Reliability of phenotypic tests for identification of Acinetobacter species

0095-1137/91/020277-06$02.00/0

Copyright © 1991, American

Society

forMicrobiology

Reliability of

Phenotypic

Tests

for Identification

of

Acinetobacter

Species

PETERGERNER-SMIDT,l* INGELA TJERNBERG,2 ANDJAN URSING2

Department of Diagnostic Bacteriology andAntibiotics, Statens Seruminstitut,

Artillerivej

5, DK-2300 Copenhagen S,

Denmark,'

and Department of Medical Microbiology, Malmo General Hospital, Universityof Lund,

S-214 01 Malmo, Sweden2

Received 30 March 1990/Accepted 15 November 1990

A numerical approach was used for identification of 198 Acinetobacter strains assigned to DNA groups

accordingtotheclassificationof Tjernberg and Ursing (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989).

The matrixusedwasconstructedfrom data publishedbyBouvet andGrimont(P. J.M. BouvetandP. A. D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986) and Bouvetand Jeanjean (P. J. M. Bouvet and S. Jeanjean,

Res. Microbiol. 140:291-299, 1989). The testschosen were those of the simplified identffication scheme for

Acinetobacter species devisedby Bouvet and Grimont (P. J. M. Bouvet and P. A. D. Grimont, Ann. Inst.

Pasteur/Microbiol. 138:569-578, 1987), namely, growth at 37, 41, and 44°C, oxidation of glucose, gelatin

hydrolysis, and assimilation of14 carbonsources.Ofthestrains tested, 181represented 12 DNAgroupsin the

matrix;ataprobability levelof .0.95,78% of themwerecorrectlyidentified, 2.2% weremisidentified, and

19.8%werenotidentified. Seventeenstrains representedtwoDNAgroupsnotincluded in thematrix; nine of

themwereincorrectly assignedtoaDNAgroupby thesephenotypic tests.Because of problems of separating strainsbelongingtoDNAgroups1, 2, 3, and 13 by using the phenotypictestsproposed by Bouvet and Grimont

(Ann. Inst. Pasteur/Microbiol.), we suggest that these groups should be referred to as theAcinetobacter

calcoaceticus-A. baumannii complex.

Organisms of thegenusAcinetobacterareubiquitous and

areoftenpresentin clinical specimens. The diversity of the

genus is reflected in the different phenotypic groups and

DNA homology groups that have been defined (1, 10).

However, because of insufficient criteria foridentification, thecustomarypracticehas been to regard allacinetobacters asmembers of asingle species, Acinetobacter calcoaceticus

(11). In the Approved Lists ofBacterial Names (17) an

additional name, A.

Iwoffii,

was included for

non-glucose-oxidizing strains.

UsingDNA-DNAhybridization, Bouvet and Grimont (2)

delineated 12 DNA groups ("genospecies") of

acinetobac-ters, of which all but two (DNA groups 8 and 9) could be

differentiated by using 28 phenotypic tests. Four new

spe-cies,A. baumannii, A. haemolyticus, A.johnsonii, and A.

junii,

were proposed, and the

descriptions

ofA. calcoace-ticus andA.

Iwoffii

were emended. Yet another species, A. radioresistens, was described in 1988 by Nishimura et al.

(15); itwaslater showntobe identicalto DNAgroup 12(20).

InaDNA

hybridization

study done byTjernbergandUrsing

(20) which included 198Acinetobacter strains, most of the

strains could be identified as members ofthe DNA groups

describedbyBouvetand

Grimont,

althoughthree new DNA

groups were described. Tjernberg and Ursing numbered their DNA groupsaccordingto the classification ofBouvet and

Grimont

(2), and the new groups were numbered 13

through 15; however,asthey couldnot

reproduce

the results

of Bouvet and Grimont concerning DNA groups 8 and 9,

they omittedDNAgroup 9 intheirsystem. Ina recentpaper,

Bouvet andJeanjean (4)reported five DNA groups

(which

theynamed groups 13through 17) of

proteolytic

Acinetobac-terstrains. Ofthesegroups, number 13

corresponds

toDNA

group 14ofTjernbergandUrsing (20). Whenwerefertothe

* Correspondingauthor.

