Classification and identification of Flavobacterium species by carbon source utilization

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0095-1137/87/071285-06$02.00/0

Classification and Identification of Flavobacterium

Species by

Carbon Source Utilization

D. RASOAMANANJARA,' B. KOROSEC,2AND H. MONTEILl*

Institute de Bactériologie de la Faculté de Médecine, Université Louis Pasteur, 67000Strasbourg,' and Centre de Calcul

du Centre Nationaldela Recherche

Scientifique,

67200

Strasbourg,7

France

Received2March1987/Accepted 8 April 1987

Carbon substrates used as the sole source of carbon and energy were tested for the classification and identification of species of Flavobacterium: Flavobacterium meningosepticum, F. breve, F. odoratum, F. multivorum, F. thalpophilum, and Flavobacterium sp. group IIb. Hierarchical classification and stepwise discriminant analysis revealed three F. meningosepticum, two F. breve, two F. odoratum, and two Flavobacteriumsp. group llbsubgroups. Glucose, histidine,asparagine,tryptophan,maltose,citric acid, and glycinewere selectedasthemostusefulsubstratestodifferentiatebetween thegroups andsubgroups.

The various species of the genus Flavobacterium have

been separatedintodistinctgroups onthe basis of DNA (3,

10), although few phenotypic features differentiate them absolutely (8). Our studywasintendedtodetermine theuse

ofvarious carbonsubstratesasthe solesourceofcarbon and

energy by Flavobacterium meningosepticum, F. breve,

Flavobacterium sp.groupIIb, F. odoratum,F.multivorum,

and F. thalpophilum. Theadvantages of such methods for the taxonomy and identification of members of the family

Enterobacteriaceae andsomemembersof the Vibrionaceae

wereconfirmed by Véron (15) and Véron and Le Minor (16, 17),who found that theuseprofiles enabled the classification

and, afterrestricting the number of substrates used, identi-fication of thespecies.

MATERIALS ANDMETHODS

Bacterial strains. Thefollowing strains weretested: 13 F. breve (A40838, A54615, B33982, B38674, B44444, B49835, B54566, B58375, B64246, B79158, B97415, NCTC 200/75, and NCTC 666/76), 59 F. meningosepticum (A49822,

A74007, A76407, B3332, B4167, B14430, B14441, B15859, B21632, B22418,B23643,B26345, B26724, B26972, B29828, B30560, B31688, B32446, B32472, B34145, B35810, B36981, B37907, B38594, B38611, B38744, B37566, B39452, B40182, B40193, B40470, B40471, B40705, B41047, B41312, B41667, B41693, B41972, B43337, B43831, B44667, B44827, B46136, B46356, B47181, B47466, B47812, B58347, B60339, B63822, B64709, B68800, B69139, NCTC 10016, NCTC 10585, NCTC 10586, NCTC 10587, NCTC 10588, and NCTC 10589), 21 F. odoratum (A16987, A46456, A66955, A67866, A75632, B65910, B77131,B78400, B83305, B87050, B90332, B91164, K1031, K1518, K1689, K1713, K1778, K1807,

K1830,K1899, andK1908),27Flavobacteriumsp.groupIIb (A28673, A70302, B1906, B9533, B10509, B12318, B15647, B20450, B23418, B24318, B32512, B37362, B46332, B46955, B48260, B49942, B54046,B56247, B57831, B69546, B77095, B77833, B82341, B91354,B92876, B94787, andF157),10 F.

multivorum (JKID, JKIJ, K1210, K1213, K1218, K1222,

K1231, LK3A, NCTC 11033, and NCTC 11034), 1 F. *Correspondingauthor.

thalpophilum (B86348), 9 Flavobacterium sp. (B38661, B40189,B50420, B50919, B52476, B59489, B90332, NK2C,

and P3), and6 strainsthat wecalled F. meningosepticum-like(A37574, A41986, A43935, A58537, B34262, andB47824)

because they belonged to F. meningosepticum serological groupsJ and L, whereas other members of theseserogroups were found to have affinities with F. breve or were not

identified (8). Strain designations beginning with K were received from M. J. Pickett. Those designations beginning

withNK, JK,and LK, from P. H. Hartman and others,were isolated at Strasbourg University Hospital from sputum, bronchial secretions, catheter tips, aspiration fluids,blood,

urine, ulcers, abcesses, and vaginal and anal swabs and identifiedby cultural and biochemical tests as specifiedby Richard and Monteil (14). All strains were kept in brain heart-glycerol-horse serum (10:1:1, vol/vol/vol) at -18°C.

