INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY,act. 1993, p. 645-658 Vol. 43, No. 4 Qo20-7713/93/040645-14$02.00/0 Copyright 0 1993, International Union of Microbiological Societies

Classification of Citrobacteria by DNA Hybridization: Designation of Citrobacter famzeri sp. nov., sp. nov., sp. nov., sp. nov., sp. nov., and Three Unnamed Citrobacter Genomospecies DON J. BRE”ER,’* PATRICK A. D. GRIMONT,, ARNOLD G. STEIGERWALT,l G. R. ELISABETH AGERON,, AND CONRADINE F. RIDDLE4 and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, and Nosocomial Infections La boratoly Branch, Hospital Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; Unitk des Entkrobactkries, Institut National de la Recherche Scientijique Unitk 199, Institut Pasteur, 75724 Paris, Ceda 15, France2; and Division of Biochemistly, Walter Reed Amy Institute of Research, Washington, D. C. 203O7-51OO3

DNA relatedness studies (hydroxyapatite method) were done on 112 strains of citrobacteria. By using the recommended definition of a genomospecies 11 genomospecies were identified in the Citrubucter. These genomospecies were separable by their biochemical profiles. Citrubucter Koseri (C’ubucter diversm) and Citrubucterumalonaticus proved to be homogeneous , as previously described. C. amlonuticus biogroup 1, as described by Farmer et al. (J. Clin. Microbiol. 21:46-76,1985), was shown to be a separate homogeneous species, which was named Citrobucterfumeri sp. nov. The Citrubucterfieundii complex was quite heteroge- neous. C.freundii sensu stricto, as represented by the type strain, contained only 9 of 66 strains in this complex. The remaining 57 strains were members of seven genomospecies. Genomospecies 5, containing 21 strains, was named Citrubacter yuungue sp. nov. Genomospecies 6, containing 15 strains, was named Citrubucter bruukii sp. nov. Genomospecies 7 and 8, each containing six strains, were named Citrubucter werkmunii sp. nov. and Citrobactersedlakii sp. nov., respectively. Genomospecies 9,10, and 11, each containing three strains, were not named.

The genus Citrobacter and the species Citrobacterfreun- genomospecies detected on the basis of DNA relatedness dii were designated in 1932 by Werkman and Gillen (13). levels were reexamined. (Genomospecies is a word that we (8, 9) (Citrobacter diversus [4], Levinea have coined to use instead of genospecies. A genomospecies malonatica [14]) and (5) (Levinea is a species defined by DNA relatedness [12]. Genomic amalonatica [ 141) are the other previously recognized Citro- species is another synonym for genomospecies and geno- bacter species. Strains of the latter two species were initially species. The term genomovar was used by Rossello et al. “to identified as indole-positive, H,S-negative biogroups of C. denote genomic groups of a nomenspecies” [lOa]; “var” is Ji-eundii.C. koseri also differs from C. freundii by its failure traditionally used for differentiation below the species level, to grow in medium containing KCN and from both C. and we therefore prefer the term genomospecies or genomic freundii and C. amalonaticus by its ability to utilize mal- species.) The traditional diagnostic laboratory tests were onate and to produce acid from D-adonitol. augmented by a battery of carbon source utilization tests to It has long been evident that a significant number of allow biochemical identification of each species and differ- Citrobacter isolates from clinical specimens do not entiation from all other species in the genus. exhibit the biochemical characteristics typical of these three Eleven genomospecies were identified. Genomospecies 1 previously described species (7). DNA relatedness studies through 3 corresponded to C. freundii, C. koseri, and C. have also revealed substantial heterogeneity within strains amalonaticus, respectively. Genomospecies 4, which corre- identified as C. freundii (3). Farmer et al. created the name sponded to C. amalonaticus biogroup 1, was named Citro- C. amalonaticus biogroup 1 for strains, usually isolated from bacter fameri sp. nov. Genomospecies 5 through 8 were human feces, that differed from C. amalonaticus by their named Citrobacteryoungae sp. nov., Citrobacter braakii sp. ability to usually ferment melibiose, a-methyl-D-glucoside, nov., Citrobacter werkmanii sp. nov., and Citrobacter sed- raffinose, and sucrose and their inability to utilize citrate (7). lakii sp. nov., respectively. Genomospecies 9 through 11, The purpose of this study was to determine, by calculating each containing three strains, were not named. levels of DNA relatedness, whether biochemically atypical Citrobacter strains in the culture collections of the Centers MATERIALS AND METHODS for Disease Control and Prevention (CDC) and the Institut Pasteur represent biotypes of previously described species Strains. The strains used in this study are listed in Table 1. or additional, undescribed species. Biochemical characteris- All strains were originally sent for identification to diagnostic tics of the previously described species and each new reference laboratories at the CDC or the Institut Pasteur. For consistency, CDC strain numbers are used in the Results and Discussion. The following type strains were used: C. * Corresponding author. amalonaticus (L. amalonatica) CDC 9020-77 (= ATCC

