Enterobacter asburiae Foundin Clinical Specimens, Reassignment

JOURNAL OF CLINICAL MICROBIOLOGY, June 1986, p. 1114-1120 Vol. 23, No. 60095-1137/86/061114-07$02.00/0Copyright ©D 1986, American Society for Microbiology

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)

1114

on April 11, 2019 by guest

http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

ENTEROBACTER ASBURIAE SP. NOV. 1115

TABLE 1. Enterobacter asburiae sp. nov. strains studied

Strain no. Location of Source° Other clinicalsender information

0803-73 N. J. StoolNot givenBloodUrineStoolBloodBloodUrineBloodUrineNot givenSputumUrineSynovial fluidThroatStool

StoolPenisNot given

SputumFinger woundIndustrial washingmachine atmouse farm

Lochia exudate

1626-78 Maine Urine3089-78 Maine Urine

0235-79 D.C. Skin0791-79 Wash. Urine

1029-79 Mass. Skin

1058-79 N. Y. Urine1494-79 N. Y. Stool0565-80 Md. Sputum(=ATCC35954)

0570-80 N. Y. Urine1060-80 La. Wound1266-80 Oreg. Stool1388-80 N. Y. Urine1698-80 Conn. Foot wound2730-80 Calif. Urine

2738-80 Colo. Stool

0014-81 Mo. Gall bladder(=ATCC35955)

0379-81 N. Y. Ulcer exudate0380-81 N. Y. Finger wound0767-81 Aqz. Exudate

0793-81 Maine Sputum1073-81 N. Y. Sputum1174-81 Wis. Stool1501-81 Mass. Urine

2766-81 N. Y. Not given2767-81 N. Y. Sputum2905-81 Wash. Sputum2915-81 N. Y. Not given

Male

Female86-yr-old male1-yr-old45-yr-old75-yr-old female90-yr-old female61-yr-old maleFemale

76-yr-old male7P-yr-old male

MaleFemale with diarrheaand fever

26-yr-old male1-yr-old M'ale

72-yr-old-maleFemale

22-yr-old female,post-C-section in-fection

44-yr-old male61-yr-old male, uri-

nary tract infectionChild, boil65-yr-old male, uri-

nary tract infection68-yr-old female, cel-

lulitis on footFemaleFemaleMale

28-yr-old27-yr-old f4-mo-old n

Male85-yr-old f

cystitis<1-yr-old

arrhea69-yr-old f

cholycy!

71-yr-old fPenrose

Male

Male23-yr-old f

nary tra

Male

plale.emale

TABLE 1-Continued

Strain no. Location of Other clinicalsender information

2966-81 N. Y. Urine3002-81 N. Y. Hand wound9265-81 N. Y. Sputum9266-81 N. Y. Not given0128-82 Canada Blood, tracheal 8-mo-old male, non-

aspirate fatal sudden infantdeath syndrome

0358-82 Canada Urine 6-mo-old female0663-82 Calif. Stool Male0835-82 Maine Blood 75-yr-old female,

septicemia1545-82 Calif. Peritoneal fluid 17-yr-old male1812-82 Calif. Stool 19-mo-old male, diar-(=ATCC rhea; Campylo-35956) bacterfetus also

isolated1869-82 Va. Endometrium 31-yr-old female0017-83 P. R. Urine 85-yr-old male, pros-

tate cancer0115-83 Mass. Abscess 59-yr-old female,

small bowel resec-tion, colostomy

0333-83 Tex. Stool 6-mo-old female0570-83 Hawaii Blood Female, abdominal(=ATCC aortic aneurysm35957)

0882-83 Nev. Sputum 71-yr-old male,chronic obstruc-tive lung disease

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.

3794-730534-750888-751232-751442-751449-752562-752717-753213-753314-751825-761889-761987-762634-773568-77

3660-770156-780208-78

UnknownCalif.N. J.N. Y.Del.Del.Pa.Del.Va.Conn.Colo.Wash.Ala.Va.Wis.