The strainswere checked forpurityonblood agar.

Precau-tions were taken to avoid contamination of media by un-wanted growthsubstances. After the usual cleaning, glass-ware wassoakedfor 2days in sulfochromic acid and rinsed well intap waterand then indistilledwater.

Mineral medium. Themineral baseof Owens and Keddie (12) with the following compositionwasused (ingrams per

liter of distilled water):

K2HPO4

(0.52),

KH2PO4

(0.375),

(NH4)2SO4 (0.5),

CaCI2

(0.05), MgSO4. 7H20 (0.2), NaCI (0.1), FeCI3

.6H20

(0.01), MnSO4.

4H20

(0.002), and

Na2MoO4-2H20 (0.002),pH 6.8. ExceptforFeCI3 - 6H20,

whichwasasolid,eachcomponentof the mediumwasfrom concentratedstock solutions.The mineralbase washeated untilboiling, thencooled to room temperature and filtered through filter paper. Agar (2.4%, wt/vol; Pastagar, Institut PasteurProduction)wasthen addedto themineral medium andsterilized for 30 min at110°C.

Growth factor requirements. The growth factor

require-ments were determinedby using the mineral medium with

the following carbon sources (wt/vol): 0.1% glucose, 0.1% acetate,and 0.1%ethanol. Itwassupplementedwithvarious combinations of growth factors andwithyeastextractashes

as used by Grant and Pramer (5). Yeast extract (Difco

Laboratories) was dried to constant weightat 70°C, trans-ferred to clean evaporating dishes, and then

placed

in a

furnace, in which the temperature was

gradually

raised to

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1286 RASOAMANANJARA ET AL.

450°C. Sampleswereashedovernight. Different ashconcen-

of

total

rations were added to the mineralmedium.

Carbon substrates.

Filter-sterilized

carbon sources were

added to the basal medium cooled to 50°C, and 25 ml was 100

placedinpetridishes andcooled.Among the substrates used byVéron(15)whicharesolubleat roomtemperatureorafter

heating between 37 and 80°C were the following 35

çarbon

9

substrateswhichallowed growth for atleast one species: at 2

g/iiter,

L-alanine, L-arabinose, L-asparagine, L-arginine, L-cellobiose, L-cysteine, D-fructose, D-galactose,D-glucose,

glycine, L-glutamine, D-lactose, L-histidine, L-leucine, L-iy- '0 sine, D-maltose, D-mannose, L-ornithine, L-proline,

L-serine, starch,D-sucrose, D-trehalose,L-threonine, L-tryptO-phan, D-xylose; at 1g/liter, adipic acid, citric acid, ethanol, glycerol, isobutanol, methionine, meso-inositol, pyruvic acid; and at 0.25 g/liter, phenol. Cysteine did not allow

60

growth

ofany strain butwas considered a

negative

control

test.

Replicating

method.Each strainwas

suspended

indistilled

sterilewater(opticaldensity, 1.3 at 600nm), and all strains * 50

-were placed on the different agar media with a Steers U

replicator. -O

Interpretation of growth. Growth intensity was visually _

40-codedfrom0to5tobeasdiscriminatingaspossible. Growth a

was interpreted after2,4, 6, 8, and 14 days of incubation at

300C. . 30

Statistical

analysis.

(i) Hierarchical

classification.

Hierar-chical ascendant

clustering by aggregation

according

tothe. _.._._._._.. variance was

performed

on the whole

sample,

using

the î 20

usualeuclidiandistance between two strains ortwo classes to build classes as homogeneous as possible. The

within-classvariance defined the homogeneity oftheclasses.A new -_

vaoai

100 El E2 E3

FIG. 2. Dendrogram of hierarchical aggregation clustering of

90 group E (58 strains).

classwasformedateachstepbyaggregation ofthenearest

70 twoclasses, and the

difference

between the variance ofthe

new class and the sum of the variances of the two former

60 classeswascalledthe level of

clustering

(7).

