Received 22 February 1999/Returned for modification 24 April 1999/Accepted 18 May 1999

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1 JOURNAL OF LINIAL MIROBIOLOGY, Aug. 1999, p Vol. 37, No /99/$ opyright 1999, American Society for Microbiology. All Rights Reserved. Biochemical Identification of itrobacter Species Defined by DNA Hybridization and Description of itrobacter gillenii sp. nov. (Formerly itrobacter Genomospecies 10) and itrobacter murliniae sp. nov. (Formerly itrobacter Genomospecies 11) DON J. BRENNER, 1 * AROLINE M. O HARA, 2 ATRIK A. D. GRIMONT, 3 J. MIHAEL JANDA, 4 ENEVOLD FALSEN, 5 EVA ALDOVA, 6 ELISABETH AGERON, 3 JIRI SHINDLER, 6 SHARON L. ABBOTT, 4 AND ARNOLD G. STEIGERWALT 1 Meningitis and Special athogens Branch, Division of Bacterial and Mycotic Diseases, 1 and Hospital Environment Laboratory Branch, Hospital Infections rogram, 2 National enter for Infectious Diseases, enters for Disease ontrol and revention, Atlanta, Georgia 30333; Unité des Entérobactéries, Institut National de la Recherche Scientifique, Unité 199, Institut asteur, aris, France 3 ; Microbial Diseases Laboratory, Division of ommunicable Disease ontrol, Department of Health Services, Berkeley, alifornia ; ulture ollection, University of Göteborg, Department of linical Bacteriology, S Göteborg, Sweden 5 ; and Department of linical Microbiology, National Institute of ublic Health, rague 10, zech Republic 6 Received 22 February 1999/Returned for modification 24 April 1999/Accepted 18 May 1999 Recent work describing six named species and two unnamed genomospecies within itrobacter has enlarged the genus to 11 species. DNA relatedness and phenotypic tests were used to determine how well these species can be identified. One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion. By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level. Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other itrobacter species. arbon source utilization tests were used to identify 86 of the strains. Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other itrobacter species. Additional strains of itrobacter genomospecies 10 and itrobacter genomospecies 11 were identified, allowing these species to be formally named as itrobacter gillenii sp. nov. and itrobacter murliniae sp. nov., respectively. Five named species and three unnamed genomospecies were recently added to the genus itrobacter on the basis of DNA relatedness and biochemical studies (2). Based on this new classification, Janda et al. (8) identified 235 itrobacter strains from the collection of the Microbial Diseases Laboratory, alifornia Department of Health Services (DHS), Berkeley. Additional strains of unnamed itrobacter genomospecies 9 were subsequently identified and studied, resulting in this species being named itrobacter rodentium (13). The study reported herein had two goals. We sought to determine the extent to which the biochemical identification of the new, as well as the traditional, species itrobacter freundii, itrobacter koseri (itrobacter malonaticus), and itrobacter amalonaticus correlated with molecular identification. We also sought to obtain additional strains of the two remaining unnamed itrobacter genomospecies and to formally name them. For these purposes, 136 itrobacter strains were identified to species level by DNA hybridization, and the data were compared with the biochemical identifications of these strains. Additional strains of each of the two remaining unnamed itrobacter genomospecies were identified, resulting in their formal description as itrobacter gillenii sp. nov. (formerly * orresponding author. Mailing address: enters for Disease ontrol and revention, , Mailstop D11, Atlanta, GA hone: (404) Fax: (404) DJB3@D.gov. itrobacter genomospecies 10) and itrobacter murliniae sp. nov. (formerly itrobacter genomospecies 11). MATERIALS AND METHODS Strains. Included in the study were 136 strains that, prior to the creation of additional itrobacter species (2), had been identified as. freundii,. koseri, and TABLE 1. Summary values a of strains assigned to itrobacter species on the basis of DNA relatedness Species No. of strains Avg Summary value b Low 60 % D % D 75. freundii koseri amalonaticus itrobacter farmeri youngae braakii werkmanii itrobacter sedlakii rodentium c itrobacter genomospecies itrobacter genomospecies a Expressed as percent relatedness to type strain. b D, divergence. c, Reaction was not carried out. 2619