Calif.R. I.Unknown

MaineConn.Maine

R. I.

0271-780682-780865-78

1497-78T(=ATCC35953)b

VOL. 23, 1986


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

1116 BRENNER ET AL.

TABLE 2. DNA relatedness of Enterobacter asburiaesp. nov. strains

Relatedness to 32PO4-labeledDNA from Enterobacterasburiae sp. nov. 1497-78

Source of unlabeled DNA Relative Relativebinding % bindingratio at Divergencec ratio atAnorb 7Jojrb

Enterobacter asburiae sp. nov.1497-78T

Enterobacter asburiae sp. nov.0380-81






























100

100

97

92

89

88

87

87

87

86

86

85

82

81

81

81

80

79

79

79

78

78

77

7AIo fi

Enterobacter agglomerans, 3123-70,75 0.5 83 Klebsiella oxytoca ATCC 13182'

Rahnella aquatilis 1327-79',75 76 Enterobacter agglomerans 5422-

69, Enterobacter agglomerans75 0.5 75 1645-71, Serratia ficaria 1165-77,

Edwardsiella tarda 3592-64,75 0.0 71 Erwinia mallotivora NCPPB

2851T, Erwinia quercina EQ 102,74 76 Ewingella americana 1468-78T,

Erwinia cypripedii EC 155,74 2.5 65 Erwinia salicis ES 102, Erwinia

nigrifluens EN 104, Enterobacter73 2.0 69 agglomerans 3482-71, Hafnia

alvei 5632-72

TABLE 2-ContinuedRelatedness to 32PO4-labeledDNA from Enterobacterasburiae sp. nov. 1497-78

Source of unlabeled DNA Relative Relativebinding % bindingratio at Divergencec ratio at600Cb 750Cb

72 2.0 65

70 1.5 66

69 1.5 63

63 5.0 5153 5.0 44

53 7.0 375148 9.0 3045 9.5 26

4342414039363636353535343434333333323131313030

25-29

8.510.0

10.5

2820

20222013221913201917181510171615il

18

20-24

{' 't Enterobacter asburiae sp. nov.0.0 100 1987-76

Enterobacter asburiae sp. nov.89 1449-75

Enterobacter asburiae sp. nov.0.0 100 1472-75

Enterobacter cloacae 1347-7185 Enterobacter (Erwinia) dissolvens

comb. nov. ATCC 23373T1.0 85 Enterobacter taylorae 2126-81T

Enterobacter taylorae 0992-7791 Enterobacter agglomerans 5378-71

Enterobacter (Erwinia) nimipressur-90 alis comb. nov. ATCC 9912T

Enterobacter taylorae 2338-8190 Enterobacter agglomerans 6003-71

Enterobacter gergoviae 0604-77T90 Enterobacter taylorae 0482-74

Klebsiella pneumoniae 275 Enterobacter agglomerans 0219-71

Escherichia vulneris 2898-7375 Shigella flexneri 24570

Enterobacter aerogenes 1627-6689 Escherichia hermannii 0980-72T

Salmonella typhimurium LT284 Enterobacter agglomerans 4388-71

Buttiauxella agrestris 1176-81T84 Klebsiella planticola 4245-72'

Enterobacter sakazakii 4562-70'0.0 81 Enterobacter agglomerans 1600-71

Kluyvera ascorbata 0408-7879 Koserella trabulsii 0329-73

Citrobacterfreundii 0406-6179 Cedecea davisae 3278-77T

Obesumbacterium proteus 4302-7482 Escherichia adecarboxylata 0435-78T

Escherichia blattae 9005-74T80 Enterobacter amnigenus 1325-79T,

78 Enterobacter intermedium 9011-82, Serratia marcescens 0868-57,81 Enterobacter agglomerans 1741-

71, Escherichia coli K-12,80 Enterobacter agglomerans 2780-

70, Enterobacter agglomerans78 1429-71, Erwinia rhapontici ER

106, Erwinia amylovora EA 178,Cedecea lapagei 0485-76T

J. CLIN. MICROBIOL.

Continued Continued


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


TABLE 2-ContinuedRelatedness to 32PO4-labeledDNA from Enterobacterasburiae sp. nov. 1497-78

