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Enterobacter asburiae sp. nov., a New Species Found in ClinicalSpecimens, and Reassignment of Erwinia dissolvens and Erwinia
nimipressuralis to the Genus Enterobacter as Enterobacterdissolvens comb. nov. and Enterobacter nimipressuralis comb. nov.
DON J. BRENNER,'* ALMA C. McWHORTER,2 AKEMI KAI,3 ARNOLD G. STEIGERWALT,'AND J. J. FARMER I112
Molecular Biology Laboratory' and Enteric Bacteriology Section,2 Division of Bacterial Diseases, Center for InfectiousDiseases, Centers for Disease Control, Atlanta, Georgia 30333, and Department of Microbiology, Tokyo Metropolitan
Research Laboratory of Public Health, Tokyo 160, Japan3
Received 17 January 1986/Accepted 8 March 1986
Enterobacter asburiae sp. nov. is a new species that was formerly referred to as Enteric Group 17 and thatconsists of 71 strains, 70 of which were isolated from humans. Enterobacter asburiae sp. nov. strains gavepositive reactions in tests for methyl red, citrate utilization (Simmons and Christensen's), urea hydrolysis,L-ornithine decarboxylase, growth in KCN, acid and gas production from D-glucose, and acid production fromL-arabinose, cellobiose, glycerol (negative in 1 to 2 days, positive in 3 to 7 days), lactose, D-mannitol,a-methyl-D-glucoside, salicin, D-sorbitol, sucrose, trehalose, and D-xylose. They gave negative reactions in theVoges-Proskauer test and in tests for indole, H2S production, phenylalanine, L-lysine decarboxylase, motility,gelatin, utilization of malonate, lipase, DNase, tyrosine clearing, acid production from adonitol, D-arabitol,dulcitol, erythritol, i(myo)-inositol, melibiose, and L-rhamnose. They gave variable reactions in tests forL-arginine dihydrolase (25% positive after 2 days) and acid production from raffinose (69% positive after 2days). Thirty-four Enterobacter asburiae sp. nov. strains were tested for DNA relatedness by the hydroxyapatitemethod with 32P04-labeled DNA from the designated type strain (1497-78, ATCC 35953). The strains were 69to 100% related in 60°C reactions and 63 to 100% related in 75°C reactions. Divergence within relatedsequences was 0 to 2.5%. Relatedness of Enterobacter asburiae sp. nov. to 84 strains of members of theEnterobacteriaceae was 5 to 63%, with closest relatedness to strains of Enterobacter cloacae, Erwinia dissolvens,Enterobacter taylorae, Enterobacter agglomerans, Erwinia nimipressuralis, and Enterobacter gergoviae. Allstrains tested were susceptible to gentamicin and sulfadiazine, and most were susceptible to chloramphenicol,colistin, kanamycin, nalidixic acid, carbenicillin, and streptomycin. All strains were resistant to ampicillin,cephalothin, and penicillin, and most were resistant or moderately resistant to tetracycline. Enterobacterasburiae sp. nov. strains were isolated from a variety of human sources, most prevalent of which were urine(16 strains), respiratory sources (15 strains), stools (12 strains), wounds (11 strains), and blood (7 strains). Theclinical significance of Enterobacter asburiae is not known. As a result of this and previous studies, proposalsare made to transfer Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacterdissolvens comb. nov. and Enterobacter nimipressuralis comb. nov., respectively.
Enteric Group 17 was defined in 1978 as a group ofbiochemically similar strains isolated from a variety ofhuman sources (14). Strains of this group were received atthe Enteric Bacteriology Laboratory Section at the Centersfor Disease Control (CDC) beginning in 1973. Before thedesgination of Enteric Group 17, these strains had beenreported as unidentified or atypical Citrobacter or Entero-bacter strains (14). Several of these had been sent to CDCdescribed as resembling Yersinia species. We now show thatthese strains represent a single new species in the genusEnterobacter, which we herein describe and name Entero-bacter asburiae sp. nov.During the course of this study, we again confirmed the
fact that the species classified as Erwinia dissolvens andErwinia nimipressuralis were closely related to Enterobacterspecies both phenotypically and by DNA relatedness (25).We now propose that these species be transferred to thegenus Enterobacter as the new combinations Enterobacter
* Corresponding author.
dissolvens and Enterobacter nimipressuralis, respectively.These new combinations are used in the remainder of thispaper.