2 2620 BRENNER ET AL. J. LIN. MIROBIOL. TABLE 2. omparison of conventional biochemical reactions of citrobacteria a % ositive strains b Test T (n 429). freundii. koseri. amalonaticus. youngae. braakii (n 9) (n 49) (n 16) (n 17) (n 16) (n 17) (n 21) (n 39) (n 15) (n 24) Indole itrate (Simmons) 88 (93) 78 (89) 80 (90) (95) 74 (90) 87 (100) 75 (88) H 2 S production (triple sugar iron) 93 (96) 78 (89) 59 (63) (81) 74 (85) (45) Urease 79 (87) 44 (56) 57 (73) 50 (69) 59 (71) 88 (94) 88 (94) (67) 38 (58) Arginine deaminase 47 (88) 67 (100) 51 (100) 94 (100) 94 (100) 81 (100) 76 (94) 52 (90) 54 (90) 67 (100) 54 (100) Ornithine decarboxylase 15 0 (11) 12 (14) (100) 94 (100) Motility (100) 88 (94) KN (100) 90 (100) 0 (6) 0 (6) (100) 92 (95) (100) Malonate (5) 0 0 D-Glucose (gas) Acid produced from: Lactose 37 (91) 78 (89) 86 (92) 69 (94) 71 (88) 38 (100) 50 (100) 24 (90) 18 (90) 80 (87) 81 (86) Sucrose 17 (18) (78) Dulcitol Salicin 3 (24) 0 (11) 6 (16) 6 (88) 6 (88) 12 (94) 18 (94) (7) 0 (8) Raffinose 17 (18) 44 (89) 73 (86) (13) 17 (21) ellobiose 60 (99) 44 (77) 35 (82) (100) 26 (77) 73 (93) 67 (96) -H 3 -glucoside 5 (14) 11 (33) 12 (22) 44 (94) 41 (88) 6 (19) 6 (18) 0 (5) 0 (5) 33 (46) 25 (38) Esculin 1 (2) 0 0 (10) 0 (31) 0 (29) 0 (25) 0 (35) (4) Melibiose NT d (98) (100) 88 (100) Glycerol 99 (100) (38) 41 (47) 90 (100) 92 (100) Sodium acetate 77 (91) 44 (56) 78 (82) 88 (94) 88 (94) (76) 64 (85) 53 (93) 54 (79) NO 3 3NO ONG c (96) a Most tests were incubated at 36 1 ; the exceptions were gelatin liquefaction (22 ) and DNase (25 ). The methyl red and Voges-roskauer results were read after 2 days of incubation. All other tests were incubated for 7 days. Results are expressed as percentages of strains positive within 48 h; the values in parentheses are the percentages of strains that gave delayed (3 to 7 day) 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 (one strain was delayed positive), acid production from D-sorbitol (one. freundii strain and one. youngae strain were delayed positive, and one. koseri strain and one. werkmanii strain were negative), acid production from L-arabinose, acid production from L-rhamnose, acid production from maltose (one. amalonaticus strain and one. youngae strain were delayed positive, and one. youngae strain was negative), acid production from D-xylose (one. freundii strain was delayed positive, and one. freundii strain and one. braakii strain were negative), acid production from trehalose, acid production from mucate (two. freundii strains, two. youngae strains, and one genomospecies 10 strain were delayed positive), acid production from tartrate (two. youngae strains, one. freundii strain, and one. braakii strain were negative), and acid production from D-mannose. The following tests, except as noted, were negative for all strains tested: Voges-roskauer, phenylalanine deaminase, lysine decarboxylase (one. braakii strain was positive), gelatin liquefaction (one. rodentium strain was delayed positive), acid production from myo-inositol (two. freundii strains was delayed positive, and two. freundii strains and one. werkmanii strain were positive), acid production from erythritol, lipase, DNase, oxidase, and pigment. b T, traditional biochemical percentages for. freundii sensu lato, taken from reference 5;, biochemical percentages obtained in a previous study (2);, combined percentages for the strains in reference 2 and those reported in this study. c ONG, o-nitrophenyl- -D-galactopyranoside. d Not tested.. amalonaticus on the basis of biochemical reactions. Eighty-six strains were sent to the diagnostic laboratories at the enters for Disease ontrol and revention (D) from 1972 to 1986; 