Source of unlabeled DNA Relative Relativebinding % bindingratio at Divergencec ratio at60oCb 75OCb

Erwinia carotovora ATCC 495, 15-19Yersinia intermedia IP 48T,Yersinia enterocolitica 497-70,Enterobacter agglomerans 6070-69, Yersinia kristensenii IP. 1474,Morganella morganii ATCC25830T, Xenorhabdus luminescens9016-80'

Yersinia pestis 1122, Erwinia ananas 10-149570-82T, Erwinia uredovora 9574-82', Providencia rettgeri 1163',Yersinia ruckeri 4535-69, Yersiniafrederiksenii 867, Erwiniachrysanthemi SR-32, Erwiniastewartii 9573-82', Providenciarustigianii 2896-68, Tatumellaptyseos H36T, Erwiniacatnegieana EC 186, Yersiniapseudotuberculosis P62,Providencia alcalifaciens 3370-67,Xenorhabdus species 2 1426-81,Proteus mirabilis PR 14, Yersiniaaldovae 0669-83T, Erwiniaherbicola 9571-82T, Erwiniamilletiae 9572-82T

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

Ampicillin (10 ,ug) 6-12 8 2.7 0Carbenicillin (100 ,ug) 6-30 23 6.1 65Cephalothin (30 ,ug) 6-10 6 1.1 0Chloramphenicol (30 ug) 14-28 23 3.3 94Colistin (10 ,ug) 10-14 12 1.2 88Gentamicin (10 ,ug) 16-23 20 1.8 100Kanamycin (30 ,ug) 16-22 20 1.3 94Nalidixic acid (30 ,ug) 12-24 20 2.9 76Penicillin (10 U) 6-10 6 1.0 0Streptomycin (10 ,ug) 13-18 15 1.5 65Sulfadiazine (250 p.g) 20-27 23 1.9 100Tetracycline (30 p.g) 12-24 i8 3.1 41

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

Strain G+CM

Enterobacter asburiae 0271-78 55 ± 0.8Enterobacter asburiae 1497-78Tb 55 ± 1.1Enterobactér asburiae 0380-81 56 ± 0.7Enterobacter asburiae 0565-80 57 ± 0.4

Escherichia coli K-12 51 ± 0.6Enterobacter agglomerans 5378-71 51 ± 0.2Enterobacter cloacae 1347-71 54 ± 1.0Enterobacter taylorae 2126-81T 55 ± 0.4Enterobacter (Erwinia) dissolvens 55 ± 0.5comb nov. ATCC 23373T

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-

tions.b T, Type strain.

VOL. 23, 1986


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

1118 BRENNER ET AL.

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

Acid from:Adonitol (66)L-arabinoseD-arabitolCellobioseDulcitolErythritolD-galactose (33)Glyceroli(myo)-InositolLactoseMaltoseD-mannitol (66)D-mannose (64)Melibiose (66)a-CH3-D-glucoside (67)RaffinoseL-rhamnoseSalicinD-sotbitolSucroseTrehaloseD-xylose

Esculin hydrolysis (67)Acid from mucateTartrate (Jordan's)Acetate utilizationLipase (corn oil) (67)DNase, 25°C (63)NO3 -- NO2Oxidaseo-Nitrophenyl-3-D-galactopyranoside

(64)

58 Enterobacter asburiae sp.e type strainCumulative %bacteria posi-tive ai (days)a:

1 2 7

o1001212

94 99 1000 0 02 2 2

74 85 93o0 0 06 25 65

91 94 941 1 16 6 6O 0 1

75 96 961 3 12

100 100 10087 90 90

o o .099 99 990 0 0

99 100 1000 0 00 0 0

100 100 1000 4 900 0 12

66 75 100100 100 10097 97 97100 100 1009 9 987 94 9769 69 727 7 7

97 100 10099 100 100100 100 100100 100 10096 96 9682 94 9937 49 6818 26 2657 87 970 0 0