645 aP

W TABLE 1 (Part I).Levels of DNA relatedness among citrobacteria w Labeled DNA from: !2 3 Citrobacter C. diversus C. amalonaticus C. freundii C. freundii Citrobacter Citrobacter % Source of genomospecies 5 strain genomospecies 6 strain CDC 9020-77T genomospecies 4 strain CDC 621-MT CDC 1242-78 &gztFi6 unlabeled DNA CDC 3613-63T CDC 2991-81T CDC 460-61T CDC 4686-86 2 (CDC no.) % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- edness D" edness edness D edness edness D edness edness D edness edness D edness edness D edness D edness edness D edness at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 60°C at 75°C at 60°C at 75°C C. diversus 3613-63= 100 0.0 100 40 12.0 48 12.5 19 41 12.0 40 12.0 23 48 12.5 45 13.5 21 46 11.5 19 705-79 94 0.5 52 4439-72 90 1.0 85 39 43 10.5 51 4110-85 89 1.0 43 C. koseri 88 0.5 41 39 12.0 48 8132-86T C. diversus 1381-70 87 0.5 43 1249-78 87 1.0 86 42 96-80 87 1.0 42 L. malonatica 84 1.0 40 8127-86T C. diversus 138-79 83 0.0 40 2921-81 83 0.5 39 1066-70 82 0.5 37 2292-70 82 0.5 38 2824-81 82 1.0 80 38 9420-77 82 1.0 37 0.5 78 4698-86 81 41 39 10.5 48 11.5 C. amalonaticus 9020-77= 44 12.0 100 0.0 100 72 8.0 52 40 13.5 37 13.5 21 48 12.5 44 14.0 18 45 12.5 17 3314-73 92 1.5 73 8.0 43 3288-76 88 0.5 42 805-73 83 0.5 35 1444-80 82 0.0 85 39 5680-72 45 82 0.5 81 40 10.5 48 1636-61 39 12.5 82 0.5 81 38 12.5 2050-74 82 0.5 38 1620-73 82 1.0 38 1637-61 42 12.0 82 1.5 80 32 11.0 3311-78 39 11.0 80 1.0 41 s 3318-75 40 12.5 79 0.0 79 38 13.0 3320-75 79 0.0 78 36 804-73 78 1.0 36 802-73 77 1.5 72 37 5679-72 39 75 1.0 72 36 10.0 41 Citrobacter genomospe- cies 4 2991-81T 41 11.5 59 6.0 48 100 0.0 100 34 13.5 37 13.0 19 42 13.0 40 12.5 39 12.0 14 1604-79 69 100 1.0 99 46 1263-79 65 6.0 53 100 1.0 94 40 725-77 68 6.5 53 98 0.5 100 42 2369-78 62 98 0.5 95 38 415-80 68 98 3.5 88 43 1402-82 62 92 0.5 94 38 3295-78 64 54 92 3.5 82 40 624-79 57 89 0.5 85 34 3249-74 40 63 6.5 50 87 2.5 86 40 12.0 44 2604-78 61 86 3.0 78 38 2727-78 59 84 3.0 75 36 530-79 61 84 3.5 76 36 433-80 57 76 2.0 76 35 C. freundii 62 1-64= 44 12.0 38 13.0 43 13.5 100 0.0 100 72 4.0 65 71 7.5 54 61 9.5 58 4689-86 38 12.5 90 1.0 87 67 3.0 64 4691-86 88 0.5 86 69 3.5 63 100 0.0 60 5.0 61 4 69 0 -8 6 39 12.0 85 0.5 84 69 3.0 64 4688-86 35 11.5 81 0.5 82 67 3.0 65 68 7.5 48 63 5.5 63 1243-78 75 4.0 67 92 1.0 90 71 9.0 47 2929-78 71 4.0 62 73 3.0 70 78 3.5 63 61 5.5 56 1242-78 68 3.0 60 100 0.0 100 74 4.0 63 67 5.5 55 892-61 37 34 68 3.5 63 82 1.5 78 63 Citrobacter genomospe- cies 5 460-6 1 43 12.0 36 13.0 37 13.0 61 6.5 57 6.5 46 66 6.5 100 0.0 100 59 7.0 42 5 118-60 67 5.5 52 62 8.0 43 98 0.5 89 4692-86 41 12.5 69 5.5 54 97 0.5 92 3062-62 67 7.0 53 97 1.0 91 2974-50 68 7.5 49 63 7.5 43 95 0.5 2611-80 65 6.0 52 62 7.5 48 95 2.5 87 8107-85 69 5.5 48 95 2.5 85 827-81 63 6.5 49 62 8.0 49 93 2.0 87 1917-82 65 5.5 50 59 7.5 45 93 3.0 86 4675-60 63 7.0 50 89 1.0 83 Q 8077-84 40 36 61 5.5 55 58 7.0 43 88 1.5 86 9 8109-85 65 6.0 45 87 2.0 t2 6440-59 63 7.0 52 85 3.5 84 k 3313-75 35 61 5.5 44 55 7.0 38 84 1.5 75 8111-85 63 7.0 55 7.0 40 84 4.0 70 8 8112-85 56 6.5 51 6.5 39 83 1.0 cn cd 4694-86 34 61 7.5 48 83 3.5 68 m 2548-79 58 6.0 46 83 4.5 66 a m 5245-60 54 6.0 82 1.0 81 cn 8108-85 50 5.0 80 2.0 74 8106-85 56 6.0 53 6.5 39 76 4.5 67 m Continued on following page 3 W TABLE 1-Continued w m Labeled DNA from: 3 Citrobacter Citrobacter Citrobacter C. diversus C. amalonaticus C. freundii C. fieundii Source of CDC 3613-63= CDC 9020-77T genomospecies 4 strain CDCfieundii 621-UT cDc 1242-78 CDC 4691-86 genomospecies 5 strain genomospecies 6 strain m unlabeled DNA CDC 2991-81T '- CDC 460-61T CDC 4686-86 cl (CDC no.) % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- edness D" edness edness D edness edness D edness edness D edness edness D edness edness D edness D edness edness D edness at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 60°C at 75°C at 60°C at 75°C Citrobacter genomo- species 6 4686-86 40 13.5 64 63 5.0 52 65 8.5 44 100 0.0 100 3 158-63 39 65 6.0 54 64 5.5 56 66 89 1.0 84 2990-58 41 67 5.5 5054 65 5.5 55 65 89 1.0 83 3358-77 71 5.56.5 60 69 4.5 60 72 7.0 47 86 0.5 88 4687-86 40 14.0 42 14.0 69 5.0 59 66 4.5 55 71 8.0 46 84 1.5 75 8105-85 40 13.5 57 70 7.5 44 79 2.5 75 8110-85 68 5.0 54 65 5.0 58 67 9.0 45 78 2.5 71 8O-5gT 35 37 13.0 59 5.5 67 6.0 59 9.0 77 2.5 70 9115-81 37 66 6.0 55 63 5.5 57 64 77 3.0 71 4685-86 40 13.0 41 13.5 68 5.0 59 64 4.5 55 69 8.0 45 75 1.0 68 8113-85 65 5.0 54 62 5.0 53 67 8.5 47 75 2.5 68 61 4.5 52 8114-85 65 5.0 56 64 7.0 48 74 2.5 72 989-73 35 4.5 51 60 5.5 50 63 6.5 71 2.5 66 64 64 2028-79 35 62 5.0 49 70 2.0 67 34 59 5.0 52 60 69 2.5 64 4508-86 60 5.0 52 Citrobacter genomo- species 7 876-58T 67 7.5 57 61 7.0 45 34 59 7.0 3323-75 36 13.5 44 12.2 7.0 45 64 6.5 49 64 40 59 7.5 46 2444-78 66 62 59 7.0 41 37 60 7.5 631-77 37 6.5 60 56 7.0 41 2083-81 59 64 7.5 52 65 40 65 6.5 51 2238-81 37 6.5 51 56 7.0 49 60 Citrobacter 58 genomo- species 8 4696-86T 43 10.5 45 46 11.0 12.0 42 13.0 41 41 12.0 3188-73 43 47 49 35 10.5 46 190-81 40 12.0 20 43 43 11.5 40 40 14.0 T 3659-74 37 37 44 37 11.5 40 4697-86 36 10.0 36 41 32 11.5 35 33 12.0 m 305-74 35 40 43 31 12.5 39 3 33 w b ;;1 T! r0 VOL. 43, 1993 NEW CITROBACTER SPECIES 649