26 of these strains were from the Microbial Diseases Laboratory of the DHS, having been received from 1970 to 1994; 13 strains were from the ulture ollection, University of Göteborg (UG), Göteborg, Sweden; eight strains were from the National Institute of ublic Health (NIH), rague, zechoslovakia; and three strains were from the Division of Toxicology and Division of omparative Medicine, Massachusetts Institute of Technology (MIT), ambridge, Mass. Biochemical tests. The methods used for the conventional biochemical tests done at the D and for the carbon source utilization tests (using Biotype strips [BioMérieux, La Balme les Grottes, France]) carried out at the Institut asteur have been described previously (2, 6, 7). In the present study, Biotype-100 strips were incubated at 30 for 4 days. The D strains were identified both by conventional biochemical tests done at the D and by carbon source utilization tests carried out at the Institut asteur. The NIH, UG, MIT, and the DHS strains were also identified by conventional biochemical tests at D. The DHS strains were also identified by conventional biochemical tests at the DHS (8). In all cases, identification on the basis of either conventional biochemical tests or carbon source utilization tests was made by using the differential tables previously published by Brenner et al. (2). DNA hybridization. The methods used for DNA extraction and purification and the hydroxyapatite hybridization method for determining levels of DNA relatedness have been described previously (2, 4). Reactions were carried out at 60 (for optimal DNA reassociation) and at 75 (for stringent DNA reassociation). ercent divergence was determined by thermal elution of reassociated DNA on the assumption that each 1 decrease in thermal stability within a reassociated DNA duplex was due to approximately 1% of nucleotide bases within that sequence being unpaired. ercent divergence was calculated to the nearest 0.5%. All DNA relatedness reactions were carried out at least twice. RESULTS AND DISUSSION The standard used to identify strains was the genetic definition of a species on the basis of DNA relatedness, as recommended by Wayne et al. (14). This recommendation states that DNAs from strains of a given species are at least 70% related at optimal conditions for DNA reassociation (60 incubation temperature in this study) and that divergence (unpaired bases) within related nucleotide sequences is 5% or less. We used the additional criterion that DNA relatedness of strains within a species remains above 60% in reactions carried out under stringent incubation conditions (75 incubation temperature in this study). A strain was assigned to a given species when the relatedness of its DNA to labeled DNA from the type strain of that species fulfilled the species definition. The DNA

3 VOL. 37, 1999 IDENTIFIATION AND DESRITION OF ITROBATER SEIES 2621 TABLE 2 ontinued itrobacter species b. werkmanii. sedlakii. farmeri. rodentium Genomospecies 10 Genomospecies 11 (n 13) (n 14) (n 14) (n 3) (n 3) (n 3) (n 8) (100) 83 (100) 0 (36) 0 (36) 0 (100) 0 (67) 33 (100) 50 (100) (100) (100) (100) 63 (75) (92) (100) 17 (83) 67 (100) 50 (88) (33) (100) 23 (77) (100) 4 (100) (83) 67 (100) 63 (100) (8) 17 (50) 17 (50) 0 (93) 0 (93) 0 (100) 0 (100) 0 (67) 17 (83) 0 (67) 13 (63) (83) 8 (85) (8) 17 (50) 17 (50) 0 (36) 0 (36) 0 (100) 0 (83) 0 (33) 17 (50) 0 (100) 0 (100) (100) 38 (75) (33) 67 (100) 67 (83) (100) 79 (100) 0 (100) 0 (50) 0 (33) 0 (17) 33 (100) 75 (100) (100) 83 (100) relatedness data are summarized in Table 1. Relatedness values obtained with 131 of the 136 strains fully conformed to the molecular definition of a species. Of the five exceptions, two were closest to. amalonaticus, two were closest to itrobacter braakii, and one was closest to itrobacter youngae. They each fulfilled two of the three criteria (percent divergence of less than 5 and relatedness of above 60% in 75 reactions), but their relatedness in 60 reactions was slightly under 70%. While it is possible that these five exceptions represent one