O O100o

98 98 100

Reaction oftype strain,ATCC 35953b

+(57

+

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

1 2 7

Citrate (Christensen's) (59) 86 95 98 +Tyrosine clearing (37) 0 0 0Yellow pigment, 25°C 0 0 0

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

J. CLIN. MICROBIOL.


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


TABLE 6. Characteristics for differentiating Enterobacter asburiae sp. nov. from Enterobacter, Citrobacter, and Yersinia species

Positive reaction in the following testa:Organism Methyl Voges- Ornithine Motility Growth in Malonate Melibiose a-CH3-D- L-rhamnose Sucrose

red Proskauer (Moeller's) (22°C) KCN glucoside

Enterobacter asburiae 100 9 94 6 96 1 9 94 7 100sp. nov.

Enterobacter aerogenes - + + + + [+] + + + +Enterobacter agglomerans V V - [+] V V V - [+] [+]Enterobacter amnigenus V + V [+] + + + V + VEnterobacter cloacae - + + + + V + [+] + +Enterobacter dissolvens - + V V + + + ND + +Enterobacter gergoviae - + + + - + + - + +Enterobacter intermedium + + + [+] V + + + + VEnterobacter nimipressuralis - + + + + + + ND +Enterobacter sakazakii [-] + + + + [-] + + + +Enterobacter taylorae [-] + + + + + - V + VCitrobacter amalonaticus + - + + + - - - + [-]Citrobacter diversus + - + + - + - V + [-]Citrobacter freundii + - [-] + + [-] V - + VYersinia aldovae + + + + - - - - +Yersinia enterocolitica + - + + - - - - - +Yersinia frederiksenii + - + + - - - - + +Yersinia intermedia + [-] + + - - [+] [+] + +Yersinia kristensenii + - + + - - - -

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-

VOL. 23, 1986


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

1120 BRENNER ET AL.

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.

LITERATURE CITED1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck.

1966. Antibiotic susceptibility testing by a standardized singledisk method. Am. J. Clin. Pathol. 45:493-496.

2. Brenner, D. J., A. C. McWhorter, J. K. Leete Knutson, andA. G. Steigerwalt. 1982. Escherichia vulneris: a new species ofEnterobacteriaceae associated with human wounds. J. Clin.Microbiol. 15:1133-1140.

3. Brenner, D. J., C. Richard, A. G. Steigerwalt, M. A. Asbury,and M. Mandel. 1980. Enterobacter gergoviae sp. nov.: a newspecies of Enterobacteriaceae found in clinical specimens andthe environment. Int. J. Syst. Bacteriol. 30:1-6.

4. Burkholder, W. H. 1948. Genus I. Erwinia Winslow et al., p.463-478. In R. S. Breed, E. G. D. Murray, and A. P. Hitchens(ed.), Bergey's manual of determinative microbiology, 6th ed.The Williams & Wilkins Co., Baltimore.

5. Burkholder, W. H. 1957. Genus VI. Erwinia Winslow et al.,1917, p. 349-359. In R. S. Breed, E. G. D. Murray, and N. R.Smith (ed.), Bergey's manual of determinative microbiology,7th ed. The Williams & Wilkins Co., Baltimore.

6. Carter, J. C. 1945. Wetwood of elms. 111. Nat. Hist. Surv. Bull.23:407-448.

7. Dye, D. W. 1969. A taxonomic study of the genus Erwinia. IV."Atypical" erwinias. N. Z. J. Sci. 12:833-839.

8. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneap-olis.

9. Edwards, P. R., and M. A. Fife. 1952. Capsular types ofKlebsiella. J. Infect. Dis. 91:92-104.

10. Edwards, P. R., and M. A. Fife. 1962. Eleven undescribedArizona serotypes isolated from man. Antonie van Leeu-wenhoek J. Microbiol. 28:402-404.