MATERIALS AND METHODS
Bacterial strains. There are 71 Enterobacter asburiae sp.nov. strains in the CDC collection (Table 1). Ail strains weremaintained in semisolid Trypticase soy agar (BBL Microbi-ology Systems, Cockeysville, Md.) at room temperature (18to 28°C). This medium contained Trypticase peptone, 15 g;Phytone peptone, 5 g; NaCI, 5 g; agar, 4 g; and distilled water,1,000 ml. Ail incubations were at 36 + 1°C unless otherwisenoted.Media and biochemical tests. Biochemical tests were done
on 68 Enterobacter asburiae sp. nov. strains at 36 ± 1°Cunless otherwise specified. Commercial media were usedwhenever possible. The biochemical tests were done by themethods of Edwards and Ewing (8) with some previouslydescribed modifications (19, 20).G+C content of DNA. The guanine-plus-cytosine (G+C)
4000-83 Conn. Hand wound 16-yr-old male4052-83 N. Mex. Throat 1-mo-old male, upper
respiratory tractinfection
0316-84 N. Y. Stool Male0713-84 Md. Sputum 71-yr-old male, pneu-
monia4500-84 N. C. Urine 60-yr-old female, uri-
nary tract infectiona All clinical specimens were from humans.b T, Type strain.
nale contents of Enterobacter asburiae sp. nov. DNAs and otherDNAs wçre determined spectrophotometrically by thermaldenaturation (21), with Escherichia coli B DNA included as
female, a control.
DNA hybridization. )NA hybridization was used to deter-femaie, di- mine relatedness of the 34 strains of Enterobacter asburiae
sp. nov. listed in Table 2. Unlabeled DNA was isolated and~tema e. purified as previously described (2). DNA from strain1497-78 (later designated the type strain of jnterobacterasburiae sp. nov., ATCC 35953) was labeled in vitro with32PO4 by nick translation by Ihe method of Rigby et al. (22)
Femqje, and as recommended in thé instructions furnished with adrah commercial nick translation reagent kit (number 8160, Be-
thesda Research Laboratories, Inc., Gaithersburg, Md.).The relatedness of labeled DNA from the type strain tounlabeled DNAs from 33 other Enterobacter asburiae sp.
Femalfetion nov. strains and to stock DNAs from 84 strains of Entero-bacteriaceae was determined by the hydroxyapatite methodas previously described (2).
Antimicrobial susceptibility tests. Antimicrobial suscepti-bility was determined on 17 Enterobacter asburiae sp. nov.strains by the disk method of Bauer et al. (1). The antimi-
Continued crobjal agents and concentrations used are listed in Table 3.
Providencia stuartii 0132-68, Moel- 5-9lerella wisconsensis 2896-78T,Xenorhabdus nematophilus 9012-80, Erwinia rubrifaciens ER 105,Erwinia tracheiphila ET 106a 32P04-labeled DNA from Enterobacter asburiae sp. nov. 1497-78 was
reacted with unlabeled DNA from the same strain (homologous reaction),from a series of other Enterobacter asburiae sp. nov. strains, and fromrepresentative strains of Enterobacteriaceae. Each reaction was done at leasttwice. Average reassociation in homologous reactions was 53% beforenormalization. Control reactions, in which labeled DNA was incubated in theabsence of unlabeled DNA, showed an average binding to hydroxyapatite of1.5%. The control value was substracted before normalization.
b Relative binding ratio = (percent DNA bound to hydroxyapatite inheterologous reactions)/(percent DNA bound to hydroxyapatite in homolo-gous reactions) x 100.
C Percent divergence was calculated on the assumption that a 1% decreasein thermal stability of a heterologous DNA duplex compared with that of thehomologous duplex was caused by 1% of the bases within the duplex thatwere unpaired. It was calculated to the nearest 0.5%.
