Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole cell fatty acid analysis

(1)

CopyrightC1991, AmericanSociety forMicrobiology

NOTES

Differentiation of Bacillus

anthracis from Bacillus

cereus

by

Gas

Chromatographic

Whole-Cell

Fatty

Acid

Analysis

DEANLAWRENCE, STEFAN HEITEFUSS, ANDHORST S. H. SEIFERT*

Institutefor Tropical Animal Health of theGeorg-August-University, Kellnerweg 6, 3400Gottingen, Federal

Republic of Germany

Received 14January1991/Accepted4April1991

Three strains of Bacillus anthracis andsevenstrains of Baciluscereusweregrown oncomplex medium and

on synthetic medium. Gaschromatographic analysis of whole-cell fatty acids of strains grown oncomplex

medium gavenearly identical fatty acidpatterns. Fatty acid patterns of strainsgrown onsyntheticmedium

showedahighcontentofbranched-chain

fatty

acids.Significant differences between the fattyacid patternsof thetwospecieswerefound. Odd iso/anteisofattyacidratioswereaboutequalinB.anthracisstrains, whereas

inB. cereusstrains the fractions of iso acidswereatleast twiceashighasthefractions of anteiso acids. The method described herein is used inourdiagnostic laboratorytohelp differentiate between thesetwospecies.

One of the specific tasks of the Institute for Tropical AnimalHealth is theidentification of specimens of bacterial pathogens. From tropical countrieswereceive fieldsamples

containing mostlyendospore-formingbacteria(Bacillus and Clostridium species), whichareidentifiedby gas chromato-graphic(GC) analysis of long-chain fatty acidsor

fermenta-tionproducts (5, 17).

Clostridium species are easily differentiated. Incontrast, Bacillusanthracis and B.cereusareverysimilarspecies that

differ inveryfew characteristics (4). Several methods have

been developed to obtain a good differentiation between

thesetwospecies (1, 2, 11, 12, 15,16, 18). However,noneof

thesemethods provides themeansforanabsolutelycorrect differentiation. We needaquick,simple, and reliable method

of differentiation to use in our epizoological studies of

anthraxdisease inlivestock andgameinAfrica.

The use of GC identification of bacteria is becoming

widespread in many diagnostic laboratories (14). The

method is similar to the method we developed for the

identification of Clostridium species (5). It was the aim of

this worktoestablishacomputerprogramfordifferentiation

of B. anthracis and B. cereus by GC whole-cell fatty acid

analysis.

Kaneda (7, 8) analyzed the fatty acids of two Bacillus thuringiensis strains and two pathogenic B. anthracis strains. He found that B. thuringiensis, likeB. cereus, has

higher relative proportions of i13:0, a13:0, and i14:0 acids thandoesB.anthracis. However, thiswasnotdemonstrated withavirulent strains.

Microorganisms.B.anthracis andB. cereusstrains used in

this study were obtained from the German Collection of

Microorganisms orwereisolates orreference strains of the

InstituteforTropical Animal Health. All strainsarelisted in

Table 1.

Culture media.Reinforcedclostridial medium(RCM)

con-tained the following ingredients (in grams per liter): meat

*Correspondingauthor.

extract, 10.0;peptone, 10.0;yeast extract,3.0; glucose, 5.0; starch, 1.0; NaCl, 5.0; sodiumacetate, 3.0; cysteine hydro-chloride, 0.5; agar,0.5 (all fromMerck, Darmstadt, Federal Republic ofGermany). The pHwasadjustedto6.8, andthe medium wasautoclaved for 15 minat 121°C.