or more new species, we decided that they were close enough to the species definition to merit provisional assignment to the species to which they were most closely related. All 136 strains were identified by conventional biochemical tests done at D. The 87 strains from the D collection were also identified by carbon source utilization tests. ersonnel carrying out identification by any method were blinded to the results obtained with other methods. One hundred nineteen of 136 strains (88%) were correctly identified on the basis of their biochemical profiles. Fourteen strains (10%) were misidentified as other species in the genus itrobacter, and three strains (2%) were correctly identified as itrobacter but were not identified to species level. Seven of the misidentified strains and one of the nonidentified strains were biochemically atypical. freundii. The atypical characteristics most often seen were ornithine decarboxylase production (5 strains); indole production (3 strains); negative growth on citrate, negative fermentation of raffinose, and nonmotility (2 strains each); malonate utilization and fermentation of i-inositol (1 strain each); and negative fermentation of melibiose, negative production of H 2 S, and negative gas production from D-glucose (1 strain each). These results are encouraging, indicating that despite changes in reaction percentages and the inclusion of a large number of atypical strains, the large majority of itrobacter strains can be identified phenotypically by using conventional biochemical tests. O Hara et al. (11) investigated the abilities of five commercial identification systems to recognize the newly defined species of itrobacter by using the 112 strains identified by DNA hybridization in reference 1. Because the eight newly defined species were not included in the databases of any of these systems, most of these strains were identified as. freundii. The Vitek GNI card (BioMérieux Inc., Hazelwood, Mo.), which contains all 11 named and unnamed itrobacter species, groups seven species which can be identified by using six additional conventional biochemical tests into the itrobacter freundii complex. When evaluated by O Hara et al. (12), 12 of 16 strains in this complex were correctly identified by using the additional tests. The database of the MicroScan Rapid Neg ID3 panel (Dade Behring, Inc., West Sacramento, alif.) contains eight of the nine named itrobacter species (. rodentium is not included). When evaluated by Bascomb et al. (1), 14 of 15 strains of the newer species were correctly identified. Revised biochemical test percentages for all itrobacter species are presented in Table 2 and are compared with the biochemical table presented previously (2). It should be noted that many of the percentages presented in the previous paper were changed when the data were recalculated (Table 2, columns ). Most of the changes are small, but nonetheless, the Hospital Environment Laboratory Branch of the D considers these results to be a correction of and a replacement for those presented previously (2). While the addition of strains in the present study did not cause most reaction percentages to change significantly, there are a number of percentage shifts worthy of mention. This is especially true for. freundii, whose biochemical profile originally included strains of the 10 subse-

4 2622 BRENNER ET AL. J. LIN. MIROBIOL. TABLE 3. arbon source utilization by itrobacter species a arbon source itrobacter species b cis-aconitate trans-aconitate Adonitol Aminobutyrate Aminovalerate D-Arabitol Benzoate aprate D-ellobiose m-oumarate Dulcitol Esculin Ethanolamine L-Fucose Gentiobiose Gentisate L-Glutamate DL-Glycerate Glycerol Hydroxybenzoate Hydroxybenzoate Hydroxybutyrate myo-inositol Ketogluconate Ketoglutarate DL-Lactate Lactose Lactulose D-Lyxose Malonate Maltitol D-Melibiose O-Methyl- -galactoside O-Methyl- -galactoside O-Methyl-D-glucose O-Methyl- -Dglucoside O-Methyl- -Dglucoside alatinose henylacetate henylpropionate L-roline ropionate rotocatechuate utrescine D-Raffinose L-Sorbose Sucrose D-Tagatose D-Tartrate L-Tartrate meso-tartrate Tricarballylate D-Turanose L-Tyrosine Xylitol