The

hierarchical

s classificationis

represented by

a

dendrogram.

(ài) Stepwise discriminant analysis. Stepwise discriminant

analysis (1,4) enables the

selection

of the variables for which the linear combination leads to the best separation of the 40 s

classes

previously defined, using

the minimum number of variables. The stepwise

procedure

for selecting the variables

- ,

XOis

based on the ratio of the variation within theclass being

considered. This ratio iscalled the F ratio. At each step, the variable is selected for which the F ratio is maximized. At

Ï 20 stepzero,thevariable isselectedforwhichthe means of the

classes (as determined by variance analysis) are then most

10 1 m r different. This is the first discriminating variable. At each ~~~~~-<-~.. <

~

- - - followingstep, the F ratio iscomputed,taking thepreviously

selected variables into account. This

procedure

is

repeated

1- 2 1 2 1 2 1 2 until the F ratio is too small. A set of new coordinates,

,

,.

.I

,S , " ,

...

, expressed as a linearcombination of the selectedvariables

A B C D E (canonical variable axes), isdetermined. Each strain of the

FIG. 1. Dendrogram ofhierarchical aggregation clustering ofall studiedclasses was plotted on the first two canonical axes.

150strains. CAHVOR (Associationpour ledéveloppement des analyses

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TABLE 1. Reaction of groups and subgroups to carbon substrates used

Carbon- Reaction bysubgroupa

substrate

A,

A2 B1 B2 Cl C2 Di D2 E

L-Leucine d (35) +(100) d (46) d(63) +(100) +(100) +(100) +(100) +(100)

L-Lysine - (0) d (40) d (29) +(100) +(100) +(100) +(100) +(96) +(98)

DL-Valine - (7) +(80) +(100) +(82) + (90) +(100) +(91) +(100) +(100)

L-Alanine - (7) d (60) d (25) +(90) + (90) +(100) +(100) +(100) +(97)

L-Histidine d (28) + (80) -(0) -(0) d(70) +(90) +(95) +(96) +(90)

L-Tryptophan -(14) + (80) -(0) d(27) +(80) +(100) +(100) +(100) +(100) L-Methionine - (14) d (70) d (22) -(18) +(90) +(100) +(100) +(100) +(97)

L-Cysteine - (0) -(0) -(0) -(0) -(O) -(0) -(0) -(0) -(0)

Glycine -(14) d (40) d (29) d(27) d(30) -(20) +(92) +(96) +(96)

L-Serine - (14) d (40) -(0) d(71) +(80) +(90) +(100) +(90) +(88)

L-Asparagine - (0) +(100) +(81) +(100) +(100) +(100) +(100) +(100) +(100) L-Proline d (42) + (90) +(89) +(100) +(90) +(100) +(100) +(100) + +(100) L-Glutamine d (35) +(100) +(87) +(90) +(90) +(100) +(100) +(100) +(100) L-Threonine - (7) +(100) d (52) d (70) +(90) +(100) +(100) +(100) +(100) L-Arginine - (7) +(100) +(88) +(100) +(90) +(100) +(100) +(100) +(100) L-Ornithine - (14) +(80) +(100) d (70) +(90) +(100) +(100) +(100) +(100) D-Trehalose - (7) d (30) +(100) +(100) +(90) +(100) + +(100) + +(100) +(100)

Starch - (7) +(100) +(100) ++(100) +(90) +(100) ++(100) ++(100) +(100)

D-Fructose - (0) -(20) +(100) +(100) +(100) d(40) + +(100) +(100) +(100)

D-Lactose - (0) -(10) +(89) +(100) d (40) d(50) + +(100) +(96) +(82)

D-Galactose - (7) -(10) +(100) +(100) d (60) +(80) + +(100) + +(100) +(100) D-Sucrose - (14) -(0) +(82) d(27) d (50) +(100) + +(100) +(100) +(98) D-Mannose - (7) -(10) +(100) +(100) +(100) d(50) +(100) + +(100) +(100)