DNAgroups of Bouvet and Jeanjean inthis paper, the group

number is preceded byB-J.

Inthe presentstudy, 198 Acinetobacter strains belonging

to 14 different DNA groups, mainly from clinical sources,

were investigated by using the simplified identification schemeofBouvet andGrimont (3). Aprobabilistic method

(13) for identification was used on the basis of a matrix

constructed from phenotypic data about Acinetobacter

strainsobtained byBouvet andGrimont(2) and Bouvet and

Jeanjean (4).

MATERIALS ANDMETHODS

Strains. Thestudy included198Acinetobacterstrains that were assigned to 14 DNA groups (Table 1). The majority

consisted of strains from clinical sources in Sweden,

Den-mark, and The Netherlands. The type andreference strains

wereobtainedfromtheculturecollections indicatedby their

designations. Of the strains, 119 were selected from a

previous DNA study of acinetobacters (20) and 37 were

selectedfromaprotein profile study ofacinetobacters(7).At least one strain of each DNA group has been investigated bothbyusandbyBouvet andcoworkers(2, 4).The strains were stored in glycerolbroth at-70°C.

DNArelatedness. Two

hybridization

methods were used.

Strains selected fromthe

previously

mentioned DNA

study

(20) were investigated

by

means of the

hydroxyapatite

methodofBrenner etal. (6)asmodifiedby Lind and

Ursing

(14).

Hybridization

was

performed

in 0.28 M

phosphate

bufferfor18hat25 to

30°C

below Tm

(thermal

denaturation

midpoint).

The

remaining

strains were

assigned

to DNA groupsby a

quantitative

bacterialdot filtermethod

(19).

In

this method,

hybridization

was

performed

in 2x SSC (lx

SSC is 0.15MNaCl

plus

0.015 Msodium

citrate)

for 24 hat

25°C below Tm. The same reference strains were used for

both methods, and the reference DNAs were labeled with

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TABLE 1. Listof Acinetobacter strains examined

Strains"

ATCC17902, ATCC23055Tb (A. calcoaceticus),42,b 59,b 132, 64,' 66,' 67,'68,' 74C(LMD 22.17)

ATCC 9955, ATCC 17904"bATCC17978"bCCUG 19096Tb (ATCC 19606T; A. baumannii),65b 1,b 107" 133"b 144b 147, 1,c13,C 18,c24,' 27,c 28,'c 29,' 60,' 77' (LMD 82.54), 78' (NCTC 7844), Ac 1141,d 189,e ab2444,eab2445,e

50853-82e

ATCC17922," ATCC 194,b40,b 41,b 55,b62b 79,b M,b128b 143b 162b 176" 204,b212,b 31,' 36,' 37,C 59,C61,'c 63'

ATCC17906Tb (A.haemolyticus),ATCC 17907,b ATCC 19002,b19, 32b 61" 81" 159" 188 197,b199,b 38,c 39,C 40,c

41,' 69,c 11,e106,e u119,e 204,e274e

ATCC 17908 (A.junii), 22", 23, 27, 53a," 74a, 74b,b 80, 96, 113:3, 117, 124, 127, 138b140,b 155a,b 177,b 178b

189, 46,c 53c

ATCC 17979" 39e

ATCC 17909Tb (A.johnsonii), ATCC 17923," ATCC 17946 b ATCC 17969b 68" 92" 97" 112" 134"b137"b 153"b209 b

4, 17, 51, 116,- 164, 200, 65219-84, 65321-84

ATCC 9957," ATCC 17910, ATCC 17968," ATCC 17987," NCTC5866 (A.Iwoffii),44,b

1101,b

122, 135", 145b202",

42,c 44,-45, 49,C 51, 54, 82, ulOO,e 201, 256,e 283, 284, 286, 65109-84, 86981-84

ATCC 17924" 113:2, 198"

CIP 63.46" (ATCC 11171), 51", 58b,b 174", 210"b73' (LMD 81.109),225w

FO-1Tb(IAM 13186T;A.radioresistens), SEIP 12.81, Ac 176Pmk49,f26, 50, 52, 73, 781, 125, 131, 148,