RESULTS AND DISCUSSION
DNA hybridization. Labeled DNA from Enterobacterasburiae sp. nov. 1497-78 was 69 to 100% related to 33 otherEnterobacter asburiae sp. nov. strains in 60°C reactions (theaverage was 81%). Divergence in related sequences was 0 to2.5%, and relatedness remained high in 75°C reactions (63 to100%; the average was 80%) (Table 2). Ènterobacterasburiae sp. nov. was 5 to 63% related to other species ofEnterobacteriaceae (Table 2). Closest relatedness was toEnterobacter cloacae (63%), and 40 to 53% relatedness wasseen with Enterobacter dissolvens comb. nov., Enterobactertaylorae, Enterobacter agglomerans, Enterobacter
TABLE 3. Susceptibility of 17 Enterobacter asburiae sp. nov.strains to antimicrobial agents as determined by agar diffusion
Zone dian (rtim) % BacteriaRange Mean SD susceptible
nimipressuralis comb. nov., and Enterobacte> gergoviae.Enterobacter asburiae sp. nov. is, therefore, a single newspecies in the genus Enterobacter.G+C content. Four Enterobacter asburiae sp. nov. strains
had G+C contents of from 55 to 57 mol%, most similar tothose of Enterobacter cloacae, Enterobacter taylorae, andEnterobacter dissolvens comb. nov. (Table 4).Grimont et al. (personal communication) found 5 addi-
tional hybridization groups within strains considered to beEnterobacter cloacae or Enterobacter cloacae-like, one ofwhich was very similar to, if not identical to, Enterobacterasburiae sp. nov.
Description of Enterobacter asburiae sp. nov. Enterobacterasburiae sp. nbv. (as.ber'ry.i. N.L. gen. n. asburiae) isnamed in honor of Mary Alyce Fife-Asbury, an Americanbacteriologist who made many important contributions tothe classification of Enterobacteriaceae, particularly in de-scribing new Klebsiella and Salmonella serotypes (9, 10, 16),new genera, and new species (3, 11-13, 18). Strains ofEnterobacter asburiae sp. nov. are gram-negative, oxidase-negative, nonmotile, fermentative, nonpigmented rods withthe general characteristics of the family Enterobacteriaceaeand of the genus Enterobacter (Table 5). They gave positivereactions in tests for methyl red, citrate utilization (Simmons
TABLE 4. G+C content of Enteirobacter asburiae sp. nov.strains and strains of other enteric bacteria
Enterobacter gergoviae 604-77 58 ± 0.2Enterobacter (Erwinia) nimipressuralis 59 ± 0.6comb. nov. ATCC 9912Ta The values given represent the mean of from three to eight determina-
TABLE 5. Biochemical reactions of 6nov. strains and of th<
Test (no. of strains tested when <68)
IndoleMethyl redVoges-Proskauer, 360CCVoges-Proskauer, 220C (33)CCitrate (Simmons)H2S on triple sugar iron agarH2S on peptone iron agar (58)UreadPhenylalanineL-lysine (Moeller's)L-arginine (Moeller's)L-ormithine (Moeller's)Motility, 36°CMotility, 220C (35)Gelatin, 22°CGrowth in KCNMalonateD-GlucoseAcidGas
and Christensen's), urea hydrolysis (may be delayed; seefootnote to Table 5), L-ornithine décarboxylase (Moeller's),growth in KCN, hydrolysis of esculin, reduction of nitrate tonitrite, O-nitrophenyl-f3-D-galactopyranoside, acid and gasfrom the fermentation of D-glucose, and fermentation ofL-arabinose, cellobiose, D-galactose, glycerol (delayed), lac-tose (may be delayed), D-mannitol, D-mannose, a-methyl-
TABLE 5-ContinuedCumulative %bacteria posi- Reaction of
Test (no. of strains tested When <68) tive at (days)a: type strain,ATCC 35953b
a A blank space indicates that the test was not read at this time period.b Symbols: -, negative at the end of the appropriate incubation period; +,
positive at 24 h or at the time of the test. Numbers in parentheses indicate theday the test became positive.