D-Maltose -(7) -(10) +(94) +(100) +(80) +(100) + +(100) +(100) +(100) L-Arabinose - (0) -(10) +(88) +(100) d (40) +(100) +(100) +(100) +(100)

D-Xylose - (0) -(10) +(84) +(100) d (40) +(90) +(100) +(100) +(100)

D-Glucose -(0) -(10) +(94) +(100) d (40) +(100) + +(100) + +(100) +(100) D-Cellobiose - (14) -(0) +(82) +(100) d (30) +(90) +(100) +(100) +(100)

Phenol - (0) -(10) d(52) +(100) d (20) d (70) +(100) +(100) +(98)

Ethanol - (0) -(0) +(88) +(90) d (10) d(40) + +(100) +(100) +(100)

meso-Inositol - (7) -(10) d(52) d(45) -(0) d (60) +(96) +(100) +(100)

Glycerol - (0) -(0) +(81) +(90) d (60) d (60) + +(100) +(100) + +(100)

Isobutanol - (0) -(0) d(29) d(63) -(10) d (40) + +(100) + +(100) +(100) Pyruvic acid - (14) -(30) +(84) +(90) d(70) +(90) + +(100) +(100) +(100)

Citric acid - (0) -(0) -(0) -(0) -(0) -(0) +(100) -(0) -

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aThesubgroupsconsisted of strains of thefollowingFlavobacteriumspecies:AI,12strains ofF.odoratum; A2,11strainsofF.odoratum;

B1,

15strains of

Flavobacterium sp. groupIIb;B2, 10 strains of Flavobacterium sp. groupIIb;Cl,8strainsofF.breve;C2,13 F.meningosepticum-like strains;Dl,15strains ofF.multivorum;D2, 7 strains ofF.meningosepticum; andE, 59 strains ofF.meningosepticum. Reactionswereclassifiednegative (-), positive (+),andstrongly positive(+ +); d indicates thatthestrainsgavedifferent results. The percentage ofpositivestrains(scores2 to5)isgiveninparentheses.The datain boldface typearefrom the substratesmostuseful indifferentiatingFlavobacteriumspecies.

de données, Institut

Statistique

des

Universités, Paris)

and BMDP 7M(University of

California,

Los

Angeles)

programs were used for hierarchical classification and

stepwise

discriminant

analysis.

They were run on an IBM 3081

MSV/XA computer at the Centre de Calcul du Centre National de la Recherche

Scientifique, Strasbourg.

RESULTS

Theaddition ofyeastextract ashes allowed

growth

ofall the strainson mineral medium

supplemented

with

glucose,

acetate,andethanol, whereasmostofthemdidnotgrow on this medium with vitaminsor

growth

factors.Theadditionof

amino acids was not needed. Thus, the final medium was composed of the mineral

medium,

yeast extract ashes

(1

g/liter of medium), agar, andcarbonsubstrate.

The hierarchicalclassification ofthe 150 strainsis shown on the

dendrogram

(Fig. 1). By

the naked eye, the

sample

was separated into five main

clusters, A,

B,

C, D,

and E.

Clusters A, B,

C,

and D were each divided into two subgroups. Hierarchical classification was

performed

on

cluster E,

dividing

it into three

subgroups

(Fig. 2).

The

subgroups were asfollows:

(i)

thetwo

subgroups

of cluster

A,

consisting mainly

ofF. odoratum

strains, (ii)

the two

subgroups

of cluster B,one made upofFlavobacterium sp. group IIb strains andtheother made upof Flavobacterium

sp. group IIb strains and 1 F.

meningosepticum strain,

(iii)

thetwo

subgroups

of cluster

C,

one

consisting

of9 F. breve strains and the other made up of4 F.

breve,

1 Flavobac-terium sp. group Il b, and the 5 F.

meningosepticum-like

strains,

(iv)

theone

subgroup

of clusterD

mainly

composed

ofF.multivorum strains and another

subgroup

composed

of

5 F.

meningosepticum

strains,

1 Flavobacterium sp. group

IIb,

and 1 F. breve

strain,

and

(v)

the three

subgroups

of clusterE,one

subgroup

composed

of sixFlavobacteriumsp. group

IIb,

1 F.

breve,

and1 F.

meningosepticum

strain,

one

composed

of16 F.

meningosepticum

and4 Flavobacterium

sp. group IIb

strains,

and another

composed

of 28 F.

meningosepticum

strains.

Stepwise

discriminant

analysis

was

performed

onthefive

main

clusters,

and

eight

variables

(lysine,

histidine,

galac-tose,

glucose, ethanol,

glycerol,

isobutanol,

and citric

acid)

wereselected asthemost

discriminating.

Theresultsofthe

canonicalvariable

analysis

areshown in

Fig.

3.

Otherdiscriminant

analyses

were

performed

between the

subgroups

of clustersAand

C,

the

subgroups

of C andE,the

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1288 RASOAMANANJARA ET AL.

strainsbelongingtoGLCsubgroupsC and F(onlyonestrain of the GLCsubgroup A strain), while subgroup A2 consisted of strains belonging to GLC subgroup A (only one strain of the GLC subgroup F). These two subgroups are quite

distinct(Fig.4a). However,wedo not knowif the

subgroups

A.

e,

s,~

o

Cononicul variable 1

4 s e'

FIG. 3. Plot ofall strains on first and second canonical axes.

(The ellipses encompass the strains constituting the groups; the centers of thegroups[0] areindicated.)

two subgroups ofD, and the three subgroups ofgroup E, followedby canonical variable analyses. Between clusters A and C, the most discriminating variables were lysine, ala-nine, asparagine, sucrose, maltose, and glucose. The most discriminating variables between clusters C and E were glucose, glycine, tryptophan, ethanol, meso-inositol, and

sucrose, and between subgroups D1 and D2, it was citric acid. The differentiation between the three subgroups of cluster E and between the two subgroups of cluster B requiredtoomanyvariablestobeworthwhile. Theresultsof the canonical variableanalyses areshown in Fig. 4.

Theresults oftestsonthe carbonsourceusedaregivenin

Table 1. These results were coded from 0 to 5 for the statistical analyses, which needed well-defined resultstobe finely discriminative. Thiswaspossible onlywhen the tests were done onthe samebasalmedium and were interpreted

by thesameoperator,i.e.,while the discriminationwasvery

subtle. However, the identification does not need such an acute interpretation, and, for more practical purposes, the results are expressed here as negative (code 0 and 1 for negative to weakly positive), positive (code 2 and 3), and strongly positive (code 4 and 5).

We selected glucose, histidine, asparagine, tryptophan, maltose, citric acid, and glycine asthe most discriminating variables for allgroupsandsubgroups, andanidentification

scheme isproposed in Fig. 5.

DISCUSSION

Ourstudy showed that no specific growth factor, except

for some mineral components contained in yeast extract,

was required for the growth of Flavobacterium species. Hierarchical ascendant clustering clearly separated the five species studied into five main clusters, whicharequite

distinct (Fig. 4). F. odoratum was split into two different subgroups; these findings agreed withbase composition and DNAreassociation studies (8), electrophoretic protein

pat-terns (R. J. Owen and P. J. H. Jackmann, Newsl.

Flavobacterium-Cytophaga Group, 3:10-12, 1983), and

gas-liquid chromatographic (GLC) studies (13) that clearly showed that there were at least two subgroups of F. odoratum. Our F. odoratum subgroup

A,

consisted of

.3.

.,.

3~

el

.3~

.-s

s.

a

9

-l -. -_ -i à i4li

la

.là 4 -4 -2 * * 4 i 1

Os

.le -i -. i i i* i i*

FIG. 4. Plot ofsome subgroups on first and second canonical axes. (a) Subgroups of A and C; (b) subgroups ofC and E; (c) subgroupsof D.

6

3

o

_6

4e

e

a

e

.2

o c

uIl

-s -4

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1m STRAIN

CLUCOSE

HISTIDINE ASUINE

CITRIC ACID

+1~-F.ultlvori GLYCINE

F.mlotlcm

Flavob lrm

Flavobcterlm

sp. group IIb

NALT0U F.Odort 1

F.brl F.odoratm 2

nlngoseptlcm-llken

FIG. 5. Identification scheme for Flavobacterium species.

revealed by our studies correspond to those found by the otherstudies, because of the lack of reference strains.