5 VOL. 37, 1999 IDENTIFIATION AND DESRITION OF ITROBATER SEIES 2623 quently recognized itrobacter species (5). The significant percentage shifts include H 2 S production (formerly 96% and now 63%), sucrose fermentation (formerly 18% and now 78%), dulcitol fermentation (formerly 71% and now 24%), and raffinose fermentation (formerly 18% and now 88%). In each case, the actual percentage is probably closer to the former value, since that value is based on a 10-fold-higher number of strains (albeit including many that are not. freundii) and a much higher percentage of typical strains. The percentages obtained from the biochemical tests in this study revealed a few small, but significant, changes when compared to the percentages obtained in the recent previous study (2). This is not surprising given the small number of representative strains for most species. For. braakii, delayed growth on citrate and delayed utilization of sodium acetate, and production of ornithine, and gas from glucose changed from positive to variable, and fermentation of glycerol changed from variable to positive. For itrobacter werkmanii, production of arginine changed from rapid (within 48 h) positive to delayed positive. For. rodentium, production of urease changed from positive to delayed positive, and motility changed from negative to delayed variable. For itrobacter genomospecies 10, fermentation of salicin, raffinose, and esculin changed from negative to variable. For itrobacter genomospecies 11, growth on citrate changed from positive to delayed positive, production of urease and arginine changed from delayed positive to variable, and fermentation of melibiose changed from delayed positive to delayed variable. By using the species profiles generated in our previous study, 65 of the 86 strains (76%) tested by carbon source utilization were correctly identified. Fifteen strains (17%) were not identified to species level, and six (7%) were misidentified as other species in the genus itrobacter. After adjustments were made in the identification program for carbon sources, all strains were retested and reidentified. Upon retesting, 84 of the strains (98%) were correctly identified and two (2%) were misidentified as other itrobacter species. On the basis of carbon source utilization patterns,. freundii strains formed seven clusters (biotypes) and. braakii strains formed two clusters. rofiles for the biotypes and revised carbon source utilization patterns for all species are shown in Table 3. The. braakii biotypes were separable by analysis of their abilities to utilize 4-aminobutyrate, lactose, D-lyxose, maltitol, 1-O-methyl- -galactoside, 3-phenylpropionate, and propionate.. freundii biotypes were separable on the basis of their profiles for use of the following carbon sources: trans-aconitate, 4-aminobutyrate, 5-aminovalerate, dulcitol, ethanolamine, 1-glutamate, myoinositol, maltitol, 3-O-methyl-D-glucose, 1-O-methyl- -D-glucoside, palatinose, 3-phenylpropionate, propionate, putrescine, sucrose, meso-tartrate, and D-turanose. Additional strains of each unnamed itrobacter genomospecies were identified on the basis of DNA relatedness and biochemical studies. Three additional strains confirmed to be itrobacter genomospecies 9 were obtained from David B. Schauer. As with the three initially reported strains of this species, the additional strains were all from mice and were causative agents of transmissible murine colonic hyperplasia (13). The six strains of itrobacter genomospecies 9 were recently described as the new species. rodentium (13). Eleven additional strains of itrobacter genomospecies 10 were identified, for a total of 14, and seven additional strains of itrobacter genomospecies 11 were identified, for a total of 10. The names itrobacter gillenii sp. nov. and itrobacter murliniae sp. nov. are proposed below for itrobacter genomospecies 10 and for itrobacter genomospecies 11, respectively. Where known, the sources of