C The percent positive varies considerably depending on the test conditions.At 22 or 37°C, after 2 or 4 days, 12% are positive in the O'Meara test. In the a-naphthol test, 79%o were positive in 2 days at 22 or 37°C; 82% were positive in4 days at 37°C, and 94% were positive in 4 days at 22'C.
d Most of the strains hydrolyzed urea with our method, but the AmericanType Culture Collection found five of five strains (American Type CultureCollection strain designations in Table 1) negative with their medium andmethod.
glucoside, salicin, D-sorbitol, sucrose, trehalose, and D-xylose. They gave negative reactions in tests for indoleproduction, in the Voges-Proskauer test (see footnote toTable 5), in tests for H2S production, phenylalaninedeaminase, L-lysine decarboxylase (Moeller's), gelatin hy-drolysis, malonate utilization, lipase (corn oil), DNase,tyrosine clearing, and fermentation of adonitol, D-arabitol,dulcitol, erythritol, i(myo)-inositol, melibiose, and L-rhamnose. Variable reactions were obtained in tests forL-arginine dihydrolase (Moeller's), acid from mucate, L-tartrate fermentation (Jordan's), acetate utilization, and fer-mentation of raffinose.A number of biogroups were identified among Enterobac-
ter asburiae sp. nov. strains. Strains in these biogroups gaveone or more of the following atypical test reactions: negativefor urea hydrolysis, ornithine, growth in KCN, gas fromglucose, fermentation of L-arabinose, D-mannitol, alpha-methyl-glucoside, raffinose, and D-xylose, and positive inthe Voges-Proskauer test, tests for motility, and tests forutilization of malonate and fermentation of melibiose andL-rhamnose.
Several Enterobacter asburiae sp. nov. strains were re-ceived at the CDC as suspected Yersinia or Citrobacterspecies. Tests that differentiate Enterobacter asburiae sp.nov. from species in these genera are shown in Table 6. Itspositive methyl red test and negative Voges-Proskauer testand tests for motility, utilization of malonate, and fermenta-tion of melibiose and L-rhamnose serve to differentiateEnterobacter asburiae sp. nov. from all named Enterobacterspecies (Table 5). Tests of differential value in separatingEnterobacter asburiae sp. nov. from species of Enterobac-teriaceae are described elsewhere (14, 15).
All Etiterobacter asburiae sp. nov. strains were suscepti-ble to gentamicin and sulfadiazine and resistant to ampicillin,cephalothin, and penicillin. Variable susceptibility was seento the other antimicrobial agents tested (Table 3).The sources of Enterobacter asburiae sp. nov. strains and
accompanying clinical information are given in Table 1. Thestrains came from 24 states, the District of Columbia, PuertoRico, and Canada. Where the sex of the patient was identi-fied, 27 of 50 isolates were from males. Where age was given,24% of strains were isolated from people aged O to 2 years,26% of strains were isolated from people aged 16 to 44 years,and 50% of strains were isolated from people aged 59 to 90years. Sixteen of the strains were from urine, 15 were fromrespiratory sources, 12 were from stools, 11 were from
a Actual percent positive reactions from Table 4 are given for Enterobacter asburiae sp. nov. For the other organisms (symbols): -, less than 10% positive; [-J,10 to 25% positive; V, 26 to 74% positive; [+], 75 to 89% positive; +, 90% or more positive; ND, not done. Ail results were from a 48-h incubation.
wounds or exudates, 7 were from blood, and 1 each wasfrom the endometrium, gall bladder, lochia, penis, peritonealfluid, synovial fluid, and environment. Enterobacterasburiae sp. nov. is widely distributed geographically in theUnited States and is frequently encountered, at least withrespect to the more recently described species of Enterobac-teriaceae (14, 15). Its presence in seven blood cultures(mentioned above) at least suggests clinical significance.When isolated from other sources, however, its clinicalsignificance is uncertain and requires further study.The type strain of Enterobacter asburiae sp. nov. is
1497-78 (=ATCC 35953). It was isolated from a lochiaexudate from a 22-year-old woman in R.I. Its biochemicalcharacteristics are those of the species as shown in Table 3.The G+C content of its DNA is 55 ± 1.1 mol%.