25407), C. diversus (a synonym of C. koseri) CDC 3613-63 (= ATCC 27156), L. malonatica (a synonym of C. koseri) CDC 8127-86 (= ATCC 25408), C. koseri CDC 8132-86 (= ATCC 27028), and C. freundii CDC 621-64 (= ATCC 8090). The following strains were obtained from the Institut Pasteur: 13-61 (= CDC 4692-86), 9-73 (= CDC 4697-86), 1-75T (= dm CDC 4696-86T) (T = type strain), 7-76 (= CDC 4698-86), 1-83 (= CDC 4687-86), 72-84 (= CDC 4689-86), 80-84 (= CDC 4691-86), 84-84 (= CDC 4688-86), 92-84 (= CDC 4694-86), 102-84 (= CDC 4690-86), 1-85 (= CDC 4685-86), 3-85 (= CDC 4693-86), 5-84 (= CDC 4695-86), and 37-85 (= CDC 4686-86). The following strains were previously studied by Crosa et al. (3) (the designations used in the study of Crosa et al. are given in parentheses): CDC 2974-50 (= Bethesda 7), CDC 4675-65 (= 4675-60 sic), CDC 8105-85 (= Pllc), CDC 8106-85 (= Bethesda lA), CDC 8107-85 (= Bethesda 2A), CDC 8108-85 (= Bethesda 3A), CDC 8109-85 (= Bethesda 4A), CDC 8110-85 (= Bethesda 6), CDC 8111-85 (= Bethesda 8A), CDC 8112-85 (= Bethesda 9A), CDC 8113-85 (= Bethesda 29A), CDC 8114-85 (= 3796), and CDC 8115-85 (= H310a). Biochemical tests. The methods used for biochemical test- ing at the CDC (Table 2) have been described previously (6, 10). All test preparations were incubated at 36 2 1°C for 7 days unless otherwise specified. Carbon source utilization tests were done at the Institut Pasteur by using Biotype strips (BioMkrieux, La Balme les Grottes, France) that contained 99 pure carbon sources. The strips were inocu- lated by using Biotype medium 1 according to the manufac- turer's instructions. The carbon sources used are shown in Table 3. Preparations were incubated at 30°C for 4 days. DNA hybridization. To obtain cells for DNA extraction, Citrobacter strains were grown in brain heart infusion broth at 36 2 1°C with shaking until they reached the late logarith- mic phase. The methods used for extraction and purification of DNA and the hydroxyapatite hybridization method for determining levels of DNA relatedness have been described previously (2). DNAs were labeled enzymatically in vitro with [32P]dCTP by using a nick translation reagent kit (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) as directed by the manufacturer.

RESULTS DNA relatedness studies were done on 112 strains identi- fied as members of the genus Citrobacter. These strains were categorized into 11 genomospecies (Table 1). Genomospe- cies 2 contained 16 strains, all of which had been previously identified biochemically as C. kosen'. Included in genom- ospecies 2 were the type strains of C. diversus, C. koseri, and L. malonatica, all of which are subjective synonyms. Genomospecies 3 contained 16 strains that had been previ- ously identified as C. amalonaticus; it included the type strain of C. amalonaticus, which is also the type strain of L. amalonatica. Genomospecies 4 contained all 14 strains biochemically identified as C. amalonaticus biogroup 1. Genomospecies 1 contained the type strain of C. freundii, but included only 9 of 66 strains previously identified bio- chemically as C. freundii or as closest to C. freundii. The remaining 57 C. freundii-like strains were members of seven genomospecies; 21 strains were in genomospecies 5, 15 strains were in genomospecies 6, 6 strains each were in genomospecies 7 and 8, and 3 strains each were in genom- ospecies 9, 10, and 11. Conventional biochemical reactions for each genomospe- cies are given in Table 2. It is possible to identify each TABLE 1 (Part 2). Levels of DNA relatedness among citrobacteria W Labeled DNA from: E 21 Z Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter M Source of unla- genomospecies 6 strain genomospecies 7 strain genomospecies 8 strain genomospecies 8 strain genomospecies 9 strain genomospecies lO?train genomospecies 11 strain beled DNA CDC 80-58= CDC 876-58T CDC 4696-86T CDC 4697-86 CDC 1843-73= CDC 4693-86 CDC 2970-59T m (CDC no.) % Relat- c1 % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- edness D edness edness D edness edness D edness edness D edness edness D edness edness D edness edness D edness at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at75"C at60"C at 75°C at 60°C at 75°C at 60°C at 75°C C. diversus 3613-63= 38 40 45 47 10.5 36 12.0 42 11.0 40 11.0 705-79 4439-72 4110-85 C. koseri 8132-86T 46 12.5 21 C. diversus 1381-70 1249-78 96-80 L. mabnatica 8127-86T C. diversus 138-79 2921-81 1066-70 2292-70 2824-81 9420-77 4698-86 C. amalonaticus 9020-77T 36 53 54 13.0 56 10.0 38 11.5 22 41 12.0 39 12.0 3314-73 3288-76 805-73 1444-80 5680-72 1636-61 2050-74 1620-73 1637-61 3311-78 3318-75 3320-75 804-73 802-73 5679-72 W VOL. 43, 1993 NEW CITROBACTER SPECIES 651