11. Ewing, W. H., B. R. Davis, and M. A. Fife. 1972. Biochemicalcharacterization of Serratia liquefaciens and Serratia rubidaea.Center for Disease Control, Atlanta.

12. Ewing, W. H., and M. A. Fife. 1972. Enterobacter agglomerans(Beijerinck) comb. nov. (the Herbicola-Lathyri bacteria). Int. J.Syst. Bacteriol. 22:4-11.

13. Farmer, J. J., III, M. A. Asbury, F. W. Hickman, D. J. Brenner,and the Enterobacteriaceae Study Group. 1980. Enterobactersakazakii: a new species of "Enterobacteriaceae" isolated fromclinical specimens. Int. J. Syst. Bacteriol. 30:569-584.

14. Farmer, J. J., III, B. R. Davis, F. W. Hickman-Brenner, A.McWhorter, G. P. Huntley-Carter, M. A. Asbury, C. Riddle,H. G. Wathen-Grady, C. Elias, G. R. Fanning, A. G.Steigerwalt, C. M. O'Hara, G. K. Morris, P. B. Smith, and D. J.Brenner. 1985. Biochemical identification of new species andbiogroups of Enterobacteriaceae isolated from clinical speci-mens. J. Clin. Microbiol. 21:46-76.

15. Farmer, J. J., III, G. R. Fanning, B. R. Davis, C. M. O'Hara, C.Riddle, F. W. Hickman-Brenner, M. A. Asbury, V. A. LoweryIII, and D. J. Brenner. 1985. Escherichiafergusonii and Entero-bacter taylorae, two new species of Enterobacteriaceae isolatedfrom clinical specimens. J. Clin. Microbiol. 21:77-81.

16. Fife, M. A., A. C. McWhorter, and P. R. Edwards. 1962. Tennew Arizona serotypes isolated from animals and animal foodproducts. Antonie van Leeuwenhoek J. Microbiol. 28:369-372.

17. Graham, D. C. 1964. Taxonomy of the soft rot coliform bacte-ria. Annu. Rev. Phytopathol. 2:13-42.

18. Grimont, P. A. D., F. Grimont, J. J. Farmer III, and M. A.Asbury. 1981. Cedecea davisae gen. nov., sp. nov. and Cedecealapagei sp. nov., new Enterobacteriaceae from clinical speci-mens. Int. J. Syst. Bacteriol. 31:317-326.

19. Hickman, F. W., and J. J. Farmer III. 1978. Salmonella typhi:identification antibiograms, serology, and bacteriophage typing.Am. J. Med. Technol. 44:1149-1159.

20. Hickman, F. W., J. J. Farmer III, A. G. Steigerwalt, and D. J.Brenner. 1980. Unusual groups of Morganella ("Proteus")morganii isolated from clinical specimens: lysine-positive andornithine-negative biogroups. J. Clin. Microbiol. 12:88-94.

21. Marmur, J., and P. Doty. 1962. Determination of the basecomposition of deoxyribonucleic acid from its thermal denatur-ation temperature. J. Mol. Biol. 5:109-118.

22. Rigby, P. W. J., M. Dieckmann, C. Roades, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

23. Rosen, H. R. 1922. The bacterial pathogen of corn stalk rot.Phytopathology 12:497-499.

24. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath. 1980.Approved lists of bacterial names. Int. J. Syst. Bacteriol.30:225-420.

25. Steigerwalt, A. G., G. R. Fanning, M. A. Fife-Asbury, and D. J.Brenner. 1976. DNA relatedness among species of Enterobacterand Serratia. Can. J. Microbiol. 22:121-137.

26. Waldee, E. L. 1945. Comparative studies of some peritrichousphytopathogenic bacteria. Iowa State Coll. J. Sci. 19:435-484.

J. CLIN. MICROBIOL.


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

Enterobacter asburiae Foundin Clinical Specimens, Reassignment

Documents

Transcript of Enterobacter asburiae Foundin Clinical Specimens, Reassignment