isolation of the strains used in the present and in the previous studies (2) are shown in Table 4. It seems clear that all itrobacter species other than. rodentium are predominantly isolated from human clinical specimens. Description of itrobacter gillenii sp. nov. itrobacter gillenii (gil.len i.i. N.L. gen. n. gillenii, to honor George Francis Gillen, an American microbiologist, who, along with. H. Werkman, studied the fermentative production of trimethylene glycol from glycerol and proposed the genus itrobacter [15]). Formerly called itrobacter genomospecies 10. It is negative for the production of indole and ornithine decarboxylase, positive for utilization of malonate, and delayed positive for growth on citrate and usually for production of arginine dihydrolase. Other biochemical characteristics useful for differentiation are negative reactions for production of urease and fermentation of dulcitol and the inability to utilize gentisate, 3-hydroxybenzoate, 3-O-H 3 -D-glucose, L-sorbose, and tricarballylate as sole carbon sources (2). omplete results of routine biochemical reactions are given in Table 2, and complete carbon source utilization reactions are given in Table 3. Known sources of isolation are human stool (nine strains); human urine, human blood, and animal stool (one strain each); and the environment (two strains). There is insufficient information to speculate on the clinical significance of. gillenii. The type strain is D (AT and UG 30796), which was isolated from a human stool in France. Description of itrobacter murliniae sp. nov. itrobacter murliniae (mur.lin i.ae. N.L. gen. n. murliniae, to honor Alma. McWhorter-Murlin, an American microbiologist, who, during her 39-year career at the enters for Disease ontrol and revention, made substantial contributions to our knowledge of Salmonella serotyping and itrobacter and to the taxonomy of a number of species in the family Enterobacteriaceae [3, 4, 9, 10]). Formerly called itrobacter genomospecies 11. It is positive or delayed positive for production of indole and growth on citrate, usually delayed positive for production of arginine dihydrolase, and negative for production of ornithine decara ercentages of strains which gave positive reactions within 96 h. All strains, except as noted, utilized the following carbon sources: N-acetyl-D-glucosamine, D-alanine (except one. farmeri strain), L-alanine, L-arabinose, L-aspartate (except one. youngae strain), citrate (except one. freundii biotype F strain), D-fructose, fumarate, D-galactose, D-galacturonate (except one. gillenii strain), D-gluconate, D-glucosamine (except for one. freundii biotype G strain), D-glucose, D-glucuronate, 2-ketogluconate (except for one. rodentium strain), D-malate (except for one. gillenii strain), L-malate, maltose (except for one. youngae strain), maltotriose (except for single strains of. youngae,. farmeri, and. gillenii), D-mannitol, D-mannose, mucate, L-rhamnose, D-ribose (except for one. youngae strain), D-saccharate (except for single strains of. youngae and. gillenii), L-serine (except for two. farmeri strains), D-sorbitol (except for one. freundii biotype strain), succinate, D-trehalose, and D-xylose (except for single strains of. freundii biotype F and biotype G). All strains, except as noted, failed to utilize the following carbon sources within 96 h: L-arabitol, betaine, caprylate (except for one. freundii biotype strain), i-erythritol, glutarate (except for one. werkmanii strain), histamine, L-histidine, HQ- -glucuronide (except for one. braakii strain), itaconate (except for one. werkmanii strain), D-melezitose (except for three. freundii biotype A strains), quinate, trigonelline, tryptamine, and tryptophan. b 1,. freundii biotype A; 2,. freundii biotype B; 3,. freundii biotype ; 4,. freundii biotype D; 5,. freundii biotype E; 6,. freundii biotype F; 7,. freundii biotype G; 8,. braakii biotype A; 9,. braakii biotype B; 10,. werkmanii; 11,. sedlakii; 12,. youngae; 13,. farmeri; 14,. koseri; 15,. amalonaticus; 16,. rodentium; 17,. gillenii; 18;. murliniae.