Chemically defined RM mediumwasdeveloped by Leppla

(13) to increase yields of anthrax toxin. It contained the following ingredients at the indicated final concentrations (milligrams per liter), with all amino acids being of the L configuration: tryptophan, 35; glycine, 65; tyrosine, 144; lysine hydrochloride, 230; valine, 173; leucine, 230; isoleu-cine,170; threonine, 120; methionine, 73; aspartic acid, 184; sodiumglutamate, 612; proline, 43; histidinehydrochloride, 55; arginine hydrochloride, 125; phenylalanine, 125; serine, 235; NaCl, 2,920; KCl, 3,700; adenine sulfate, 2.1; uracil, 1.4; thiamine hydrochloride, 1.0; cysteine, 25; KH2PO4, 460; Tris, 9,060; glucose, 5,000; CaCl2-2H20, 7.4;

MgSO4. 7H20, 9.8;MnCl2 4H20, 1.0; NaHCO3, 8,000 (all fromSigma Chemie,Deisenhofen,FederalRepublic of Ger-many). For sterilization, the medium wasfiltered after the

pHwasadjustedto7.2; 50-ml sampleswereplaced in 100-ml

Erlenmeyer flasks.

Incubation. Each strainwasgrownfor 24 h in 5 ml of RM

mediumat37°C under aerobic conditions. Thiswasdoneto adapt field strainstoanewmedium.After 24hof growth, 5

mlof thesuspensionwaspipetted into 50 ml of the medium

and incubated for 48 h at37°C under aerobic conditions. Isolation offatty acids. After 48 h ofincubation, the cells

were harvested by centrifugation, washed with saline to

removethe medium, and thenlyophilized overnight inaBeta

Ilyophilizer(Heraeus-Christ, Osterode, Federal Republic of Germany). Lyophilized cells (approximately 100 mg [dry

weight])were saponified inacapped tube by adding1 mlof a solution containing 50% 7.5 M NaOH-50% methanol

(high-pressure liquid chromatography grade; Merck) and heating for 30 min at 100°C in a boiling-water bath. Fatty

acidsweremethylated by adding 2 ml ofasolution

contain-ing 55% 6 N HCI-45% methanol and then incubating for 10

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NOTES 1509

TABLE 1. Origins of Bacillus strainsa

Speciesand strain Source Originalno.

B. anthracis

Steme Wellcome Sterne

Zambia TAH Fieldstrain

18-74 TAH CN 18-74

B. cereus

1661 DSM DSM 487

1664 DSM DSM 2301

1688 DSM DSM 351

1689 DSM DSM 360

1690 DSM DSM 508

1691 DSM DSM 626

1692 DSM DSM 2299

TAH, Institute for Tropical Animal Health,

Gottingen,

FederalRepublic ofGermany; DSM, German Collection of Microorganisms, Braunschweig, Federal Republic of Germany; Wellcome, Wellcome Research Foundation Laboratories. B. anthracis Sterne is an avirulent strain that is used for vaccination studies at our institute. B. anthracis Zambia is an isolate from an elephant fromZambia,Africa.

min at80°C. The fatty acid methylesters wereextractedwith 1.25 mlof50%hexaneand50% diethyl ether (high-pressure liquid chromatography grade; Merck). The organic layerwas washed with3 ml of0.3 NNaOH. Approximately250,ul of the upper layer was placed in autosampler vials for GC analysis.

GC. GCanalysis of fatty acid methyl esters was carried out with a Perkin-Elmer gas chromatograph (Sigma 2000) withanautosampler(AS 8300) and aflameionization detec-tor on a PVMS-54 capillary column (length, 25 m; film thickness,0.32

Ftm;

innerdiameter,0.2mm)(Bodenseewerk Perkin-Elmer, Uberlingen, Federal Republic of Germany). Theflowrateof the

carrier

gas(N2)was3ml/min, the split ratiowas 100:1,and theinjection volumewas1 ,ul. The GC data were integrated and quantified as percent total peak area with a Perkin-Elmer LCI-100 laboratory

computing

integrator connected to an IBMpersonalcomputer. Whole-cellfatty acids from each strainweresaponified, methylated, andchromatographed atleast twotimes.

The temperature programoftheGCoven wasasfollows: the temperature was initially

100°C;

it was raised to 220°C with a ramp rate of

3°C/min,

raised to280°C with a ramp rate of30°C/min, and thenheld for3 min. The runtimewas45 min, and the temperature ofthe injector and detector was

350°C.

A commercial mix of27bacterial

fatty

acid

methyl

esters

(Supelco,

Bad

Homburg,

Federal

Republic

of Ger-many)wasusedas acalibration standard.

Components

that werenot in thecalibration standardweretentatively identi-fied by calculation of the

equivalent

chain

length

(ECL)

according to the following equation: ECLX =

[(RT,

-RTn)/(RTn+1

-

RTn)I

+ n, wherex isanunknown compo-nent,

RT,

istheretentiontimeofx,

RTn

is theretention time of

Cn:O

(methylesterbefore unknown

component),

RTn+1

is the retention time of

C(n+1):O

(methylester after unknown component), andnis thenumber of C atoms.

Calculation of similarities. Acomputer program written in "C" on an IBMpersonalcomputerwasusedtoevaluateGC data. The similarities between the fatty acid

profiles

ofthe strains were estimated by calculating the Bravais-Pearson coefficient of linear correlation(3) with the

following

equa-tion: r = {[N x £xy] - [(£x) x

(£y)]}/A[N

x2 -

(£x)2]

[N

£y2-(£y)2],

wherex andy are variables and Nis the numberof observations(components).xand y

correspond

to

0

0 20 40

Retention time (min)

FIG. 1. Chromatogramof whole-cellfattyacids fromB. anthra-cis Zambiagrownonsyntheticmedium.

pairs

ofpercentage

peak

areasfor each

peak

inturnina

pair

of

chromatograms.

An r value of +1 indicates

complete

positive

correlation of

analyses,

andan rvalueof0indicates

no correlation. Peaks of unknown

identity

were not taken into account because their total amount was never

higher

thanabout 1% of total

peak

area.

Fattyacidpatterns. Two

original

chromatograms

of strains grownon

synthetic

mediumareshown in

Fig.

1

(B.

anthracis

Zambia)

and

Fig.

2

(B.

cereus

1661).

The

fatty

acids that

weredetected in all examinedstrains arelisted inTable 2. All values are the means of at least two

analyses

oftwo

experiments.

The

fatty

acidpatternsofB.anthracis andB. cereus strains grownonRCM are alsolistedin Table 2. B. anthracis andB. cereus strains both

produced nearly

iden-tical

fatty

acid

patterns

when grown on

complex

medium. Themost

predominant

fatty

acids were hexadecanoic acid

(42.60

to63.52% of the

total)

andtetradecanoic acid

(8.87

to

21.06% of the

total).

Branched-chain

fatty

acids made up 11.17 to29.73% ofthe total.

The

fatty

acid distributions inB. anthracis andB. cereus

strainsgrownon

synthetic

mediumarealso

given

in Table 2. The contents of branched-chain

fatty

acids were much

higher (67.51

to

84.30%)

than those of strains grown on

complex

medium. The fractions of hexadecanoic and

tet-radecanoicacidsweremuchlower

(10.67

to20.36%and 1.90 to

3.72%, respectively).

Similarity

of strains. (i) Growthon

complex

medium.

Sim-ilarities of the

fatty

acid

profiles

of the strains grown on

complex

medium

(RCM)

were estimated

by

calculation of

VOL. 29,

1991

Ia %D 1.4

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0 U) vD v4 CX o 00o

0

Al4

a1I

l

1

II.

I

'I

FI

0

is ..

I

1Tt

-amw"

_II

I

a ..,

at

InF

20 CO co la 0 S 013 U, la C) ~0 *-u .S .0 U., CO C4 cql Q *4 la 'e .!2 ;i Ev

7ll

--40 eus

Retention time (min)

FIG. 2. Chromatogram of whole-cellfatty acids fromB. cer

1661 grownonsynthetic medium.

the coefficient of linear correlation. The lowest correlation coefficientbetween allstrains of thetwo

species

was0.946. Thismeanshigh similarityof thefattyacidprofiles. Differ-entiation isnot

possible.

(ii)

Growth

onsynthetic medium. Similarities of the

fatty

acid

profiles

of the strainsgrownon

synthetic

medium

(RM)

werealso estimatedbycalculation of the coefficient of linear correlation (Table 3). B. anthracis strains had correlation coefficients of at least 0.946, and the fatty acid

profiles

showed

high similarity.

Mostofthe

fatty

acid

profiles

ofthe B. cereus strains showed high similarity with correlation coefficients between0.969 and0.993;someof the correlation coefficients

(0.801

to

0.932)

indicated higher diversityof the fatty acid profiles. The correlation coefficients betweenB. anthracis and B. cereus were neverhigherthan 0.872.

Ratiosofbranched-chainfatty acids.Themost significant difference between the fatty acidprofilesof thetwospecies aretheratios of the odd iso and anteisofattyacids(Table 4).

B. anthracis strains had odd iso-branched-chain acid/ corresponding anteiso acid ratios between 0.64 and 1.03. Thisindicates that the fractions of iso acids were almost the sameasthe fractions of the anteiso acids.

In B. cereus strains the fractions of the iso acidswere at least twice as high as the fractions of the corresponding anteisoacids(ratiosof2.23to8.65).

Kaneda

(8)

found that thesynthesisofthefattyacidsi13:0, a13:0, and i14:0 in B.

thuringiensis

and B. cereus was

CO -S 'C CO Ct 4. 'C l) CO ci

-o

₀

0 rN"0 0 )6 Ca - )

C14 eao00 0R - 0~0 ~ 1. - NQO

U C 46ci140 l - ~Ci ~ci;61

1:4 7~~~~~~~-4~ R

Q 0% 00 00 00 00 Ul

U1

U C-00

arC 0%1 .r 0'r 'rC00 0%c7\m uf -Ct N- 00t\1~O0

W)0R\1000 N1- N 4\~0 'rC11 W) 0CD 0r-4

'-4 '-4

.-4C)r-4Cf-C)It1 CDS- aCD 00 ')r-4000')C

. rC - IC,4 0 N 0% -4t-4~0~0CC a0--4crC CD000D N (IND 'rCf0% )C)\ crC %0 CfC0 N0

r-l 'C W O %OS0'-4 q"~ NW n0

'-40

00C~

41C-Si CrC CrC CrC

'l

CrC CrC\

r-cnrC4N 0C-Soo0 0ar-4Cc 0 0 CNC)I -C~N)N

N ;C,- 4CS6000000 C-S 0% C-SIcr N~ Nl 0000

-4C-S C-SI

cr00 '-4IR~o\C 00 rC 0% 0%Nt-a' 000 C-SI1

a-0%O0ON00v)00~0) N- "t-00Nr rt-NNC'

cr It arC 000cC) CrC 0 n 00enrC 4N- CC-SI 0)N

-~C 4e;cC-S C-'ic ;C-4 C)V4 CrC -SN IC-Sel4 CrC 0 )C-S CrN Cr %NrC a- C-I t

'-4 0%CC C-S 0 0 0'-4It0%N C-SC-SI arC CrC 0It (0

C-SI rcl;C-SI el el;00 C-SI CO 4 - CC00 C-S 0

'-4 I

C-SC

0%t CD0N -4C-S enCtr 00'-4 crC-4 N- CC \CC CfC '-40C) 0tC000% '-4\CC '-4'-4 '-4 Cr arC Cr 0C C

00C (SC '-0 - rC rC Cr 4el;C 0 C-Si 000;

'-4 cC41 r-4C-S

C-SI 'C arC CDS0 crC CC arC 0%n 4Cl-SIS 00 N- C-SC lq 00Nl

00IR CrC NW) tt -4C-SI ON\O ON00 N-lc a-4%O tn N- Wr

'-4 t

C-S 10%CC C-S C-SI C-S O%N 0%ON CrCcn 0cN) C-SI enCrC C'-4c%a, 0= 0% N- \cOcrC N ON- %000'-4cCD'

-Cf)rC '-4

'-4C-SI Nt '40clir4C-0 C-SIC-S el;C-S C-SI 4'-400 '-4

r-4

'-40w00 0 ONNON%0000 * ot Cf-rC00'r

00 0~el 0%Cdr4ONt -00 tn000CD0- " 00C-SI cr05000C 00'-4 0 Itar;C% 000c

r- r-4 r-

7-'--4'-4 ON '-4 ON\C00% crC ON\05~o C 000 arC CCCren

00C-S SC rC elSON '- ON arC 'C%O N- cC W)0DIR N

Cf)'Ite48C)e CDa O~oC0 '40)C-S 00C-S

\C0,-N N-O 0CfCCrC 000 crCNcM r-4C-SIeC-S0 * 00irC 0%Cr)C-0 n -*crW'rC 0>O%N CrCen W)C 5050

0%~500550,R4 '= C-SI 00 N- a-,0C-y,N 00enC rC 00

N1 '-400000%)00%N0%-40050C-S 00 - 0f cCD

r- r-4 ,-4

r-- r--S-SIN 0%aN 00w -* NC-S NCC0% 00\0Not

0 41 00 C-I00 trC '-4CrC CrC0 CrCl -4000 C-SI

"NM .4-4tn o r,oo006

4W-~~~~r-~~~~

r-COa-4,4,-.-mrS.

4. -4r4

r4r-Y7

S_r.₀

C' r. a 42 0 0 a) cts CO a) A t -

liint

L

k

i

ll. c I I c

allm tnN 'IOtn tn0 ON tnmm IRt 'IttinaN

en

r-4(= allvi6 .6 C; .6 C'i 6 06 4vi 06 vi 6m6 C14P-4 V-4

40 14 a .. 14 I@ 0 .4 0 0 1-4 01 a 0 iz 14 a .4 C14 14 0 ..4 i I

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NOTES 1511 TABLE 3. Similarities of fatty acid profiles of B. anthracis and B. cereus strains grown on synthetic

medium-Linear correlation of fatty acid profile of:

Speciesand B.anthracis B.cereus

Sterne Zambia 18-74 1661 1664 1688 1689 1690 1691 1692

B. anthracis

Sterne 1.000 0.946 0.971 0.798 0.633 0.714 0.743 0.815 0.680 0.872

Sambia 1.000 0.977 0.683 0.527 0.701 0.738 0.753 0.714 0.783

18-74 1.000 0.748 0.569 0.704 0.728 0.785 0.681 0.836

B. cereus

1661 1.000 0.932 0.929 0.914 0.974 0.849 0.988

1664 1.000 0.904 0.864 0.934 0.801 0.902

1688 1.000 0.993 0.975 0.975 0.926

1689 1.000 0.969 0.986 0.918

1690 1.000 0.922 0.978

1691 1.000 0.854

1692 1.000

a Numbers in boldface type indicate correlation coefficients higher than 0.900.

different fromthat in B. anthracis. The difference was small but clear. These fatty acids added up to nearly 6% of the total fatty acidcontents of B. cereus but only made up about 0.1 to 2% of the total fatty acid content ofB. anthracis. However, this was only tested on one virulent strain, which cannotberepresentativeand could not beconfirmed byour results. We found that the contents of these three fatty acids were very similar in all tested strains.

According to Kaneda (7-10), the content of branched-chained fatty acids can be increased by adding branched-chainamino acidstothemediumasprecursorsforfatty acid synthesis. Forexample, the addition of isoleucine resultsin an increase of odd anteiso branched-chain fatty acids, and thecontentofeveniso acids is increased by adding valineto the medium.

Fatty

acid synthesis is catalyzed by two syn-thetases with differentspecifities toward thechain initiator. The differences in thefatty acidpatternsof thetwospecies might be explained by thedifferent substrate specificities of thefatty acidsynthetase complexes. Thiswillbetheobject of further investigations. The method described here is currently beingtested on further reference andfieldstrains and other species ofthe genus Bacillus, especially B. thur-ingiensis, which is also similar to B. cereus (4). Early investigations withone strain ofB. thuringiensisresulted in fatty acidpatterns similartothose ofB. cereus.

In mostdiagnostic laboratories working with GC methods, bacterial strains are usually grown on a standard complex

TABLE 4. Ratios of odd isoandanteisofatty acids of B.anthracis and B. cereus strains grown on synthetic medium

Species and i13:0/a13:0 i15:0/a15:0 i17:0/a17:0 strain

B. anthracis

Sterne 1.03 0.99 0.90

Sambia 0.78 0.80 0.74

18-74 0.85 0.95 0.64

B. cereus

1661 3.05 2.86 2.99

1664 8.07 6.23 8.65

1688 4.37 3.03 4.07

1689 3.07 2.28 3.51

1690 2.93 2.25 3.12

1691 3.90 2.35 4.45

1692 2.51 2.23 2.23

medium. It was shown in this report thatdifferentiation ofB. cereusfromB. anthracis is notpossible underthese condi-tions. However, growthon synthetic RM medium leads to small butsignificant differencesin the fatty acidpatternsof the two species. This characteristic might be valuable for differentiationof avirulentB.anthracisstrains; itmightalso helptoavoid animaltests forpathogenicity.

The computer programs used in this study were written by AndreasHeine, andthe bacteriological workwasassistedby Ute Sukop. Their contributions aregratefully acknowledged.

REFERENCES

1. Ascoli, A. 1911. Die Prazipitation bei Milzbrand. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1Orig. ReiheA 58:63-70.

2. Cherry, W. B., and E. M. Freeman. 1959. Staining bacterial smearswith fluorescentantibody.V.Therapid identificationof Bacillus anthracisinculture andin human andmurine tissues. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig.Reihe A 175:582-597.

3. Drucker, D. B. 1976.Gas-liquid chromatographic chemotaxon-omy. Methods Microbiol.9:51-125.

4. Gibson, T., and R. E. Gordon. 1984. Bacillus, p. 529-550. In H. D. Bergey, N. R. Krieg, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & WilkinsCo., Baltimore.

5. Heitefuss,S. 1991. Dissertation, UniversitatGottingen, Gottin-gen, FederalRepublic ofGermany.

6. Jensen,J.,and H.Kleemeyer.1953.Die bakterielle Differential-Diagnose des Anthrax mittels einesneuen spezifischenTestes ("Perlschnurtest"). Zentralbl. Bakteriol. Parasitenkd. Infek-tionskr.Hyg. Abt. 1 Orig.ReiheA159:494-500.

7. Kaneda,T. 1967. Fattyacids in the genusBacillus. I. Iso-and anteiso-fattyacidsascharacteristic constituentsoflipidsof10 species.J. Bacteriol. 93:894-903.

8. Kaneda,T.1968.Fatty acidsinthegenusBacillus.II.Similarity inthefatty acidcompositionof Bacillusthuringiensis, Bacillus anthracis,andBacilluscereus.J. Bacteriol. 95:2210-2216. 9. Kaneda, T. 1970. Factors affecting the relative ratio offatty

acidsinBacilluscereus. Can. J. Microbiol. 17:269-275. 10. Kaneda,T.1977. Fatty acids ofthe genusBacillus:anexample

of branched-chainpreference. Bacteriol. Rev.41:391-418. 11. Knisely,R. F. 1965. Differentialmedia fortheidentification of

Bacillus anthracis. J. Bacteriol. 90:1778-1783.

12. Knisely,R.F. 1966.Selectivemedium for Bacillus anthracis. J. Bacteriol. 92:784-786.

13. Leppla, S. 1986. Production and purification of anthrax toxin. MethodsEnzymol. 165:103-116.

VOL.29, 1991

on April 12, 2020 by guest

http://jcm.asm.org/

(5)

14. Leyrer, J.1987. Identifizierung vonBakterienmittels Gaschro-matographie. ForumMikrobiol. 9:341-343.

15. Logan, N. A., and R. C. W. Berkeley. 1984. Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130:1871-1882.

16. Schmid, P., J. Machleid, C. Witzmann, and R. Bohm. 1990. Spezifitat monoklonaler Antikorpergegenvegetative Zellenvon

B. anthracis. Tagungsbericht der Deutschen Veterinarmedi-zinischenGesellschaft, Marburg,Federal Republic ofGermany.

17. Seifert, H. S. H., H. Bohnel, J. Bunge, A. Ehbrecht, U. von

Kuehnheim, andH.Weck. 1979. EineneueMethodezur

Differ-enzierung pathogener Anaerobier: die statistischeAuswertung quantitativ erfal3ter Fettsauremustervon

Stoffwechselproduk-tender Erreger.Zentralbi. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1Orig. Reihe A 243:82-101.

18. Somerville, H. J., and M. L. Jones. 1972. DNA competition studies within the Bacillus cereus group of bacilli. J. Gen. Microbiol. 73:257-265.