KEY WORDS: lrycobâcteria; r1c-Fatfy Acids; Oxidåtion pãtterns; Radlometric

Ree. Inst. Med. ttop. São Paalo 2g(7)t9-17, jøneirc-f everc¡rc, 1987

RADTOMETFTC STUDTES ON THE OX|DAT|ON OF (l-rC) FATW ACTDS BY DRUG.SUSCEPTIBLE AND DRUG.BESISTANT MYCOBACTERIA

Eilwâldo E. C/IIuáAGO, M.D., The¡ess M. KOPAffIC, GItflt¡ K. HOPKINS, N¡noy P. CANNON & Heùy N. WAGNEXI JR,, M.D.

ABSTRACT

A

radiometric assay system has been used

to

study oxidation pattems of

(1-14C) _{fatty acid.s by drug-susceptible and drug.resistant organisms}

_of

_the genus lì(ycobacteliüm.

TV,¡o strains

of

M. tuberculosis susceptible

to all

drugs, HrRv and Erdmân,

were used, Drug-resistan¿ organisms included

in

this invesftgation were M. tu.

berculosis tl3zRv resistant to 5 ug,/ml isoniazid, M, bovis, M. aviul4 M.

intra߀llu-Iarc, M. kansesü and M. chelonei. Tt¡e organisms were inosulated in sterile leac,

tion vials containing liquid ?Hg medium, 10% .ADC €nrichment and 1.0 uoi of one

of

the (114C) fatty acids (butyric, hexanoic, octanoig decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic), Vials v/ere incubated

at

3?C and the laooz envolved'veas measuæd daily for 3 days with a Bactec R'301 instrument.

Although each individual organism displayed a different pattern of fatty

oxi-dation, these pattems were not distinctive enough

for

identiJtcation of the

orga-nism. No combination

of fatly

acids nor preferential oxidation

of

long chain or

of

short châin

fatw

acids were able

to

separate susceptible from resistant

or-ganisms. ¡'ulther investigation with a laryer number

of

drug susceptible

myco-bacteria including assimilation studies and oxidation

of

other substætes may be

required

to

achieve a distinction between drug-susceptible and drug-resistant my-cobacteria.

KEY

WORDS: lrycobâcteria; r1c-Fatfy Acids; Oxidåtion pãtterns; Radlometric system; Drug-resistance.

INTBODIJCTION

fhe

idea

of

using carbon-l4 labeled fatw

acids as substrates to study Í¡ycobaterial

meta-bolism resulted from an incidental finding in one

of

the

experiments

with M.

lq¡ra€mu-rium s.

It

was observed

that

po\ßorbate 80

markedly enìanced

the

oxidation

of

(U-t4C) acetate by this organism.

It

was assumed il¡ât oleic acid, which is part of the chemical

struc-ture

of

polysorbate 80, was ¡esponsible for

increâsed oxidation rates

of

(UJ4C) acetate. An

investigation

of

the

oxidation rate

of

(llaC) oleic acid revealed a r4CO2 _ploduction

compå-rable

to

thât

of

(U-t4c) acetate and 1ed

to

a study

ol

the entire series

of

(lJaC) fatt¡¡ acids. Because some radiometric similarities had aheady been found with M. lelfiaemuriuIlr End

M. tuberculosis a, _{the study}

_of

_the (ltac) fatty

F¡om the Division of Nucleâr Meauc¡¡e, The Joh¡s Ifopki¡s Medlcal Instltutlons, Baltimorq MÊÌylaDd 212115, U.A.A.

Sùppofied in paÌt by USPHS Glarlt GM 10548.

¡ddF-ai for coEespoúdelrc€: Edfl¡aldo E. Cam.aryo, M.D., Centlo de Medicina Nucle3Ì ¿la Unive¡sialåde do São P¿ulo. CalJ€

CAMAEGO, E. E,; KOPÂJIIC, T. M.; I{OPKINS, c. K.; C¡¡ÍNON, N. p. & WAGNER JR., lr. N. _- Raauomet c stucties

on the oxia¡âtion of (1l C) far¿y âciats Þy dftg.sÙsceptibte and at¡ug-rcsrsta¡r oÌ.cobacte a. Rev. rnst. Med. rrol'.