152, 157b 160, 163, 184, 187, 195, 266, 271, 70819-85

ATCC 17903b 89" 100, 165,b62,' 65,' Ac 2041,dAc2284,"Ac 2285d Ac2376,dAc2624,d Ac2627,d 353,e53893-82,e

53937bbe

ATCC 17905, , 10"l,114"

118,, 151a

aATCC,AmericanType CultureCollection,Rockville, Md.; CCUG,CultureCollection, UniversityofGoteborg,Goteborg, Sweden; CIP, Collectionde

l'Institut Pasteur, Paris,France; IAM,Culture Collection of the Institute ofAppliedMicrobiology, UniversityofTokyo, Tokyo,Japan;LMD,CultureCollection, Laboratorium voor Microbiologie, Delft, The Netherlands; NCTC, National Collection ofType Cultures, London, United Kingdom; SEIP, Service des

Enterobacteriesdel'Institut Pasteur, Paris,France;T, type strain.The reference strain for each DNAgroupused isunderlined. b Strains selected from those described in reference20.

' Strains received from L. Dijkshoorn (7). Strains received from L.Dijkshoorn. e Clinical isolates from Denmark.

fStrain received from M.M.Adam, NationalInstitute ofHygiene, Budapest, Hungary.

125I according to the method of Selin et al. (16). The hybridizationparameterAT,,,(differenceinthermal

denatur-ation midpoint between homologous and heterologous du-plexes) was used because it could be estimated by both

methods. The mean andrange ofAT,,, for the DNAgroups

areshown in Table 2.

Phenotypictests. Testsfor growthat 30(control), 37, 41,

and44°CwereperformedinBacto brain heart infusion(BHI)

broth (Difco) with a water bath. Tubes containing 5 ml of brothwereinoculatedwithadropofanovernightculture in

the same broth. Glucose oxidation was tested in Hugh and

Leifson's medium containing 1% glucose (open tube) (9).

Gelatin liquefaction was tested by the classical gelatin stab method(12). Testsfor hemolysisweredoneon5%sheep and human blood agar plates. The assimilation tests were

per-formed in a fluid medium containing a mineral base as

described by Stanier et al. (18) with the addition of an appropriate carbon(C)sourcein a0.1% (wt/vol) concentra-tion. Thefollowing C sources were used: DL-lactate,

DL-4-aminobutyrate, trans-aconitate, citrate, glutarate,aspartate, azelate, ,-alanine, L-histidine, D-malate, malonate, hista-mine, L-phenylalanine, and phenyl acetate. (Phenyl acetate

is the popular name of two different agents: acetic acid

phenyl ester and benzeneacetic acid. We used the phenyl ester.)Inaddition, strains ofDNAgroups1, 2,3, 10, 11,and

13 were tested for assimilation of levulinate, citraconate, 4-hydroxybenzoate, and L-tartrate; these tests were

sug-gestedinthebiotyping ofDNAgroup2 (3). Tubes 11mmin

diameterwith 3 ml of substrate were used. The pH of the solutions was 7.0.

Asuspension oftheteststrainwasmade bymixinga10-,ul

loopful ofa plate culture incubated overnight in 10 ml of 0.9% NaCl; 10

RI

of this suspension was added to the

assimilation media. Solid tube media were inoculated from the same suspension with a straight inoculation wire. The incubation temperaturewas30°Cfor all testsexceptthetest forgelatinaseproduction, which wasperformedat22°C. All reactionswerereadvisually after1and 2days of incubation.

TABLE 2. Hybridization data for 14 Acinetobacter DNA groups included in this study

DNAgroup (no.of AT,,, range, mean (no. of strains tested) by:

strainstested) Filtermethodb HA method'

1(10) 0.0-1.9, 1.2(5) 1.8-5.6,3.6(4)

2(25) 0.0-2.7, 1.0(18) 0.7-3.0, 1.7 (10) 3(20) 0.0-2.9, 1.6 (7) 0.5-4.0, 2.0(11)d 4(21) 0.0-1.4,0.7(10) 0.2-2.2,1.1 (13)d

5(21) 0.0-0.4,0.2(2) 0.0-3.4, 1.6(18)

