D.L. is indebted to the Nationaal Fonds voor Geneeskundig Onderzoek, Belgium, for research and personnel grants; K.K. is were obtained for 0. urethralis: 0000044 and 0000045 (after 2 indebted to the Nationaal Fonds voor Wetenschappelijk Onderzoek days of incubation). for a research grant; and R.R. is indebted to the Instituut tot 0.urethralis has been isolated from urine, the urinary Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid tract, and also the ear. Pathogenicity is probably low. en Landbouw for a scholarship. The G+C content of DNA is 46 to 47.5 mol% (T, method). Type strain is ATCC 17960 (= CDC 7603, CCUG 13463, LITERATURE CITED LMG 5303) (listed as Acinetobacter lwofii in the ATCC 1. American Type Culture Collection. 1985. Catalogue of bacteria, catalogue [l]).This strain was isolated from an ear. Charac- phages and rDNA vectors, 16th ed. American Type Culture Collection, Rockville, Md. teristics of the type strain are given in Table 6. Bgvre, K. Neisseriaceae Description of Oligella ureolytica sp. nov. Oligella 2. 1984. Family VIII. Prdvot 1933,119, p. 288-301. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual ureolytica (ur'e.o.ly.ti.ca. M.L. n. urea, urea; Gr. adj. of systematic bacteriology, vol. 1. The Williams & Wilkins Co., lyticus, dissolving; M.L. adj. ureolytica, dissolving [hydro- Baltimore. lyzing] urea). 3. Bflvre, K., and N. Hagen. 1981. The family Neisseriaceae: This species is proposed to accommodate the CDC group rod-shaped species of the genera Moraxella, Acinetobacter, IVe strains. Description is the same as that of M. urethralis Kingella, and Neisseria, and the Branhamella group of cocci, p. 210 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

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