6 2624 BRENNER ET AL. J. LIN. MIROBIOL. TABLE 4. Sources of isolation for itrobacter isolates Taxon No. of strains isolated from a : Stool Urine SF Blood Wound Sputum Other Animal Environment. koseri amalonaticus farmeri freundii youngae braakii werkmanii sedlakii rodentium 6. gillenii murliniae a Other includes two isolates from bile and one isolate from mastitis; Environmental includes four isolates from soil, two isolates each from water, sewage, and cheese, and one isolate from an industrial fermentor. SF, cerebrospinal fluid. boxylase. Other biochemical tests useful for differentiation are acid production from dulcitol and esculin (delayed), growth on sodium acetate (usually delayed) but not on malonate, and the ability to utilize dulcitol, D-lyxose, 1-O-H 3 - -galactoside (delayed), and L-tyrosine, but not malonate and protocatechuate, as sole carbon sources. omplete results of routine biochemical reactions are given in Table 2, and complete carbon source utilization reactions are given in Table 3. Known sources of isolation are human stool (five strains); human wound (two strains); and human blood, human urine, and food (one strain each). There is insufficient information to speculate on the pathogenicity of. murliniae. The type strain is D (AT and UG 30797), which was isolated from an unknown source in Illinois. AKNOWLEDGMENTS E.A. and J.S. were supported in part by grant IGA from the Internal Grant Agency of the zech Ministry of Health. REFERENES 1. Bascomb, S., S. L. Abbott, J. D. Bobolis, D. A. Bruckner, S. J. onnell, S. K. ullen, M. Daugherty, D. Glenn, J. M. Janda, S. J. Lentsch, D. Lindquist,. B. Mayhew, D. M. Nothaft, J. R. Skinner, G. B. Williams, J. Wong, and B. L. Zimmer Multicenter evaluation of the MicroScan rapid gramnegative identification type 3 panel. J. lin. Microbiol. 35: Brenner, D. J.,. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and. F. Riddle lassification of citrobacteria by DNA hybridization: designation of itrobacter farmeri sp. nov., itrobacter youngae sp. nov., itrobacter braakii sp. nov., itrobacter werkmanii sp. nov., itrobacter sedlakii sp. nov., and three unnamed itrobacter genomospecies. Int. J. Syst. Bacteriol. 43: Brenner, D. J., A. McWhorter, A. Kai, A. G. Steigerwalt, and J. J. Farmer III Enterobacter asburiae: a new species found in human clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov. J. lin. Microbiol. 23: Brenner, D. J., A.. McWhorter, J. K. Leete Knutson, and A. G. Steigerwalt Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. lin. Microbiol. 15: Ewing, W. H Edwards and Ewing s identification of Enterobacteriaceae, 4th ed., p Elsevier, New York, N.Y. 6. Farmer, J. J., III, M. A. Asbury, F. W. Hickman, D. J. Brenner, and the Enterobacteriaceae Study Group Enterobacter sakazakii: a new species of Enterobacteriaceae isolated from clinical specimens. Int. J. Syst. Bacteriol. 30: Hickman, F. W., and J. J. Farmer III Salmonella typhi: identification, antibiograms, serology, and bacteriophage typing. Am. J. Med. Technol. 44: Janda, J. M., S. L. Abbott, W. K. heung, and D. F. Hanson Biochemical identification of citrobacteria in the clinical laboratory. J. lin. Microbiol. 32: McWhorter, A.., R. L. Haddock, F. A. Nocon, A. G. Steigerwalt, D. J. Brenner, S. Aleksic, J. Böckemuhl, and J. J. Farmer III Trabulsiella guamensis, a new genus and species of the family Enterobacteriaceae that resembles Salmonella subgroups 4 and 5. J. lin. Microbiol. 29: McWhorter-Murlin, A.., and F. W. Hickman-Brenner Identification and serotyping of Salmonella and an update of the Kauffmann-White scheme. enters for Disease ontrol and revention, Atlanta, Ga. 11. O Hara,. M., S. B. Roman, and J. M. Miller Ability of commercial identification systems to identify newly recognized species of itrobacter. J. lin. Microbiol. 33: O Hara,. M., G. L. Westbrook, and J. M. Miller Evaluation of Vitek GNI and Becton Dickinson Microbiology Systems rystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli. J. lin. Microbiol. 35: Schauer, D. B., B. A. Zabel, I. F. edraza,. M. O Hara, A. G. Steigerwalt, and D. J. Brenner Genetic and biochemical characterization of itrobacter rodentium sp. nov. J. lin. Microbiol. 33: Wayne, L. G., D. J. Brenner, R. R. olwell,. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E.. Moore, R. G. E. Murray, E. Stackebrandt, M.. Starr, and H. G. Trüper Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37: Werkman,. H., and G. F. Gillen Bacteria producing trimethylene glycol. J. Bacteriol. 23: