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Cedecea Davisae Gen INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1981, p. 317-326 Vol. 31, No. 3 0020-7713/81/030317-10$02.00/0 Cedecea davisae gen. nov., sp. nov. and Cedecea lapagei sp. nov., New Entero bacteriaceae from Clinical Specimens PATRICK A. D. GRIMONT,’ FRANCINE GRIMONT,’ J. J. FARMER 111,2 AND MARY A. ASBURY’ Service des Enterobacteries, INSERM Unit 199, Institut Pasteur, F- 75724 Paris Cedex 15, France,’ and Enteric Section, Centers for Disease Control, Atlanta, Georgia 303332 We propose the name Cedecea gen. nov. for a group of organisms in the Enterobacteriaceae that were isolated from clinical sources in North America (the clinical significance of these organisms is unknown). This name was coined by two of us (P.A.D.G. and F.G.) from the letters CDC, the abbreviation for the Centers for Disease Control, where the organisms were originally discovered. Phenotypically, Cedecea resembles no other group of Entero bacteriaceae; the members of this genus are lipase positive, resistant to colistin and cephalothin, and negative for deoxyribonuclease, gelatin liquefaction, and utilization of L- arabinose and L-rhamnose. Deoxyribonucleic acid relatedness studies showed that Cedecea strains were 32 to 100% related to each other and less than 23% related to other members of the Enterobacteriaceae. We found five deoxyribo- nucleic acid hybridization groups among 17 Cedecea strains, but three of these groups contained only 1 strain (strains 001,002, and 012). Two deoxyribonucleic acid hybridization groups were named. Cedecea davisae sp. nov. (nine strains), the type species of the genus, fermented sucrose and D-XylOSe and was positive in the ornithine decarboxylase and ascorbate tests. C. davisae grew in a mineral salts medium with glucose as the carbon source only if the medium was supple- mented with 0.1 pg of thiamine per ml. The type strain of C. davisae is strain 005 (= ATCC 33431 = CDC 3278-77 = CIP 80.34) Cedecea Zapagei sp. nov. (five strains) did not ferment sucrose and D-xylose and was negative in the ornithine decarboxylase and ascorbate tests. This species grew on glucose as the role source of carbon and energy with no growth factor requirement. The type strain of C. Zapagei is strain 004 (= ATCC 33432 = CDC 0485-76 = CIP 80.35). Within species, deoxyribonucleic acid relatedness was 80 to 100% at 60°C (Sl nuclease method), and C. davisae and C. Zapagei were much more closely related to each other (32 to 52%) than to members of any other group within the family Enterobacteriaceae (1 to 21%). Despite the progress made in bacterial tax- basis of their deoxyribonucleic acid (DNA)- onomy in the last 20 years, reference laboratories DNA hybridization and phenotypic character- frequently encounter unidentified strains. These istics, Cedecea is an arbitrarily constructed strains are often kept in a collection which can name derived from the abbreviation CDC (Cen- be analyzed to detect possible groups of similar ters for Disease Control). unidentified bacteria. In 1977, workers at the Centers for Disease Control gave the vernacular MATERIALS AND METHODS name 44Enteric 15” to a SOUP Of l7 uni- Bacterial swains. The Cedecea strains used in dentified Enterobacteriaceae strains which were this study are listed in Table 1. The ofthe non- hPase (Corn Oil) Positive. In 1980, we Proposed a Cedecea reference strains used in the DNA relatedness new genus, Cedecea, containing two species (ce- study have been reported previously (3,4,11). decea davisae and Cedecea Zapagei) and three Methods. A suspension of strain 005 in distilled unspecified strains (Cedecea sp.) (J. J. Farmer, water (about 0.02 ml) was deposited on a carbon- 111, p. A. D. Gkont, F. Grimont, and M. A. coated specimen grid, negatively stained with 2% (Wt/ Asbury, Abst. Annu. Meet. Am. SOC.Microbial. VOl) phosphotungstic acid, and exambed with a Sic- 1980, C123, p. 295). However, that proposal did m~~o>~&~o~~~~~~~~)e not constitute effective and valid publication of by the method of Clowes and Hayes (5). Growth on the scientific names. various compounds as carbon and energy sources was In this paper we propose the names Cedecea determined by multiple inoculation (9) onto M70 min- gen. nov., Cedecea davisae sp. nov., and Cede- imal medium (14) supplemented with 0.01 pg of thia- cea Zapagei sp. nov. for these organisms on the mine per ml, 100 pg of cysteine per ml, 0.5 pg ofpara- 317 318 GRIMONT ET AL. INT. J. SYST.BACTERIOL. TABLE1. Origins of the Cedecea strains studied Strain designations Origin“ Species This paper Centers for Disease Source Location Control C. davisae 005 CDC 3278-77 Stool New Jersey 006 CDC 0512-78 Eye New York 008 CDC 2205-78 Sputum United States 010 CDC 1395-75 Gall bladder Alabama 015 CDC 2295-78 Sputum United States 016 CDC 2296-78 Wound United States 018 CDC 2932-78 Urine United States 019 CDC 1868-79 Sputum New York 020 CDC 1790-78 Wound California C. lapagei 004 CDC 0485-76 Throat Canada 003 CDC 0818-75 Sputum Kentucky 007 CDC 0817-78 Sputum New York 009 CDC 1554-75 sputum Georgia 017 CDC 2488-78 Sputum Virginia Cedecea sp. 001 CDC 4853-73 Sputum New Jersey 002 CDC 0621-75 Foot wound California 012 CDC 3699-73 Toe Canada a All strains were from human clinical specimens. aminobenzoic acid per ml, and 0.1 to 0.2% carbon radioactive DNAs (l),to extract and purify unlabeled source. We used previously described procedures (9) DNAs, and to shear both labeled and unlabeled DNAs for the following tests: Voges-Proskauer test (Richard (2). Sheared DNAs were dialyzed overnight against modification), hydrolysis of gelatin (plate method), 0.042 M NaCl and then stored at 4OC over a layer of anaerobic growth in the presence of potassium chlor- chloroform. ate, reduction of tetrathionate in peptone water, hy- In the hybridization experiments, we used the S1 drolysis of tributyrin, Tween 40, Tween 60, Tween 80, nuclease method (6), slightly modified (10). S1 nu- DNA, chitin, and starch, o-nitrophenyl-P-D-gdacto- clease was prepared by the method of Sutton (13). pyranoside test, P-xylosidase test with para-nitro- The DNA duplexes remaining after S1 nuclease treat- phenyl-P-xyloside (Sigma Chemical Co.), pigment and ment for 40 min at 60°C were precipitated by adding odor production, growth with 0, 2, 4, 6, and 8%NaC1, 0.25 ml of ice-cold 25% (wt/vol) trichloroacetic acid and growth at 5,15,20,37,40,and 42°C. The ascorbate and then collected on membrane filters (Sartorius, test was performed with ascorbate medium (Enteric Gottingen, Germany). The filters were washed with fermentation base [Difco] 18 g; bromothymol blue, three 5-ml volumes of ice-cold 5%trichloroacetic acid, 0.04 g; sodium L-ascorbate, 10 g; distilled water to 1,000 dried, and put into vials containing 10 ml of scintillant ml; pH 7.5) This medium was dispensed in 10-ml (4 g of Koch-Light “Butyl PBD” per liter of toluene), quantities into tubes (16 by 100 mm) and autoclaved and the radioactivity was measured with an Intertech- for 10 min at 121°C; 4 ml of mineral oil was then nique model SL 4000 liquid scintillation spectrometer. added, and the medium was stored at 4°C. Tubes were The degree of polynucleotide sequence homology was discarded if the pH fell below 7.0. All other biochem- calculated by determining the ratio between the av- ical tests were performed as described by Edwards and erage counts in the nuclease-treated samples and the Ewing (7). In addition, the Voges-Proskauer test was average counts in the untreated samples. Results were performed at the Centers for Disease Control by add- normalized to the untreated samples and to the ho- ing 0.5 ml of API reagent 1 (40% [vol/vol] KOH mologous reaction, after the percentage of S1-resistant solution) and 0.5 ml of API reagent 2 (6% alpha- material in the control tube (containing only dena- naphthol in absolute ethanol) to 1 ml of a 48-h culture tured radioactive DNA) was subtracted. The temper- in MR-VP broth (Difco). This method (listed in Table ature at which 50% of the reassociated DNA became 5 as Voges-Proskauer-special) was a more sensitive hydrolyzable by S1 nuclease was determined by the method for detecting the acetoin pathway in Cedecea. Susceptibilities to antimicrobial agents were deter- method of Crosa et al. (6). The percent divergence mined by disk diffusion on Mueller-Hinton agar (In- (AT,) (3) was calculated from the difference in such stitut Pasteur Production), as recommended by an temperatures between the heterologous DNA reaction international study group (8). Susceptibility to the and the homologous DNA reaction. vibriostatic compound 0/129 was determined on Muel- The melting temperatures of 50-pg/ml DNA solu- ler-Hinton agar with disks impregnated with 0.5 mg of tions in 0.1~SSC buffer (1xSSC is 0.15 M NaCl plus 2,4-diamino-6,7-diisopropylpteridinephosphate (syn- 0.015 M trisodium citrate) were measured by M. Po- onym, compound 0/129; Institut Pasteur Production) poff, using a Gilford spectrophotometer. The guanine- per disk. plus-cytosine contents of DNAs were determined from We used previously described procedures to label melting temperatures by the equation of Owen et al. DNA with [3H]thymidine (10) to extract and purify (12). VOL. 31, 1981 CEDECEA GEN. NOV. 319 RESULTS None of the 17 Cedecea strains produced pig- ments, oxidase, indole from peptone water, H2S Phenotypic characterization. Figure 1 from triple sugar iron agar, urease, phenylala- shows an electron micrograph of a representa- nine deaminase, lysine decarboxylase, gelatinase tive strain of davisae (strain 005).
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