Proposal to transfer Erwinia dissolvens and Erwinianimipressuralis to the genus Enterobacter. Rosen (23) de-scribed an organism isolated from diseased corn as"Phytomonas dissolvens." This organism was subsequentlyplaced in the genus "Aerobacter" (now Enterobacter) byWaldee in 1945 (26). Burkholder (4) included it in the genusErwinia in the 6th edition (1948) of Bergey's Manual ofDeterminative Bacteriology. It was kept in the genusErwinia in the 7th edition (1957) of Bergey's Manual, al-though Burkholder (5) acknowledged that this was donepending further comparative studies to determine whether itshould be placed in the genus "Aerobacter" (Enterobacter).Dye (7) studied two strains designated as Erwinia dissolvens,concluding that the two were different biochemically andwere more like klebsiellae than erwiniae.
Steigerwalt et al. (25) characterized three strains desig-nated as Erwinia dissolvens both biochemically and by DNAhybridization. One of these strains, ED114, was shown to beof the genus Klebsiella. DNAs from the other strains, ATCC23373 and ED105, were 96% interrelated, with no divergenceevident in the related DNA sequences. In reciprocal DNAhybridization reactions, strains of Enterobacter cloacaewere 60 to 82% related to Erwinia dissolvens, but there was
8.5 to 10% divergence in the related sequences. The Erwiniadissolvens strains could not be phenotypically separatedfrom Enterobacter cloacae (25). Erwinia dissolvens is in theApproved Lists ofBacterial Names (24), and ATCC 23373 isits type strain. (The current American Type Culture Collec-tion catalog lists ATCC 23373 under Enterobacter cloacae,mentioning that it is the type strain of Erwinia dissolvens,and lists ATCC 9912 under Enterobacter cloacae, mention-ing that it is the type strain of Erwinia nimipressuralis.)
Further study is necessary to determine whether Erwiniadissolvens belongs in Enterobacter cloacae, but there is nodoubt that it should be classified in the genus Enterobacter.Therefore, we propose the transfer of Erwinia dissolvens tothe genus Enterobacter as Enterobacter dissolvens comb.nov. The biochemical reactions of Enterobacter dissolvensare as described previously (25). The type strain of Entero-bacter dissolvens is ATCC 23373.Erwinia nimipressuralis was isolated from elm trees with a
disease called wetwood (6). This organism is biochemicallysimilar to Enterobacter cloacae (7, 17, 25). It is negative intests for acid production from sucrose and raffii.ose, whereasEnterobacter cloacae is positive in these tests (25). Inreciprocal DNA hybridization reactions, Erwinia nimipres-suralis was 52 to 67% related to Enterobacter cloacae(divergence within related sequences, 11.5 to 12.5%) and 58to 63% related to Enterobacter dissolvens (divergence withinrelated sequences, 11.0 to 11.5%) (25). Erwinia nimipres-suralis is in the Approved Lists ofBacterial Names (24), andATCC 9912 is its type strain. There is considerable doubt asto whether this organism was the cause of wetwood andwhether it is a phytopathogen (7).The data cited above strongly suggest that this organism
should be a species closely related to, but separate from,Enterobacter cloacae. Therefore, we propose that Erwinianimipressuralis be transferred to the genus Enterobacter asEnterobacter nimipressuralis comb. nov. The biochemicalreactions of Enterobacter nimipressuralis are as describedpreviously (25). The type strain of Enterobacter nimipres-
suralis is ATCC 9912, which is referred to as Enterobactercloacae in the American Type Culture Collection catalog(see explanation concerning Erwinia dissolvens above).The documented strains of Enterobacter dissolvens and
Erwinia nimipressuralis were isolated from nonclinicalsources. We know of no clinical isolates. Because both ofthese species are very close to Enterobacter cloacae pheno-typically, it is quite possible that any clinical isolates wouldhave been reported as Enterobacter cloacae.
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