Group B was divided into two subgroups, but no

nutri-tionalfeature differentiated them.

Group C was divided into two subgroups, one with the

eight F. breve strains and the other with the five F. meningosepticum-like strains, oneFlavobacterium sp.,and

five F. breve strains, including the reference strain NCTC 666/76, although thetwoF. brevestrains NCTC 200/75 and 666/76 were found to be closely related by DNA-DNA hybridization (9). While GLC studies of Flavobacterium had separated thesetwostrains asdid carbon substrate studies,

ourprecedent hypothesis seemstobeconfirmed here (13); NCTC 666/76 might belong to another F. breve subgroup represented here by six strains. We first biochemically identified the F. meningosepticum-like strains as F.

meningosepticum becausethey had the biochemical charac-teristics of F.meningosepticum,especiallyarapidly positive O-nitrophenyl-B-D-galactopyranoside test (within 15 min) whichwasconsidered by Richard and Monteil (14) a

distin-guishing feature betweenF. breveand F.meningosepticum. The only test that distinguished them from the other F. meningosepticum strainswasslow,weak esculinhydrolysis

after48 h, whereasall the othershydrolyzed esculin strongly within24 h. In thisstudy, these strains differed fromF.breve strains by their growth in a minimal medium containing

glucose (Fig. 5).

GroupDwasdivided intotwosubgroups, onecontaining

all theF.multivorum andthe F.thalpophilumstrains andthe

othercontainingF. meningosepticum strains, including type strainNCTC 10016, whichhad beenconfirmedelsewhereas

atypical (11), and one F. breve strain which was probably misidentified. It was somewhat difficult to differentiate the

strains ofspecies F. meningosepticum and F. multivorum because all ofthem grew in most of the carbon

substrate

media, although F. meningosepticum generally grew less than F. multivorum, which is probably why some F. meningosepticum strains that grewwell wereagglomerated

with the F.multivorum strains. However, growth with citric

acid clearly distinguished F. multivorum from F. meningo-septicum, and thesesubgroupsare quite distinct (Fig. 4b).

The F. breveandF. meningosepticum strains included in

group E were probably misidentified. No nutritional or

serological

features

differentiatedthe three F. meningosep-ticumgroup Esubgroups,whereascanonicalanalysis clearly separated subgroup1fromsubgroups2and3

(Fig.

4b). GLC

analysis

revealed threeF. meningosepticum

subgroups,

but there was no concordance with the three subgroups found

here. Some Flavobacterium sp. group IIb strains were includedinthisgroup E,

probably

because oftheconfusion existingbetween thesetwo

species.

The many

discrepancies

betweenourresultsand those found inthenutritional studies

ofBruun (2)are probably due tothe addition ofCasamino

Acids and tryptophan in the media which gave results

different

from thoseobtained

using

ashesofyeastextract. We have beenable toattribute mostofthestrains used in this study to their

original

biochemical

species,

although

some of them were

split

into two or three

subgroups.

This

study confirms the

heterogeneity

within F.

odoratum,

Flavobacteriumsp. group

IIb,

andF.

meningosepticum

and revealsanintermediategroup betweenF.

meningosepticum

and F. breve (the F.

meningosepticum-like strains).

The

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1290 RASOAMANANJARA ET AL.

concordance with the classification obtained by GLC was especially marked for F. odoratum and also for the F. meningosepticum-like strains which were assigned by GLC

to F. breve, rather than to F. meningosepticum.

ACKNOWLEDGMENT

We are extremely grateful to Beatrice Lapeyrefor her technical help.

LITERATURE CITED

1. Bertier, P., and J. M. Bouroche. 1975. Analyse des données multidimensionnelles. Presses Universitaires de France S.A., Paris.

2. Bruun, B. 1983. Studies on a collection of strains of the genus Flavobacterium. 2. Nutritional studies. Acta Pathol. Microbiol. Immunol.Scand. Sect. B 91:35-41.

3. Callies, E., and W. Mannheim. 1978. Classification of the Flavobacterium-Cytophaga complex on the basis of respiratory quinones and fumarate respiration. Int. J. Syst. Bacteriol. 28:14-19.

4. Dixon, W. D. 1981. BMDP statistical software. University of California Press, Berkeley.

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