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rrrd W w TABLE 1LContinued i? Labeled DNA from: 3 w Citrobacter Citrobacter Citro bacter Citrobacter Citrobacter Citrobacter Citrobacter Source of unla- genomospecies 6 strain genomospecies 7 strain genomospecies 8 strain genomospecies 8 strain genomospecies 9 strain genomospecies 10 ?train genomospecies 11 strain 2 beled DNA CDC 80-5gT CDC 876-5gT CDC 4696-86T CDC 4697-86 CDC 1843-73T CDC 4693-86 CDC 2970-59= (CDC no.) % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- % Relat- edness D edness edness D edness edness D edness edness D edness edness D edness edness D edness edness D edness at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C at 60°C at 75°C Citrobacter genomo- species 6 4686-86 70 2.5 65 3158-63 68 3.5 62 2990-58 70 4.0 63 3358-77 4687-86 75 1.0 74 8105-85 75 2.0 69 8110-85 73 1.5 70 80-5gT 100 0.0 100 69 7.5 58 37 38 11.5 28 12.0 43 9.5 49 9.0 9115-81 73 0.5 72 4685-86 75 1.5 73 8113-85 71 1.5 68 8114-85 71 1.5 72 989-73 71 0.5 72 2028-79 69 2.5 71 4508-86 65 0.5 67 Citrobacter genomo- species 7 876-5gT 51 8.0 38 100 0.0 100 36 41 12.0 51 9.0 3323-75 57 100 0.0 100 40 2444-78 56 96 1.0 90 37 631-77 55 91 1.0 89 35 2083-81 55 88 2.5 81 40 2238-81 54 82 1.5 79 34 Citrobacter genomo- species 8 4696-86T 34 49 100 0.0 100 86 41 12.0 3188-73 36 93 1.0 94 190-81 34 89 1.0 79 3659-74 31 84 0.5 85 4697-86 29 11.0 10 84 1.0 79 100 0.0 100 305-74 31 77 0.5 76 VOL. 43, 1993 NEW CITROBACTER SPECIES 653

genomospecies on the basis of these commonly employed tests. Results of carbon source utilization tests, done on the same strains, are given in Table 3. All of the genomospecies can be differentiated on the basis of carbon source utilization reactions. Table 4 shows the conventional biochemical and carbon mmmbbd source utilization test results that are most useful for iden- tifying the Citrobacter genomospecies. Since genomospecies 2 (C. koseri) is the only Citrobacter species that can produce acid from adonitol and D-arabitol, it was omitted from Table 4 for simplicity. When only conventional biochemical tests are used, two or more tests are used for differentiating between all pairs of genomospecies except the following pairs: genomospecies 1 and 10 (only the malonate test is used), genomospecies 6 and 8 (only the malonate test is used), and genomospecies 6 and 11 (only the ornithine decarboxylase test is used). All genomospecies can be differentiated by at least two carbon source utilization tests.

DISCUSSION A genomospecies has been defined as a group of strains whose DNA are at least 70% interrelated, with 5% or less divergence in the related sequences (12). When this defini- tion was used, DNA relatedness studies identified 11 genom- ospecies in the genus Citrobacter. The results obtained confirmed the hypothesis of Farmer et al. that C. amalonat- icus biogroup 1 is a fourth species in the genus Citrobacter B (7). The biochemical and genetic heterogeneity previously observed in strains identified as C. freundii (3, 7) was confirmed and extended. In fact, other than the strains W0 bm identified as C. amalonaticus biogroup 1, strains previously identified as C. freundii or Citrobacter species were shown to belong to seven previously undescribed DNA hybridiza- tion groups or genomospecies. Each of the 11 genomospecies of the genus Citrobacter can be identified by conventional biochemical tests (Tables 2 and 4) and by carbon source utilization tests (Tables 3 and 4). A combination of conventional and carbon source utilization mmo mmm tests provides a very firm basis for identification of all citrobacteria. A total of 112 strains were included in this study. Sixteen of these were C. koseri (genomospecies 2) strains, 16 were C. amalonaticus (genomospecies 3) strains, and 14 were C. amalonaticus biogroup 1 (genomospecies 4) strains. Of the remaining 66 strains, only 9 were members of C. freundii (genomospecies 1) as defined by the type strain of this mmm species, CDC 621-64. The majority of the strains were in bbb genomospecies 5 (21 strains) and 6 (15 strains); genomospe- cies 7 and 8 each contained 6 strains, and genomospecies 9, 10, and 11 each contained 3 strains. It seems likely that C. freundii is not the most frequently isolated Citrobacter species, although to verify this it will be necessary to reidentify the strains in our culture collection on the basis of the biochemical characteristics determined in this study. Of course, especially for those genomospecies mmN mmm with small numbers of strains, it will be necessary to verify that their biochemical profiles are definitive. On the basis of the extremely limited clinical data received with the strains studied, it is impossible to estimate the clinical significance of these organisms (Table 5). Nonethe- less, the small amount of available information suggests the possibility of clinical differences among citrobacteria. C. koseri isolates were obtained from men (61%) more often than from women, whereas exactly the opposite prevalence 654 BRENNER ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Conventional biochemical reactions for Citru6acter genomospecies" % of positive strains Test Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- species 2 species 3 species 4 species 1 species 5 species 6 species 7 species 8 species 9 species 10 species 11 Indole 94 100 100 38 14 33 0 100 0 0 100 Citrate (Simmons) 100 100 7 (86) 88 76 (95) 87 (100) 100 83 (100) 0 33 (100) 100 H,S produced on triple 0 13 0 75 (88) 67 (81) 60 100 0 0 67 67 sugar-iron agar Urease 56 (69) 81 36 38 (50) 76 47 (67) 83 100 100 0 67 (100) Arginine 88 (94) 81 (100) 100 75 (100) 52 (90) 67 (100) 100 100 0 33 (100) 67 (100) Ornithine 94 (100) 94 (100) 100 0 5 93 0 100 100 0 0 Motility 94 94 (100) 100 100 95 87 100 100 0 67 100 KCN 0 (6) 94 (100) 100 88 (100) 95 (100) 100 100 100 33 100 100 Malonate 94 13 0 13 5 0 100 100 100 100 0 D-Glucose (gas) 100 94 100 100 76 93 100 100 100 100 100 Acid produced from: Lactose 75 (94) 38 (88) 21 (100) 88 (100) 24 (90) 80 (87) 17 (100) 100 100 67 67 (100) Sucrose 44 13 100 100 19 7 0 0 0 33 33 Dulcitol 38 0 0 13 86 33 0 100 0 0 100 Salicin 19 (94) 31 (94) 0 (93) 0 (13) 10 0 (7) 0 17 (50) 0 (100) 0 (67) 0 (67) Adonitol 94 0 0 0 0 0 0 0 0 0 0 Raffinose 0 0 100 50 (100) 10 7 (13) 0 0 0 0 33 Cellobiose 94 100 100 25 (75) 43 (100) 73 (93) 20 (100) 100 100 67 100 a-CH,-glucoside 40 (93) 14 (21) 92 (100) 0 (25) 0 (5) 29 (43) 0 0 0 0 0 Esculin 0 (33) 0 (29) 0 (54) 0 5 0 0 17 (50) 0 (33) 0 (33) 0 (100) Melibiose 0 0 100 100 5 78 (100) 0 100 0 67 33 (100) D-Arabitol 100 0 0 0 0 0 0 0 0 0 0 Glycerol 100 25 (31) 85 100 90 (100) 86 100 67 0 67 (100) 100 Acetate 86 (93) 93 (100) 77 (100) 50 (75) 67 (76) 50 (92) 80 (100) 83 33 (67) 0 (33) 33 (100) NO3 reduced to NO, 100 93 100 100 86 100 100 100 100 100 100 ONPG~ 100 100 100 100 95 85 100 100 100 67 (100) 100

a Most preparations were incubated at 36 5 1°C; the exceptions were the gelatin liquefaction preparation (22°C) and the DNase preparation (25°C). Most results were read after 7 days of incubation; the methyl red and Voges-Proskauer results were read after 2 days. Results are expressed as percentages of positive strains within 48 h; the values in parentheses are the percentages of strains which gave delayed-positive reactions. The following tests, except as noted, were positive for all strains tested: methyl red, acid production from D-glucose, acid production from D-mannitol, acid production from D-sorbitol (one genomospecies 2 strain was negative), acid production from L-arabinose, acid production from L-rhamnose, acid production from maltose (one genomospecies 3 strain was delayed positive and one genomospecies 5 strain was negative), acid production from ~-xylose(one genomospecies 1 strain was delayed positive), acid production from trehalose, acid production from mucate (one genomospecies 10 strain was delayed positive), acid production from tartrate (one genomospecies 1 strain and one genomospecies 6 strain were negative), and acid production from mannose. The following tests, except as noted, were negative for all strains tested: phenylalanine deaminase, lysine decarboxylase (one genomospecies 6 strain was positive), gelatin liquefaction, acid production from myo-inositol (one genomospecies 1 strain was delayed positive), acid production from erythritol, lipase, DNase, oxidase, and pigment. ONPG, o-nitrophenyl-P-D-galactopyranoside. of isolation (61% from women) was seen in C. amalonaticus positive. Produces arginine dihydrolase and ornithine decar- and C. amalonaticus biogroup 1. Most of the Citrobacter boxylase. Other biochemical reactions useful in differentia- genomospecies were isolated from nonhuman as well as tion are acid production from a-CH,-D-glucoside, melibiose, human sources; however, genomospecies 9 was isolated raffinose, and sucrose and the ability to utilize benzoate, only from animals. Human isolates of all genomospecies 4-hydroxybenzoate, maltitol, D-melibiose, 1-0-CH,-a-galac- except C. koseri were obtained predominantly from stools; toside, palatinose, protocatechuate, D-raffinose, and sucrose C. koseri was most often isolated from urine and cerebro- but not m-coumarate, dulcitol, and malonate as sole carbon spinal fluid. sources. Additional reactions useful in differentiation are It is clear that clinical data are needed on all of the given in Table 4. Full biochemical reactions are shown in genomospecies formerly grouped in C. freundii. We hope Tables 2 and 3. that the delineation of 11 genomospecies in the genus Citro- Isolated from human stools, urine, wounds, and blood. bacter, the means to identify these taxa biochemically (Ta- Presumptively pathogenic. The type strain is CDC 2991-81 bles 2 to 4), and the taxonomic proposals given below will (= ATCC 51112), which was isolated from the stool of a aid in determining both the incidence and clinical signifi- 1-year-old boy in New York. cance of these organisms. Description of Citrobacter youngae sp. nov. Citrobacter Description of sp. nov. Citrobacter youngae (young'ae. N. L. gen. n.youngae, to honor Viola farmeri (far'mer-i. N. L. gen. n. farrneri, to honor John J. M. Young, an American microbiologist, for her contribu- Farmer 111, an American microbiologist, who recognized tions to the genus Citrobacter, including the description of and biochemically defined C. amalonaticus biogroup 1 and C. amalonaticus [L. arnalonatica] [ 141). Corresponds to who has made many substantial contributions to the recog- Citrobacter genomospecies 5. Usually indole negative, de- nition and classification of new species and genera in the layed positive for citrate and arginine dihydrolase, and and other families [5-71). Informally negative for ornithine decarboxylase. Other biochemical called C. amalonaticus biogroup 1 and Citrobacter genom- reactions useful in differentiation are acid production from ospecies 4. Indole positive and citrate negative or delayed dulcitol but not from melibiose and the ability to utilize VOL. 43, 1993 NEW CITROBACTER SPECIES 655

TABLE 3. Carbon source utilization reactions of Citrobacfer genomospecies

% of positive strainsu Source Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- Genomo- species 2 species 3 species 4 species 1 species 5 species 6 species 7 species 8 species 9 species 10 species 11

cis-Aconitate 100 93 (100) 57 (71) 88 78 (91) 89 (100) 100 100 0 33 (67) 67 (100) tram- Aconi t a te 25 (63) 0 57 13 (38) 0 0 83 50(100) 0 0 0 Adonitol 100 0 0 0 0 0 (6) 0 0 0 0 0 D-Alanine 100 100 71 (85) 100 100 100 100 100 100 67 (100) 100 4-Aminobutyrate 0 (6) 0 (13) 7 (21) 25 (50) 22 (39) 17 (56) 0 (67) 50 0 0 33 (67) 5-Aminovalerate 6 (44) 33 (53) 7 (36) 38 (88) 43 (56) 50 100 33 (50) 0 0 67 (100) D-habit01 100 0 0 0 0 (4) 0 0 0 0 0 0 Benzoate 0 73 93 0 0 0 0 100 0 0 0 Caprate 0 (56) 0 (27) 0 (7) 0 0 (4) 0 0 0 (17) 0 0 0 D-Cellobiose 94 100 100 88 78 (87) 94 (100) 67 (100) 100 100 67 100 m -Coumara t e 0 0 0 88 (100) 96 100 100 67 0 33 (67) 0 (67) Dulcitol 44 0 0 13 87 33 0 100 0 0 100 Esculin 0 0 0 0 0 (9) 6 0 17 (50) 0 0 0 (33) Ethanolamine 19 (82) 33 (67) 14 0 (25) 9 (22) 0 17 (34) 17 (33) 0 0 0 (33) L-Fucose 100 100 100 100 91 (95) 94 100 100 33 67 100 Gen tiobiose 88 100 93 (100) 88 52 (78) 89 (100) 67 (100) 100 0 (33) 67 100 Gentisate 100 200 93 (100) 100 0 94 (100) 100 100 67 0 67 L-Glu t mate 100 100 86 (100) 75 (88) 83 (92) 94 (100) 83 (100) 83 (100) 67 0 (33) 100 Glycerol 100 100 100 100 100 100 100 100 0 100 100 3-Hydroxybenzoate 100 100 100 100 0 100 100 100 100 0 67 4-Hydroxybenzoate 0 100 100 0 0 0 0 100 0 0 0 3-Hydroxybutyr ate 6 (12) 20 (33) 7 100 74 (87) 44 (66) 67 (100) 100 0 0 33 (67) myo-Inosi to1 100 0 0 100 0 6 0 100 0 67 0 2-Ke togluconate 100 100 100 100 100 100 100 100 0 (67) 100 100 5-Ketogluconate 100 100 100 100 96 (100) 100 50 0 0 100 100 2-Ketoglutarate 6 7 (40) 7 (36) 13 (38) 0 (17) 22 (61) 33 (67) 33 (100) 0 33 (100) 0 (33) DL-Lactate 100 100 100 100 100 100 100 100 33 100 100 Lactose 56 (87) 20 (87) 50 (86) 88 (100) 22 (44) 78 (84) 17 (34) 100 100 67 33 100 Lactulose 0 0 7 88 0 67 (78) 17 100 0 (33) 67 33 (100) D-Lyxose 100 0 0 (7) 63 (88) 9 (52) 56 (78) 83 (100) 0 (67) 0 0 100 Malonate 81 (87) 0 0 0 0 0 50 (100) 67 (94) 67 (100) 0 0 Maltitol 100 7 93 25 0 (4) 44 (55) 0 0 0 0 0 D-Melibiose 0 7 100 88 0 94 (100) 0 100 0 100 33 (67) l-O-CH3-a-galactoside 0 0 100 88 0 (22) 100 0 100 0 67 (100) 0 (100) l-O-CH,-p-galactoside 100 7 (34) 14 (43) 100 65 (87) 83 100 100 100 67 100 3-O-CH3-~-glucose 13 87 86 (100) 63 0 94 (100) 100 100 0 0 67 l-O-CH,-a-D-@ucoside 94 (100) 13 86 (93) 25 0 39 (45) 0 0 0 0 0 l-O-~,-p-~-g~ucoside100 100 100 100 100 100 100 100 0 (33) 100 100 Palatinose 100 13 100 25 4 67 0 0 0 0 67 Phenylacetate 0 0 0 25 0 0 0 0 0 0 (33) 0 3-Phenylpropionate 0 0 0 75 96 83 100 0 0 0 (33) 0 (67) L-Proline 100 87 57 (71) 100 87 (91) 100 100 100 100 33 (100) 100 Propionate 88 80 (87) 86 88 78 (87) 72 (78) 100 83 (100) 67 (100) 0 100 Protoca t echua te 0 100 100 0 0 0 0 100 0 0 0 Putrescine 0 0 0 50 (75) 0 39 (61) 67 0 100 0 0 (67) D-Raffinose 0 0 100 75 (88) 0 6 (12) 0 0 0 67 33 L-Sorbose 0 87 100 100 100 6 83 0 0 0 100 Sucrose 44 0 100 100 9 (13) 6 0 0 0 33 33 D-Tagatose 0 0 36 (43) 13 4 0 0 0 0 0 0 D-Tartrate 0 0 0 13 0 6 100 0 0 0 0 L-Tartrate 19 (75) 33 (73) 36 (65) 0 (63) 22 (74) 17 (67) 67 (84) 17 (50) 100 0 33 (67) meso-Tartrate 0 40 (87) 7 (21) 50 78 (100) 72 (94) 100 67 (83) 100 0 67 (100) Tricarballylate 0 93 100 100 4 (17) 89 (95) 100 83 (100) 100 0 100 D-Turanose 6 (31) 0 (13) 36 0 (13) 0 11 (17) 0 0 0 0 0 L-Tyrosine 88 0 0 75 (88) 74 (87) 72 (100) 67 (100) 0 0 0 (33) 100 Xylitol 19 (57) 0 0 0 0 0 0 0 0 0 0

~~ ~ ~ Percentages of strains which gave positive reactions within 48 h. The numbers in parentheses are the percentages of strains which gave positive reactions within 96 h. All strains, except as noted, utilized the following carbon sources within 48 h: N-acetyl-D-g~ucosamine,L-alanine, L-arabinose, L-aspartate (except one genomospecies 5 strain that was negative and one genomospecies 10 strain that was positive within 96 h), citrate (except one genomospecies 4 strain that was positive within 96 h), D-fructose, fumarate, D-galactose, D-galacturonate (except one genomospecies 10 strain), D-gluconate, D-glucosamine, D-glucose, D-glucuronate, DL-glycerate (except one genomospecies 2 strain), D-malate (except one genomospecies 4 strain and two genomospecies 10 strains that were positive within 96 h), L-malate, maltose (except one genomospecies 5 strain), maltotriose (except one genomospecies 4 strain and one genomospecies 5 strain), D-mannitol, D-mannose, mucate (except one genomospecies 5 strain that was positive within 96 h), L-rhamnose, D-ribose (except one genomospecies 5 strain), D-saccharate (except one genomospecies 5 strain and one genomospecies 10 strain), L-serine (except two genomospecies 4 strains), D-sorbitol, succinate, D-trehalose, and D-xylOSe (except two strains of genomospecies 1). All strains, except as noted, failed to utilize the following carbon sources within 96 h: L-arabitol, betaine, caprate, caprylate, i-erythritol, glutarate, histamine, L-histidine, HQ-P-glucuronide (except one genomospecies 6 strain), itaconate (except one genomospecies 7 strain that was positive within 96 h), D-melezitose, quinate, trigonelline, tryptamine, and tryptophan. TABLE 4. Biochemical tests useful in differentiating Citrobacter genomospecies" W

2 E C. amalonaticus C. farmeri C. freundii C. youngae C. braakii C. werkmanii C. sedlakii Genome- Genome- Genomo- Biochemical test (genomo- (genomo- (genomo- (genomo- (genomo- (genomo- (genomo- species 8) species 9 species 10 species 11 species 3) species 4) species 1) species 5) species 6) species 7) 3 w IndoIe Citrate Urease Arginine dihydrolase Ornithine decarboxylase Motility Malonate Sucrose (acid) Dulcitol (acid) Raffinose (acid) a-CH,-D-glucoside (acid) Esculin Melibiose (acid) Salicin (acid) Acetate Carbon source utilization Benzoate - rn-Coumarate - (4 Dulcitol + Gentisate d Glycerol + 3-Hydrolzybenzoate d 4-Hydroxybenzoate myo-Inositol 5-Ketogluconate Lactulose D-Lyxose Maltitol D-Melibiose 1-0-CH,-a-galactoside 3-O-CH,-~-glucose Palatinose 3-Phenylpropionate Protocatechuate D-Raffinose L-Sorbose Sucrose D-Tartrate Tricarballylate L-Tyrosine v, a Genomospecies 2 (C. koseri) is not included in this table. This taxon is easily differentiated from all other genomospecies by its ability to produce acid from adonitol and D-arabitol and its ability to utilize these '9 substrates as sole carbon sources; it is also unable to grow in the presence of KCN. +, 83% or more of the strains are positive within 2 days; -, 17% or less of the strains are positive; d, 18 to 82% of the strains are positive within 2 days. Parentheses indicate that a test is positive between 3 and 4 days (for carbon source utilization tests) or between 3 and 7 days (for other tests). 5 z r VOL. 43, 1993 NEW CITROBACTER SPECIES 657

TABLE 5. Clinical information on Citrobacter isolates for which data were available

No. of strains No. of strains No. of strains No. of strains isolated from the isolated from isolated from: the following isolated from following human sources: of the following ages: Taxon nonhuman sources: Cerebro- Males Females Stools Urine spinal Blood Wounds Sputum Other Animals Food <2 2-15 1640 41-60 >60 fluid Y‘ Yr Yr Yr Yr

~ ~~ ~~ C. kosen’ 29 19 4 18 14 6 3 72 1 10 1 1 3 16 C. amalonaticus 18 28 32 3 5 3 1 1 713 5 8 C. amalonaticus 16 25 25 6 2 6 181 1511 biogroup 1 Genomospecies 1 1 1 5 1 1 1 11 Genomospecies 5 3 3 11 1 2 3 1 31 Genomospecies 6 4 1 7 2 111 Genomospecies 7 2 3 1 111 1 Genomospecies 8 3 1 3 1 1 Genomospecies 9 3 Genomospecies 10 1 1 Genomospecies 11 2 1 1 2

dulcitol, 3-phenylpropionate, and L-sorbose but not genti- tiation are given in Table 4. Full biochemical reactions are sate, 3-hydroxybenzoate, malonate, D-melibiose, 1-0-CH,- shown in Tables 2 and 3. a-galactoside, 3-O-CH,-~-glucose, and tricarballylate as Isolated from human stools and urine and from food. sole carbon sources. Additional reactions useful in differen- Presumptively pathogenic. The type strain is CDC 876-58 (= tiation are given in Table 4. Full biochemical reactions are ATCC 51114), which was isolated from a human stool in shown in Tables 2 and 3. France. Isolated from human stools and blood and from animals Description of Citrobacter sedkzkii sp. nov. Citrobacter and food. Presumptively pathogenic. The type strain is CDC sedlakii (sed.lak’ i.i. N. L. gen. n. sedlakii, to honor Jiri 460-61 (= ATCC 29935), which was isolated from meat Sedlak, a Czechoslovakian microbiologist, who developed a scraps in South Carolina. serotyping scheme for C. freundii [ll]). Corresponds to Description of Citrobacter braukii sp. nov. Citrobacter Citrobacter genomospecies 8. Positive for indole produc- braakii (braak‘i-i. N. L. gen. n. braakii, to honor Hendrik R. tion, arginine dihydrolase, and ornithine decarboxylase. Braak, a Dutch microbiologist, who worked on glycerol Delayed citrate positive. Other biochemical reactions useful fermentation by enteric and isolated a glycerol for differentiation are production of acid from dulcitol and fermenter that he called “Bacterium freundii” [11). Corre- melibiose but not from sucrose and the ability to utilize sponds to Citrobacter genomospecies 6. Production of in- benzoate, dulcitol, 4-hydroxybenzoate, myo-inositol, lactu- dole is variable. Positive or delayed positive for citrate, lose, malonat e, 1-0-CH, -a-galactoside, and protoca t echuate arginine dihydrolase, and ornithine decarboxylase. Other but not 5-ketogluconate and L-sorbose as sole carbon biochemical reactions useful in differentiation are lack of sources. Additional reactions useful in differentiation are acid production from salicin and sucrose and the ability to given in Table 4. Full biochemical reactions are shown in utilize m-coumarate, 1-0-CH,-a-galactoside, 3-phenylpropi- Tables 2 and 3. onate, and L-tyrosine (delayed) but not malonate and L-sor- Isolated from human stools, blood, and wounds. Presump- bose as sole carbon sources. Additional reactions useful in tively pathogenic. The type strain is CDC 4696-86 (= ATCC differentiation are given in Table 4. Full biochemical reac- 51115), which was isolated from a human stool in France. tions are shown in Tables 2 and 3. The following genomospecies, although biochemically Isolated from human stools, urine, and wounds and from separable from each other and from all other citrobacteria, animals and food. Presumptively pathogenic. The type strain each contain only three strains. While it is not likely that is CDC 80-58 (= ATCC 51113), which was isolated from a additional strains will have markedly different biochemical snake in France. characteristics, it is prudent to examine additional strains Description of Citrobacter werkmunii sp. nov. Citrobacter before naming these taxa. werkmanii (werk’man.i.i. N. L. gen. n. werkmanii, to honor Description of Citrobacter genomospecies 9. Citrobacter Chester H. Werkman, an American microbiologist, who genomospecies 9 is negative for indole production, citrate, studied the fermentative production of trimethylene glycol and arginine dihydrolase and is ornithine decarboxylase from glycerol and proposed the genus Citrobacter [13]). positive. Other biochemical reactions useful for differentia- Corresponds to Citrobacter genomospecies 7. Negative for tion are lack of motility, inability to produce acid from indole production and ornithine decarboxylase; citrate pos- melibiose and sucrose, and ability to utilize malonate but not itive and positive for arginine dihydrolase. Other biochemi- m -coumarate, glycerol, 5 - ketogluconat e, D-melibiose, 1-0- cal reactions useful for differentiation are lack of acid CH,-a-galactoside, and 3-O-CH,-~-glucose as sole carbon production from dulcitol, melibiose, and sucrose and the sources. Additional reactions useful for differentiation are ability to utilize m-coumarate, D-lyxose, malonate, 3-phenyl- given in Table 4. Full biochemical reactions are shown in propionate, L-sorbose, and D-tartrate but not dulcitol, 4-hy- Tables 2 and 3. droxybenzoate, D-melibiose, and 1-0-CH,-a-galactoside as This organism has been isolated only from rodents and is sole carbon sources. Additional reactions useful in differen- presumptively pathogenic for these animals. The type strain b58 BRENNER El’ AL. INT. J. SYST.BACTERIOL. is CDC 1843-73 (= ATCC 51116), which was isolated from a and designation of the neotype strain. Int. J. Syst. Bacteriol. hamster in Connecticut. 22: 12-18. Description of Citrobacter genomospecies 10. Citrobacter 5. Farmer, J. J., 111.1981. The genus Citrobacter, p. 1140-1147. In genomospecies 10 is negative for indole production and M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes, a handbook on habitats, isola- ornithine decarboxylase and delayed positive for citrate and tion, and identification of bacteria. Springer-Verlag, New York. arginine dihydrolase. Other biochemical reactions useful for 6. Farmer, J. J., 111, M. A. Asbury, F. W. Hickman, D. J. Brenner, differentiation are a negative urease reaction and the inabil- and the Enterobacteriaceae Study Group. 1980. Enterobacter ity to utilize gentisate, 3-hydroxybenzoate, 3-O-CH,-~-glu- sakazakii: a new species of “Enterobacteriaceae”isolated from cose, L-sorbose, and tricarballylate as sole carbon sources. clinical specimens. Int. J. Syst. Bacteriol. 30569-584. This organism has been isolated from human stools and 7. Farmer, J. J., 111, B. R. Davis, F. W. Hickman-Brenner, A. food; there is insufficient information to speculate on its McWhorter, G. P. Huntley-Carter, M. A. Asbury, C. Riddle, clinical significance. The type strain is CDC 4693-86 (= H. G. Wathen-Grady, C. Elias, G. R. Fanning, A. G. Steigerwalt, ,4TCC 51117), which was isolated from a human stool in C. M. O’Hara, G. K. Morris, P. B. Smith, and D. J. Brenner. ]France. 1985. Biochemical identification of new species and biogroups of Enterobacteriaceaeisolated from clinical specimens. J. Clin. Description of Citrubucter genomospecies 11. Citrobacter Microbiol. 21:46-76. genomospecies I1 is indole and citrate positive, delayed 8 Frederiksen, W. 1970. Citrobacter koseri (n. sp.) a new species positive for arginine dihydrolase, and ornithine decarboxy- within the genus Citrobacter, with a comment on the taxonomic llase negative. Other biochemical reactions useful for differ- position of Citrobacter intermedium (Werkman and Gillen). (entiation are acid production from dulcitol and the ability to Publ. Fac. Sci. Univ. J. E. Purkyne Brno K47:89-94. utilize dulcitol, D-lyxose, I-0-CH,-a-galactoside (delayed), 9. Frederiksen, W. 1990. Correct names of the species Citrobacter and L-tyrosine but not malonate and protocatechuate as sole koseri, Levinea malonatica, and Citrobacter diversus. Request carbon sources. for an opinion. Int. J. Syst. Bacteriol. 40:107-108. This organism has been isolated from human stools and 10. Hickman, F. W., and J. J. Farmer 111. 1978. Salmonella typhi: ’blood; there is insufficient information to speculate on its identification, antibiograms, serology, and bacteriophage typ- ing. Am. Med. Technol. 44:1149-1159. (clinical significance. The type strain is CDC 2970-59 (= J. lOa.Rossello, R., E. Garcia-Valdes, J. Lalucat, and J. Ursing. 1991. ATCC 51118), which was isolated from an unknown source Genotypic and phenotypic diversity of Pseudomonas stutzeri. in Illinois. Syst. Appl. Microbiol. 14:150-157. 11. Sedlak, J. 1973. Present knowledge and aspects of Citrobacter. REFERENCES Curr. Top. Microbiol. Immunol. 62:41-59. Braak, H. R. 1928. Onderzoekingen over Vergisting van Glyc- 12. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, erine. Ph.D. thesis. University of Delft, Delft, The Netherlands. 0. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, Brenner, D. J., A. C. McWhorter, J. K. Leete-Knutson, and R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. A. G. Steigerwalt. 1982. 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