São P¡ù¡o, 29(r):9-1?, 198?.

acid series was extended to include the latter. Pleliminary expedments showed differences in the oxidation Ëtes of fatty acids between the-se organisms and led to the hypothesis that

oxi-dation patterns

of

fatty acids might provide a ba,sis

for

differentiation of mycobacteria. For

example, a comparison of oxidation pattems of

M.

lepraæmuúum,

M.

bovis, M. _{Jtuberculosis} (It7Rv _and

_E!ùran)

_{showed sig¡ificant}

differ-ences6'7. .{lso, pr€limina.ry results with isonia-zid-¡esista"nt and isoniazid-susceptible strains of

M.

tuberculosis suggested th¿t oxidation

pat-terns

of fatty

acids and amino acids might help

to

identify susc€ptible and resistant oÍ-ganisms 8.

The present study descdbes a seties of ex,

periments

in

which oxidation

of

(1J4C) fatty

acids was used

in

an attempt

to

differentiate

susceptible and resistant strains

of

mycobac-teúa.

MATERI,ILS AND METTIODS

Preparatlon

of

Baailli: Tt¡e organisms obtain-ed from T¡udeau Mycobacterial Cultura

Collec-tion

(Saranac _Lake,

_NY.)

_included

_M.

úuber-culosis II37RV (TMC 102)

fully

susceptible; M.

tuberculosis H37Rv (TMC 303) lesistant

to

5

uglml isoniazid

(INH);

M,

tuber€ulosis Erd-man (TMC 107) ful(y susceptible; M. bovis (TMC 602) resistant to 5 ug/ml INH; M. ¿vium (TMC 706) ¡esistant to 5

ug,hl

INH, 10

ug,hl

INH,

10 ug,/ml para-amino salicylic

acid

(PAS), 2

uglml

streptomycin (SM),

5 uglml

viomysin

(vM), 5

ug,hl

kanamycin (ItM), 30 uglml

cy-close¡ine (CS),

5 uglml

ethionamide (ETA),

12 uglml ethambutol (EMB), 5 uglml rifampin

(RIF),50

ug/ml pyrazinamide (PZA),

suscep-tible to all others; M. intr¿ae ula,re (TMC 1403)

resistant

to

5

uglml

INH,

10

uglml

PAS, 2 uglml SM, 5 ug,/ml KM, 5 uglml VM, 30 ug/ml

CS,6

ug/ml

EMB,0.2

ug,/ml

RIF,50

ug,/ml PZA,. susceptible

to all

others;

M.

kansasil (TMC 1201) resistant to

I

ug/ml lNH, 2 uglml

SM, 6 uglml I'MB , 0.2 ue/ml F"IF, 25 :ulg/mtP?A,

susceptible

to all

others;

M.

chelonei (TMC 1542) resistant to

INII,

SM, P.Á.S, VM, CS, ETÀ, P"IF, pZA, susc€ptible

to

50

ug/ml

I(M.

All

organisms were received in 3

Írl

stedle serum vials containing 2 ml of b.acterial suspension in liquid ?Hg medium (BâltimoÌe Biologicd

La-boratories, BBL). These suspensions were im. mediately trânsferred

to

stedle 250

ml

eden-10

meyer tlashs

with

gauze plugs, containing 50

ml of

liquid ?II9 medium ,lvith 107¿ âlbumin-dextrose.catalase (ADC,

BBL)

enrichment and 0.05% polysobate 80. Tlle organisms were incu-bated at 3?"C

for

6

to

lt

days and then homo-genized

with

a

Soryal Omnimixer (8.5 speed

scale) twice

for

30 seconds. The numbet

of

bacteda s'as estimated using MacFarlând

ba-lium

sulfate standards 2. _{The final suspensiol}

was further diluted

with

sterile ?Hg medium

to yield from

1x

108 to 6 x 108 bacteriâ per ml,

Part of this suspension was again diluted 1:10

with

?Hg medium, divided

into

2

ml

aliquots dispensed

in

ste

le

plastic tubes and stored at -?tlc

for

future en)eriments.

Exporimental

Media:

Liquíd

?Hg

medium

\tith

lOVa ADC enrÍchment and no polysorbate \Ã'as used with all the organisrns tested. Reaction System: T'he reastion system

fol

de-tection

of

14CO2 _consisted

of

_0.8

_ml

_medium

in a

5.0

ml

multidose sterile gl¿ss vial with

0.1

ml

of bactedal suspension and 0.1

ml

(1.0

uci)

of

r'ro.substrate.

_The

_following (1-r4C)

fatty acids (Amersham Corporation) were used

with all organisms: butyric, hexanoic, octanoic,

decanoic,

lauric, myristic,

palmitic, stearicn oleic, linoleic, and linolenic. In preliminary ex-periments e, (1-14C)

fatty

_{acids were dissolved}

in

ethyl alcohol

or

methyl alcohol (lauric). A comparison

oI

14Cq output from alcohol dis-solved and bovine sen¡m albumin (BSA)

-

ca-talase complex dissolved (IJaC) fâtty acid de-monstrated a better oxidation rate when using the

latter

(Fig. 1).

In

the present study, aU

uci

of

(l-iac)

fatw

acid u¡eÎe dissolved

in

1.0

fatty

acids were prepared as follorás:

a)

250

uci of

(lrac) iatty

acid were dissolved in.1.0, ml

of

0.01 N KOH;

b)

1.0

ml of

a SOE|

solÍ-tion of BSA in lvrodds solution was added; c)

0-05

ml

of

0.1

N

HCI were added and pH

checked betv¿een 6.5 and ?.0; d) the solutionwas

Jiltercd through a 0.22

u

pore size memblane

filter

(Milliþore Corporation);

e)

0.25

ml

of stedle catalase (50,000

-

59,000 units/ml) were a.dded;

f)

sterile vrater was added to bring the

final

solution

to

desiled volume. AII speciflc activities were lowered to 25 mCi/rDM by

addi-tion of the sorresponding non-ladio¿ctive fatty

acid.

,As indicatorc

of

met¿bolic activity

of

the

organisms, l4c-formate

(25 moi/mM)

and

a

:

t

c¿.[L{RGo, E. E.; KoPAIuc, T. M.; EOPKINS, G. K.; C¡ìNON, N. P. & .WAGNER _{J8,., E. N.}

- Raitroúet¡tc stu¿tt€s

cn tho oxi¡låLion of (1-1{C) _fatiy åcids hy drug'susceplible and ùug-resistanl mycobacte a. Rev. Ist. Med. trop. Sio Paulo. 29(I):9'l?. 198?.

(U-laC) acetate (25 mCVmM) \Ã'ere used ló. Âll vials were prepaled at least

in

triplicate. Con.

trol

vials were prepared

in

the same way, but with autocloved bacte a added. Each

experl-ment $'as repeated at least, twice.

Radionetric Measuremenf:

All

vials were in-cubated at 37"C.,{n ion chamber device (Bactec

_î

R-301, Johnston Laboratories, CocJ<eysville,

!

Md.) 'was used

to

measure bactefal metabo- n

tism.

The vials were sampled daily for 3 days. -=

Results \áere obtained

as

"index

units"

(100 õ

units

₌

25 nanocuries

of

1aC _{activity). Mean} g

and standared devidation

of

the

cumulative _E

laCO, production

for

each substrate over

the

p.

entile e&erimental period wele calculated. Re.

g

sults were expressed as percent lauric acid

oxi-

9

dation.

_¡

=

SúertlitJ¡ îesting: Sterility tests were perform- õ

ed on positive samples and consisted

of

sub-culture on chocolate-agar plates and

radiome-tric

sterility testing \Ã'ith (U-l4C) glucose 10,11,

RESULTS

Figure 1 shows a comparison of raoq

out-put from

alcohol dissolved and BsA-catala.se

complex dissolved (1-r4C)

fatty

acids

with

M. ûrb€rculosis H3?Rv fully susceptible (TMC 102)

Higher ¡aCO2 _{production rates were obtained}

when the

fatty

acids

ï'ere

dissolved

in

BS.A-catalase complex. Similar results were found

with

M.

avftrm and M. tub€rcrdosis II3rRv

re-sistant

to

5 ug/ml

INH

(TMC 303).

When exposed

to

the entire

(1JC)

fatty

acids series, M. tuberculosis H3zRv fully

suscep-tible (TMC 102) sho'.ved highest oxtdation mtes

with

octanoic, decanoic, lauric,

a¡d my

stic

âcids.

Low

oxidation rates v'ere found with butyric, hexanoic, linoleic and oleic acids (Fig.

2, Table

I).

M. tuberculosis Erdman sho\ryed

preferen-tial

oxidation

with

hexanoic, lauric, palnitic

and linoleic acids. Intemediate oxidation rates

occurred Ìt/ith decanoic, myristic, stearic and

oleic a,cids. Lower oxid¿tion rates were found

with butyric, octânoic and linolenic acids. (Fig. 2, Taþle

I).

M. tub€rculosts II37RV resistant to 5 uglml

INII

(TMC 303) showed

two

distinct sets of

consecutive

fatty

acids

v¡ith

high oddation

MEfaBolrst or (t-lt)arn ncros ly M.lubdculo.ìÉ H-Rv ls ì

Fig. 1 _- Iniluence of BsA-cåtatâse complex on the oxida,

tion lates of (l.lro) fatty acrds by M. ü¡bê¡cùrosis H¡rRv

tu]ly susceptible to drugs (TMC 102) in ?Ilg ûedium.

rates: octanolc, decanoic, lauric, and myristic acids; stearic, oleic and linoleic acids.

Inter-mediate oxidation rates occr¡lred with butyric,

hexanoic, palmitic and linolenic acids (Fig. 2,

Tabþ

f).

Except

for

but¡rric, hexanoic and linolenic acids, high oxid.ation rates were found

for

all

other fatty a.cids with M. bov¡s (¡Ig. 2, Table

I),

especially decânoic and octa¡oic.

M. ka,lsasti shoeíred intermediate oxidation ¡ates with palmitic, oleic, linoleic and linolenic

acids.

Iligh

oxiaf.ation rates were found \r¡ith aU other fatw aclds, especially myristic (Fig. 3, Table

I).

Ifexanoic acid was

not

tested v¡ith this o¡ganism,

I

contrast,

lú,

chelorei showed low oxida"

CAMARGO, E. E.; KOPÁÆIC, T. M,; HOPKINS, G. K.; CANNON, N, P, & WAGNER, JR., I{. N. _ RAdiOEEiliC SIU'IIEI

on the oxialqtion of (l-1ro) fatty sctdls by ¿kug.susceptible and drug-æsist€nt ßycobacte¡la. Ree. Irst. M€d. trolÈ

SÍ¡o Påulo, 29(1):9-1?, 198?.

rAB],E

I

Oxidation sttem.s of (r.11C) _fatty aci¿ls by ¡nJcoùacteri¿'

E .e ã 9_¿ ã

*

EÉ

çTS

É

5E

5{t

Ë8 É5

É

p

5È

t243 ! 193 7243 t l8

180¡ f 315

1?86 + 350 r?51 + 298

1821 + 158

2049 È 36S

l5?8 É 210

2294 t 280 1?51 * 88

1138 + 158

1693 È 158

609 ! 90 1918 + 248 1580 f 203 2257 t 406 813 + 90

2t44 + 35+ 1625 r l8l

l1l5 r 226 2A92 t A

1151 t t3

1529 È 202

NT

2315 ! 202 1619 I 180

2?A8 r 135 ?8? + 90 2360 ! 247

u76 ! 180

2210 ! 202 2765 ). 562 152Ð + 180

1477 :r l4l ¡fI 1398 + 141

1118 + t88

15'11 + 204

t?44 + 15?

1021 + ?9

t650 I 189

880 È 204

91t + 94 927 t 283 669 :t 173" ?96 t 133 8?9 È 5? 228 I 41

626 + 151 2541 ! tA6 993 :! 191 249 ! 21

1941 :! 23? 1008 È 265 1815 i 96 1265 f 83

2330 I 216 1592 + 212 2139 i '16 $24 i 394

2t57 ! 2t6 2653 1 133 1910 r ra4 103? t ?3

2049 :t ¡51 1645 ! 239 1986 * 26? 1193 i 93

105? a 129 2334 ! 186 1261 I 134 1213 ! 145

1553 r 86 1565 + 159 l??6 t: tît 123+ r:218 ?98 i 86 r?5t È 106 196? I 210 913 + t35 669 f 86 2945 i 239 2¡01 f 191 12?6 t ?r

lr00 + 43 1141 i E0 l0?0 ! 153 570 + 104

Butyric flexenoic Octanoic Decanoic I¿urlc ¡ls¡istio Paknttic SteÀrtc Ol€ic I,i¡olelc Linolenic

' cumulativs _'{cq Foduction over 3 dsys ss met¿bollc tndex units .. _{Mean + stanaL$d deYfÊülon}

¡It = not tested

S = Susceptible to 3ll drugs (TMC 102)

R = Resistant to 5 úg/rÞ1 isoniâztd (TMC 303)

orùúr d h,Éclh!,¡êÈ.

br ! r!¡¡E¡¡¡r. Hsrir(.) _{ùÈbo,rÍÍclF.q¡.ú,}

ùü.,h d !,úclf¡d,^cB.

I

il

rr

[l

ll

Éllnll¡ill

nllrlllllllllllllln

"*Li.Li.lJ".l-.l.Ll_.Li".lJ,.Ll"Ll*l-.1."-u-.

Å'

¡lltldr

Il||t

n

, I ||Èll,

n

ü ü

LlilllU

_ll

lll

tlll

-l

L

I

11

-

l

1

_-l¡

_{l ||i| -| lt}

rlll¡rl

_'ritl

fr

llilllrllrl

_I

lll

Èt

-ruuuLlLl!|Lr!uLr

o,r. ." d h-ncrF.,,,¡. d,

br !,r!.ffi H3r¡r ri,

¡'ig. 2 _- Pattem of (1.LC) _{fatty acid} oxldation by M. lúberculostl H¡1Rv futly s$ceptible to cllugF (uppe! ¡€lt), M. tu. ùorculosls EftkE¡,Ã Ouppe¡ rtght), M, tubercDrodE .IIâ|BV resistãnt to 5 ugl¡il lsonla¿id (loÌsotr ¡eft), and ¡ú, bort€

(loPe¡ right). åll organiws wero te$ed l¡r liq1lld ?Eg medfiaE with l0% ÁDC er¡¡lchEent.

CÁMARGO, E. E.; KOP.{XTIC, T. M.; HOEKTNS, G. K.: CANNON, N, P, & WAGNER JR,., H. N. _ RådiOInEIliC STUd¡E6

oD ths oxitÞtion of (l.1ac) fatty act¿s by d¡ug'susceptihle snd drug-rcsistânt mycoþacl€lia. Aev. Inst. Med. trop.

São Pauìo. 29(l):9'U, 1987.

ú[,üxI

Å

il

ilil

uu

was chosen to elimiD¿te poorly oxidized subs.

trates.

Table

fI

shows

the

results

of

this

approach.

!'ive converdent substrates ryere chosen in Table

ff,"!r'ith

the pu4)ose

of

differentlating

the o¡ganisms used in tlds study. Ttris simpler

approach included buwric, myristic, oleic,

li-nolenic and hexanoic (Table

III).

Ho\¡rever, as can be observed in Table

II, it

was not possi-ble to separate M, tuberc.ulosts H3?Rv TMC 303

from

llL

bov¡s.

.A,s expected, t{CO2 production was not ob-seNed in the control vlals and all sterility tests

were negative.

M. aviuln oxidized ihe entire fatty acid

se-ries very avidlJ, particularly oleic and palmitic (Fig. 3, Tâble

I).

In

order to stmplify the results presented

in

Table

I

and figurcs 2 and 3, ¿n arbitrary threshold set

sl >

50Eo lauric acid oxidation

ù'.'b.,FdÉ!,h0.

o,hù" d _l,.rcl F.,e k,d.

DISCUSSION

Fatty aeid synthesizing en4¡me actidty h8s

been found

in

extracts

of

severâl m¡¡cobate-rià,73,18,?.7,24,Æ,n. T?rese studles have shoÌgD

that a

vadety

of

long chain

fatty

a.cids are

formed, from 12 to 26 carbons, by addition of two carbons to the acceptor fatty acld's

carbo-ryl

end. Acetate, malonate and NADII are

es-sential

for

these reactlons. Ttrere

is

âlso evi. dence that M, úub€¡sulosÍs accumulates lipids durlng eqronentiel grcwtb

r, with a

qmthesis

of

rrycolic aßids that psrallels the grc

rth

ra.

te3. Some

of

the short and long chain fâtty

aÆlds \Ãre¡e preferabty inco4)orated into trigly.

Linoleic acid was the best substrate with M, intracellrdare. High oxid-ation rates v¡ere

also found

with

octanoiq lauric, pÈlmitic and oleic. Intermediate rates occuûed with butyric, decanoic, stealic and linolenic. t\ilyristic was poorly oxidlzed and hexanoic \¡/as not tested

with this organism (Fig. 3, Table

I).

à!.'h dft'É4¿t¡"r'

.r¡,¡rl

db,b. d L.nclrf,,¡.H.

iltl

ltilt

UL]l]

ú¡,lt

ilill]il

t]tlut]

Fig. _{3 -} Pattem of (liao) fatty âcld oxidabloD by M. k8!åsit (uppêr l€ft), M. ctelonel (upp€r dght), M. lDtrac€Ift¡.

la¡e (Iower reft), arl.l M. ¿vtùñ (lowe¡ ¡¡ght). All organi$r¡s were tested in liquid ?IIg mechur¡ with l0% 4ÐC e ichm€nt.

rl

ntn

CÄ¡trAEGO, E. E.; KOPÂJTIC, r. M-; HOPKINS, G. K-; CÁNNON, N P. & WAGNEE JR., I1. N. _- Radiometnc $udie6

on tho oxialetion of (1.úC) f¿tiy acia¡s l¡y d¡ug.susceptible and al¡ug'¡esistânt mycobacte a. Rev. IBt. M€al. túIL Sõo P¿ùto, 29(1):9-r?, $8?.

T.{Bf,EII

(t.t{) lzlty acials with sicBiJicani oxrdâtion retes

0 0 + + + + 0 + 0 0 0

+ = > 50% acid oxrdation

0 _{= <} $7¿ laúlc oxidafion

S: &rscep¿ible to all d¡ugs (TMC 102)

& ₌ Resistanl ro 5 ug/Eil isoDiazid (TMc 303)

TABI,E III

OlidÂtion patteFs of selected (F¡C) fatty âctcls by

mycoþacteriå øt

332É

? ? ?

;E

A

Èå

Èe

åË

Ë

Ë

Ë

å

Ë

Þ! =5 E: E E > E

E

Buby¡ic My¡istic Oleic Linotênic qeÎânoic

(H3zRv) ând M. bovis

fißt

Ínüeased s¡ith the carbon châin length

to a

maximum çi'it}I de-canoic and then decreasedlz. On the other hand,

KONDO and I<.A.NAI 17, also studied the

bac-tericital effect

of

fatty

acids and found that myristic

acid

acid

had

tlle

strongest lethel

effect on M. tuberculosis H37Rv arid on M. bo.

vis: longer and shorter chain fatty acids were less toxic than

myristic.

Such toxic effects or

a

possible inhibitory effect on bacterial enzy-mes involved

in

lipogenesis

þ

probabll¡ would

not

occur

with

the small amounts

of

fatw

acids used

in

ou¡ exlreriments (approximately

4

x

10-8M per vial). ltrelefore, one could only speculate Ìvhether

or

not the eD4¡me systems

of

the organisms included

in

our expe ments are seDsitive enough to recognize potentially to-xic substances and either transform them into non-toúc triglycerides

or

excrete them as by-products.

Long chain fatty acids have been sho¡e¿n to have a stimulatory effect on oxygen uptahe by

M, tuberculosis, -á. sLnllar mechanism could be present

in

other mycobacteria and would

ex-plain the various oxidation rates observed in

our

expeliments.

ft

is

also conceivable that the mâgnitude

of

the stimulatory effect vari€s

from

one organism

to

another

for

the same

Iatty acid. Ttrerefore, the preferential oxidation

of one or more substrates over the others by

a particular mycobacteria could be due to the toxic nature of the substrate, to the stimul¿tory effect on oxygen uptâke,

to

a combination

ol

both or because the subst¡ate,

for

some

tea-son,

is

pa

icularly

suitable

for

the

energy needs

of

the organism,

Assimilation studies \Ã'ith M. lepraDn ¡rium

and M. tubolculosts H37Rv fully susceptible to

drugs 6'7 _{allswed us to identily fatty acids used}

p

marih¡

as

enerry so]Irc€s and those used primarily as ca¡bon sources. We also found

that adsorption of the fattj¡.acids of higher

mo-lecular weight such as lauric, aJ¡ristic, palÍri.

tic

and stearic could have been a determinant

of

the assimilation process.

[,ith

M.

tubercu-losis most of the substrates were used as both

carbon sources and energ¡ sources, whereas

lauric and formate were used chiefly to provi-de energy. lryith

i[.

IeplseEr¡Ìium, lãuric was

the best energy source and carbon-2,

but

not carbon-l of acetate the best carbon source, Be-cBuse assimilation studies wittr râdiochlomato. Buhdc ¡Iexanolc Ðecanoic MFistic PaIEri¿ic SteaÌic Oteic I,jnoleic tinolenic

0 0 0 ++

+

+ 0 0 NTo

NT

0 + + ++

+

+ + + ++

+

+ + + ++

+

+ + + +0

0

+ + + ++

+

+ + + ++

+

+ + + 0+

+

+ + + 0+

+

00000+

+ 0 0 N]T 0 Nf -f

t

+ + + + + + + l-(Ir¡,n,v-S) M. tuþeroulosig

(EftlmåD) M. tr¡hercu¡osl8

(Hs?Rv-R)

M. Irovts

M. k¡rsesü

M. chelo¡€i

M. intrac€llül¡!€

M. ¿vium

0 + + + 0 0 0 + + + +

++0

+00

₀₊₀

0++

+++

S ₌ susceptible to all drugs (TMC 102)

R ₌ ¡€sj.stent to 5 nglrEl isonisøid (TMC 303)

cerides re,

_or

_{into phospholipids} 2r _{arld retained}

in

the cell whereas some

of

them wete

tmns-formed into by-ptoducts a¡d excreted into the mediumó'e.

The excretion of by-products into the

me-dium may represent ¿ mechariÍsm

of

elimina-tion of toxic substances. An example

of

such a toxic effect was observed

witlì

oteic acid: in high concentration

it

inhibited the

_$owth

of tubercle bacilli,

but

in

low

concentration

it

enhanced growth

of

this organism, as did

bu-tyric

a¡d

decanoic

12.

T'his inhibitory effect

caused

by lattj¡

acids

on

M.

úub€rculosls