6 (2) NDe 2.3

7(20) 1.4-2.9,2.1(8) 1.7-4.3,3.1 (11)

8(26) 0.0-2.9, 2.2 (15) 0.0-5.0, 1.9 (11)

10(3) ND 1.0-1.1, 1.1(2)

11(7) ND 2.6-4.2,3.4(6)

12(22) 0.1-0.2, 0.1 (3) 0.0-5.4, 1.9 (18) 13(15) 0.0-1.4,0.6(10) 1.3-2.8, 1.8 (5)

14(4) ND 0.6-4.4, 2.7(3)

15(2) ND 0.4

"ATm,Difference inthermal denaturationmidpoint betweenhomologous

andheterologous duplexes.

bQuantitative bacterial dot hybridization method(19). 'HA, Hydroxyapatite.

dFortwostrains(40 and 204) of DNAgroup3 and one strain(number159)

ofDNAgroup4,theAT, wasnotdone. Thesestrains wereidentifiedto the DNAgroup level byrelativebinding atoptimaland stringenttemperatures (20).

eND,Not done.

DNA group 1 2

3 4 S 6 7 8 10 11 12 13 14 15

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TABLE 3. Phenotypic characteristics of 14 Acinetobacter hybridization groups

% Positive strains in DNA group (no. of strains tested) Test

1(10) 2(25) 3 (20) 4 (21) 5 (21) 6(2) 7 (20) 8 (26) 10 (3) 11 (7) 12(22) 13 (15) 14(4) 15 (2) Growth in BHI broth at:

440C 0 100 0 0 0 0 0 0 0 0 0 73 0 0

410C 10 100 60 48 76 0 0 8 0 0 14 100 0 50

370C 80 100 100 100 100 50 0 77 100 0 100 100 25 100

Acid from glucose 100 96 100 76 0 100 0 19 100 0 5 100 100 50

Gelatinase 0 0 0 90 0 100 0 0 0 0 0 0 100 0

Hemolysis of:

Sheep blood 0 0 0 95 38 100 0 0 0 0 0 0 100 0

Humanblood 0 0 0 95 62 100 0 0 0 0 0 0 100 0

Growth in:

DL-Lactate 100 100 100 0 100 0 100 100 100 100 100 93 100 50

DL-4-Aminobutyrate 100 100 100 95 86 0 60 88 100 71 100 93 50 50

trans-Aconitate 100 100 95 76 0 0 0 4 33 14 0 67 25 0

Citrate 100 100 100 76 86 100 90 12 100 100 0 100 100 0

Glutarate 90 100 85 0 0 0 0 0 100 100 91 93 25 0

Aspartate 100 100 100 33 5 50 75 0 100 100 9 93 25 0

Azelate 100 100 90 5 14 0 50 100 100 100 95 93 25 100

P-Alanine 90 96 95 0 0 0 0 0 100 100 0 93 75 0

L-Histidine 100 100 100 100 90 100 0 0 100 100 0 93 100 0

D-Malate 90 100 100 100 100 50 95 58 100 100 9 93 100 100

Malonate 100 100 100 0 0 0 20 0 0 14 95 33 75 0

Histamine 0 0 0 0 0 0 0 0 67 86 0 0 25 0

L-Phenylalanine 100 80 65 0 0 0 0 0 0 0 91 93 100 0

Levulinate 60 40 0 ND" ND ND ND ND 0 0 ND 0 ND ND

Citraconate 30 52 5 ND ND ND ND ND 0 0 ND 0 ND ND

4-Hydroxybenzoate 100 96 95 ND ND ND ND ND 100 71 ND 93 ND ND

L-Tartrate 50 40 90 ND ND ND ND ND 0 0 ND 0 ND ND

a ND, Not determined.

In addition, the assimilation tests and gelatinase reactions wereread until 6 daysafter the beginning ofincubation. The growth tests were scoredas positive when the medium was turbid or when an abundant sediment was seen. Reactions with unclear results were repeated. Inoculated negative controls (pure mineral base without a C source) were not used in the assimilation study, except in the case of one strain (101, a member of DNA group 14) which was able to use all C sources. Thirty-one strains were retested in all reactions.

Computer identification of strains. Computer-assisted iden-tification was done by the methods described by Lapage et al. (13). The tests selected for the computer identification

were those from the simplified identification scheme for

acinetobacters (3). The matrix used was derived from the phenotypic data of Bouvet andGrimont(2)forDNAgroups 1 to 12and Bouvet and Jeanjean(4)forDNAgroups B-J 13 to B-J 17, with modification ofthe lower and upper limitsof

the reaction probability to 0.01 and 0.99, respectively. This

was done in order to compensate forerrors in the reaction matrixor the testresults. Forall strainsthe likelihood ofthe observed test results was calculated for each DNA group in the matrix by multiplication of the probabilities for the individual tests. These likelihoods were then normalized to

give the identification scoreby dividing the likelihoodfora

given DNA group by the sum of likelihoods for all DNA groups. Inaddition,themodallikelihood fraction

(8)

for each strain in each DNA group was calculated as the likelihood for the observed results for the test strain divided

by

the maximumlikelihood possibleforastrain in that DNAgroup.

A strain was

accepted

as identified when an identification

score .0.95

(a

score

giving

a

high

identification rate

com-bined with a low rate of

misidentification)

and a modal

likelihood fraction -0.0001

(corresponding

toless than two

maximal aberranttestresults in the

matrix)

wereobtained.

RESULTS

Table 3 shows thereaction

frequencies

of all DNA groups in this

study.

The resultsfor assimilation of

phenyl

acetate are not shown and are excluded fromfurther

calculations,

since all buttwostrainswereabletousethis

phenyl

esteras

aCsource.Itwasnecessarytoobserve the assimilationtests for 6

days,

as many strains were

positive

only

after

pro-longed

incubation. This was

especially

true for strains of

DNA groups

1,

4

through 12, 14,

and 15. Thetestsusedfor

the

biotyping

ofDNA group 2

(3)

and

hemolysis

patterns

are

alsolistedin Table

3,

although

thesetestswerenotincluded

in the numerical identification.

Thecomputeridentification results obtained

by

using

the matrixareshown in Table 4.Results forDNAgroups13 and 15

(DNA

groupsnotincluded in the

matrix)

are

presented

at the bottom of Table 4. A strain was considered to be

identified

(corresponding

toaDNA

group)

ata

probability

of

.0.95.

Of 181 strains

(strains

of DNA groups 13 and 15

excluded),

141

(78%)

were

correctly

identified,

4

(2.2%)

were

misidentified,

and 36

(19.8%)

were not identified. In DNA

groups

2, 6, 7,

8,

and

12,

morethan90% of the isolates were

correctly identified;

in DNAgroups

3,

4,

5, 10,

and11, 65to

76%were

correctly identified;

and in DNAgroups 1 and 14

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TABLE 4. Identification' of 198 Acinetobacter strains with known DNA groupaffiliationby usingamatrix constructed

ofphenotypical dataavailableelsewhere(2, 4) No.(%)ofstrains

DNA

groupb Correctly Misidentified' Not Total

identified identified

1 1(10) 1(group3) 8 10

2 25 (100) 0 25

3 13 (65) 7 20

4 16(76) 5 21

5 15(71) 6 21

6 2(100) 0 2

7 18 (90) 2 20

8 24(92) 2 26

10 2 (67) 1 3

11 5(71) 2 7

12 20 (91) 1 (group8) 1 22

14d 0 (0) 2(groupB-J 16) 2 4

Total 141 (78) 4 36 181

13e 5 (group2), 3 (group3) 7 15

ise 1 (group8) 1 2

aIdentification score, 20.95; modal likelihoodfraction,

.0.0001.

bGrouping system of Tjernberg andUrsing (20).

'Groups in parentheses arethe DNAgroupstowhich thestrainswere

assigned.

dDNAgroup13of BouvetandJeanjean (4).

eNotincluded in the matrix.

(B-J 13), 10% or fewer were correctly identified. If an

identification level of-0.80 wasselected,83% oftheisolates

were correctly identified, and six strains (3.3%)were

misi-dentified (Fig. 1). At higher levels of identification

(.0.99

and

.0.999),

thenumber of strainsidentifiedwas toolow to

be acceptable.

SinceDNAgroups 13and 15 were notrepresented inthe

matrix,strainsbelongingtothese groupsshouldideallyyield

a result of "not identified." However, 5 of 15 group 13

strains wereidentifiedas DNAgroup 2strains, and 3 strains

were identified asmembers of DNA group 3. Of two group

15strains,one wasassignedto DNA group 8. The rest of the

strains in these groups were not identified (Table 4). The

effect ontheoverall identification rate is shown in Fig. 1.

Strains identified (%)

0.8 0.9 0.95 0.99

Identification level(logscale)

Strains misidentified(7)

0.999

FIG. 1. Percentageofidentified(*)andmisidentified(O)

Acine-tobacter strains atdifferent identification levels. (aand b) Strains

belongingtoDNAgroups13 and 15areexcluded.(candd)Strains

belongingto DNAgroups 13 and 15are included.

DISCUSSION

When comparing the results of this study with those of Bouvetand Grimont (2, 3), it is essential to keep in mind that different collections of strains have been investigated, all of

ourstrains have been assigned toaDNA group (the identi-fication scheme of Bouvet and Grimont [2] is basedon 255

strains, of which 74 weregenotypically characterized), and

interlaboratorydifferences oftestresults could be expected

because of differences intestmethods and media.

Our resultswerereproducible. Only the day on which the

reactions turnedpositive varied. This is in accordance with

thefindings ofBouvet andGrimont (3).

In apilot study, wefound that the reactions forgrowthat

differenttemperatures weredependent on thetest medium.

When comparing BHI broth with serum broth (Statens

Seruminstitut, Copenhagen, Denmark), we found that

strains generally grew at ahigher temperature in the latter

broth. BHI broth was preferred because it gave better

discriminatory results (unpublished results). Bouvet and

Grimont (2, 3) and Bouvet and Jeanjean (4) used a

trypto-casein soy broth (Diagnostics Pasteur). Thus, differences betweenstudies in thetestresults forgrowth at 37, 41, and

44°Caretobeexpected.

The responsesfortheassimilationtests wereverysimilar forDNA groups1,2, 3,and 13(Table 3).Thissimilarityis in accordance with the observations that these four groups are

genotypically more closely related to each other than to

other DNA groups (20). Within this complex, the abilityto

grow at different temperatures

previously

has been

given

weight as adifferential test(2). Strains ofDNA group 1and

3 could,inourlaboratory,be separated only partially bythe

temperature tests and, in thebiotyping system,

by

the test

for assimilation of levulinate

(Table

3). All but one ofour

group 1 strains assimilated D-malate, which is at variance with the results ofBouvetandGrimont (2),who

regarded

a

negative D-malate test as animportant feature of DNA group

1. In DNA group 2, all strains were correctly identified. However, therewere nounequivocaltests separatingDNA group2from DNA group13, whichwas not recognized by Bouvetand Grimont (2). One-third ofthe group 13 isolates

were identifiedas members ofDNA group 2. In the

identi-fication scheme used for biotyping of group 2 (3), i.e.,

assimilation ofphenylalanine, levulinate, citraconate,

4-hy-droxybenzoate, and L-tartrate, all ofourclinical strains of

DNAgroup 13 would have the reaction pattern ofbiotype 9

(+-,

-, +, and -, respectively). Our group 13 reference strain(ATCC 17903)washighly atypical,asitgrewonlywith

citrateasthe sole C source.

Ina recent paperby Bouvetet al. (5), species, biotypes,

and phage types were determined for 120 Acinetobacter

strains and compared with cell envelope protein profiles.

Identification atthe species level was done by a simplified

identificationscheme (3).Thirty-sevenof these strains

(orig-inating from a study by Dijkshoorn et al. [7]) were also

included in the present study with the same strain

designa-tions. For nineof the strains, a lack of correlation between

species assignment and DNA group existed. Two DNA

group1strains (64 and 68),four DNA group 3 strains (31, 36, 59,and63), andtwoDNA group 13 strains (62 and 65) were identified as A. baumannii (DNA group 2), and one DNA group 1 strain (number 67) was identified as Acinetobacter

species3 (DNA group 3). Reassignment of these strains to

the proper DNA groupimproves the correlation of the cell

envelope protein pattern and the DNA group. This also

10

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C~~-'

8

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emphasizes the problems in separating DNA groups 1, 2, 3, and 13 by phenotypic tests.

Hemolytic strains were found in DNA groups 4, 5,6, and 14 (B-J 13), and, for all of these four groups except group5, hemolysis of human and sheep blood correlated with a positive gelatinase test. Of the strains in DNA group 5, considered nonhemolytic by Bouvet and Grimont (2), 38% hemolyzed sheep blood and 62% hemolyzed human blood, although none of these strains were gelatinase positive. This result has been discussed in a previous report (20). The four strains of DNA group 14 (B-J 13) were either misidentified to DNA group B-J 16 or not identified. This is a reflection of the phenotypic similarity of DNA groups B--J 13 to B-J 17 (4).

Strains of DNA groups 7 and 8 were correctly identified at levels of 90% and 92%, respectively. The inability of strains in DNA group 7 to grow at

37°C

was confirmed; however, similar strains also were found in several other DNA groups (1, 6, 8, 11, and 14 [B-J 13]). DNA group 15, a new DNA group describedby Tjernberg and Ursing (20), could not be separatedfrom DNA group 8 in the phenotypic tests (Table 3).

In the study of Bouvet and Grimont (2), only strains of DNA groups 10 and 11 were able to utilize histamine. These two groups were separated mainly by the results of the glucose oxidation-fermentation test. In our study none of the group 11 strains were able to grow at37°C, and one strain of group 14 (B-J 13) also utilized histamine.

Strains of DNA group 12 (A. radioresistens) wereeasy to recognize with the matrix derived from the data of Bouvet and coworkers (2, 4). The type strain, FO-1, however, was highly atypical and was misidentified. Nineteen of our 22 group 12 strains, including the type strain, FO-1, were not able to grow at

41°C.

The description of A. radioresistens (15) was based on three strains, all of which werereported to grow at

42°C.

The tests for identification of acinetobacters selected by Bouvet andGrimont (3) seemappropriate; for several DNA groups, with the exception of DNA groups 1 and 14 (B-J 13),

the majority of strains were correctly identified. When

strains of DNA groups not recognized by Bouvet and coworkers (2, 4) were identified, several were misidentified as members of groups in the matrix.

If the strains were identified with a matrix based on our own results, 78.9% could be correctly identified and 21.1% could not be identified by using the identificationcriteriathat were used for the matrix of Bouvet et al. (data not shown). Identification problems occurred mainly between DNA groups 1 and 3, 2 and 13, and 8 and 15.

The need for species identification of acinetobacters in routine clinical microbiology can be questioned. In noso-comial outbreaks, the simplified identification scheme (3) can, however, like protein profiles, be useful in typing strains without any reference to DNA groups or nomenspe-cies. When referring to strains of DNAgroups 1, 2, 3, and 13, it seems more appropriate at present to use the

expres-sion A. calcoaceticus-A. baumannii complex,andif there is a need for differentiation within this complex the biotyping system suggested for A. baumannii (3) maybe used.

ACKNOWLEDGMENT

We thank Lenie Dijkshoorn, Erasmus University, Rotterdam,

TheNetherlands, for the donationofsomeof thestrainsincludedin this study.

ADDENDUM

After

the completion of this study, we were notified by P. J. M. Bouvet, Pasteur Institute, that his group used benzeneacetic acid in their phenyl acetate assimilation reac-tions.Since we used the phenyl ester of acetic acid, this may explain the difference between our studies in the phenyl acetate reactions. Inclusion of benzeneacetic acid

assimila-tion in theidentification scheme may be helpful in

discrimi-nating strains ofDNA group 8fromstrains of DNA group 7

and strains in the A. calcoaceticus-A. baumannii complex

from strains ofDNA groups 10 and 11. REFERENCES

1. Baumann, P., M. Doudoroff, andR. Y. Stanier. 1968. A study of

the Moraxella group. II. Oxidative-negative species (genus Acinetobacter). J. Bacteriol. 95:1520-1541.

2. Bouvet, P. J. M., and P. A. D. Grimont.1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacterjohnsonii sp. nov., and Acinetobacter junii sp.

nov. and emended descriptions ofAcinetobacter calcoaceticus

andAcinetobacterIwoffii. Int. J. Syst. Bacteriol. 36:228-240.

3. Bouvet, P. J. M., and P. A.D. Grimont. 1987. Identification and

biotyping of clinical isolates ofAcinetobacter. Ann. Inst.

Pas-teur/Microbiol. 138:569-578.

4. Bouvet, P. J. M., and S. Jeanjean. 1989. Delineation ofnew proteolytic genomic species in the genusAcinetobacter. Res.

Microbiol. 140:291-299.

5. Bouvet, P. J. M., S.

Jeaijean,

J.-F. Vieu, and L. Dijkshoorn.

1990. Species, biotype, and bacteriophage typedeterminations compared with cell envelope protein profiles for typing

Acine-tobacter strains. J. Clin. Microbiol. 28:170-176.

6. Brenner, D. J., G. R.Fanning, A. V. Rake, and K. E. Johnson. 1969. Batch procedure for thermal elution ofDNA from

hy-droxyapatite. Anal. Biochem. 28:447-459.

7. Dijkshoorn, L., M. F. Michel, and J. E. Degener. 1987. Cell envelope protein profiles of Acinetobactercalcoaceticus strains isolated in hospitals. J. Med. Microbiol. 23:313-319.

8. Dybrowski, W., and D. A. Franklin.1968.Conditional probabil-ity and the identification of bacteria: a pilot study. J. Gen. Microbiol. 54:215-229.

9. Hugh, R., and E. Leifson. 1953. The taxonomic significance of

fermentative versus oxidative metabolismofcarbohydrates by various gram negative bacteria. J. Bacteriol. 66:24-26. 10. Johnson, J. L., R. S. Anderson, and E. J. Ordal. 1970. Nucleic

acid homologies amongoxidase-negative Moraxella species. J. Bacteriol. 101:568-573.

11. Juni, E. 1984. Genus III.Acinetobacter Brisou and Prevot1954,

727AL, p. 303-307. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore.

12. Koch, R. 1884. KonferenzzurErorterung derCholerafrageam 26. Juli 1884 inBerlin. Berliner Klin. Wochenschr. no. 31, 32, and 32a. [Reprinted in Gesammelte Werke von Robert Koch, Bd. II, 1. Teil, p. 20-60. Verlag von Georg Thieme, Leipzig, 1912.]

13. Lapage, S. P., S. Bascomb, W. R. Wilcox, and M. A. Curtis. 1973. Identification ofbacteria by computer: general aspects and perspectives. J. Gen. Microbiol. 77:273-290.

14. Lind, E., and J. Ursing. 1986. Clinicalstrains of Enterobacter agglomerans(synonyms:Erwinea herbicola,Erwineamilletiae) identified byDNA-DNA hybridization. Acta Pathol.Microbiol. Immunol. Scand. Sect. B 94:205-213.

15. Nishimura, Y., T. Ino, and H. Iizuka. 1988. Acinetobacter radioresistens sp. nov. isolated from cotton and soil. Int. J. Syst. Bacteriol. 38:209-211.

16. Selin, Y. M., B. Harich, andJ.L.Johnson.1983.Preparationof labelednucleicacids(nick translationandiodination)forDNA homology and rRNA hybridizationexperiments. Curr. Micro-biol. 8:127-132.

on April 12, 2020 by guest

http://jcm.asm.org/

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

18. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobicPseudomonads: ataxonomic study. J. Gen.Microbiol. 43:159-271.

19. Tjernberg, I., E. Lindh, and J. Ursing. 1989. A quantitative bacterial dot method for DNA-DNAhybridizationand its corre-lationtothehydroxyapatite method. Curr. Microbiol.18:77-81. 20. Tjernberg, I., andJ. Ursing. 1989. Clinical strains of Acineto-bacterclassified by DNA-DNAhybridization. APMIS 97:595-605.

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http://jcm.asm.org/