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Zbl. Bakt. Hyg. A 265, 385-392 (1987)
Uptake of Selected Antibacterial Agents in Mycobacteriumavium
HUGO L. DAVID, SABINE CLAVEL-SERES, FRANCOISE CLEMENT,and KHYE-SENG GOH
Service de la Tuberculose et des Mycobacteries, Institut Pasteur, 75724 Paris 15, France
With 2 Figures' Received August 8, 1986 . Accepted January 7, 1987
Summary
The antibacterial action of 64 drugs against Mycobacterium avium ATCC 15769 wasscreened in Middlebrook 7H9 liquid medium. The most active drugs were ansamycin,rifampicin, clofazimine, and pristinamycin. The antibacterial action of the selected drugswas confirmed by testing clinical isolates on Middlebrook 7H10 agar medium.
The antibacterial actions were not related with the hydrophobicities and molecularweights of the drugs. However, all the active drugs were highly hydrophobic molecules oflow polarity. These drugs dissolved into the lipids forming the outer layers of the bacterialenvelope and they appeared to interact with the surface amphiphils.
Zusammenfassung
Die antibakterielle Wirkung von 64 Arzneimitteln gegen Mycobacterium avium ATCC15769 wurde in Middlebrook-7H9-Fliissignahrboden iiberpriift. Am wirksamsten warenAnsamycin, Rifampicin, Clofamizin und Pristinamycin. Die antibakterielle Wirkung der soausgewahlten Mittel wurde durch Prufung klinischer Isolate auf Middlebrook-7H10-AgarNahrboden bestiitigt.
Die antibakterielle Wirkung der einzelnen Mittel zeigte keine Beziehung zu ihrer Hydrophobizitat und ihren Molekulargewichten. Es handelte sich aber bei allen wirksamenMitteln urn stark hydrophobe Molekiile niedriger Polaritat. Diese Mittel losten sich in dendie aufere Schicht der Bakterienhiille bildenden Lipiden und schienen eine Wechselwirkungmit den Oberflachen-Amphiphilen aufzuweisen.
Introduction
The characteristic natural multiple drug-resistance in Mycobacterium avium wasattributed to the structure of the outermost layer in their cell walls (13). The proposedmechanism was one of exclusion whereby the drugs did not diffuse across the wall toreach their targets (1, 13). Revealed in transmission electron microscopy using thecytochemical method of Luft (7) the outermost layer appeared as a polysaccharide-rich
386 H. L. David, S. Clavel-Seres, F. Clement, and K.-S. Goh
structure (11) which was conceived to be made of the various amphiphatic complexlipids extractable using organic solvents (2, 4, 8). Consequently we hypothesized thatto be effective the drugs should interact with the amphiphils as a first step in theirdiffusion across the wall. To test the hypothesis we screened the antibacterial action of64 drugs having widely different structures, molecular weights, and hydrophobicitiesagainst M. avium ATCC 15769. The uptake of the most active drugs was then examined and an attempt was made to verify their interaction with the surface amphiphils. The purpose of the investigation was the hope that an understanding of druginteractions with surface amphiphils might help designing molecular structures usefulin targetting of other chemoptherapeutic agents as a means of circumventing the natural multiple drug-resistance in M. avium.
Materials and Methods
Bacteria
M. avium ATCC 15769T (=TMC 706T) was selected for these investigations. The bacteria were grown in a medium containing the 7H9 Middlebrook base (Difco, USA) supplemented with 0.5% (w/v)of Nutrient Broth (Difco, USA), and 0.5% of glycerol (7H9-NBG medium). During the investigations, the bacteria were maintained in this medium as shakecultures at 37"C.
Drug-susceptibilities. The antibacterial action of the 64 drugs listed in Table 1 wasscreened using the complete Middlebrook 7H9 medium containing increasing concentrations of the drugs. The Minimal Inhibitory Concentrations (MIC's) were defined as the drugconcentrations causing complete inhibition of growth after 14 days of incubation at 37"C.
The drugs selected from the above screening were retested on Middlebrook 7HI0 agarmedium containing increasing concentrations of the drugs. The bacterial suspensions werediluted and plated as usual (3, 5). From the size of the inoculum estimated from the controlplates, the survivors at each drug concentration were then calculated. For completeness, inaddition to the ATCC 15769 strain we also tested 10 recent clinical isolates that wereidentified using currently recommended procedures (3, 5).
Drug-uptake. To examine drug-uptake the selected drugs were added to about 200.0 mgwet weight of bacteria. At regular intervals after the addition the extracellular concentrationof the drugs was measured using a Gilford 250 Spectrophotometer. The experiments wereconducted as follows. The bacterial mass was recovered from actively growing cultures inthe 7H9-NB-G medium, and was washed once in sterile 0.5 M phosphate buffer saline, pH7.0. To the cell sediment containing about 200.0 mg of bacteria 10.0 ml of the drug solutionin buffer was added. The concentrations of the drugs were 10.0 ug/rnl for rifamycin SV,rifampicin, and clofazimine, and 100.0 u/ml for ansamycin and pristinamycin. Immediatelyafter the addition, and 60, 120, and 180 min therafter 2.0 ml aliquots were transferred tocentrifuge tubes and were immediately centrifuged. The supernatants were filtered using0.45 I-l Millipore filters, and the concentration of the drugs in the filtrates was estimatedfrom their absorbancies measured at 475 nm, 475nm, 500 nm, 475 nm, and 360 nm for,respectively, rifamycin SV, rifampicin, ansamycin, clofazimine and pristinamycin.
Thin-layer chromatography
Thin-layer chromatography (TLC) of the selected drugs, and of chloroform-methanol(2:1, v/v), extracts ofthe bacteria treated with the same drugs for 4 h, was performed on K6Silica Gel plates from Whatman Chemical Separation Inc. (USA). Rifamycin SV, rifampicin,ansamycin and clofazimine are pigments and were detected by the direct observation of theplates; pristinamycin was detected from its quenching when viewed under ultraviolet lightillumination.
Tab
le1.
Ant
ibac
teri
alac
tio
nag
ains
tM
.av
ium
AT
CC
15
76
9o
f64
dru
gs
and
anti
biot
ics,
inre
lati
on
toth
eir
hydo
phob
icit
ies
and
mo
lecu
lar
wei
ghts
Sol
ubil
ity
inaq
ueou
sso
luti
ons
Mo
lecu
lar
wei
ghts
<1.
0M
inim
alin
hib
ito
ryco
nce
ntr
atio
ns
(ug/
ml,
w/v
)
1.0
-10
.01
0.0
-50
.0>
50
.0
Non
eto
po
or
Slig
htto
go
od
Hig
h
<2
50
25
0-5
00
50
0-1
00
0
>1
00
0
<2
50
25
0-5
00
50
0-1
00
0
>1
00
0
<2
50
25
0-5
00
50
0-1
00
0
>1
00
0
Clo
fazi
min
e
Rif
ampi
cin;
Pri
sti
nam
ycin
.A
nsam
ycin
.
Sul
fam
etho
xazo
le-t
ri
met
hro
pri
m;
Gri
seof
ulvi
n.R
ifam
ycin
;M
ikam
yci
n.
Am
ikac
ine;
Stre
ptom
ycin
.
Sul
fadi
azin
e;D
-cyc
lo
seri
ne.
Gen
tam
yci
n;
Ofl
oxac
ine;
No
rflo
xac
ine;
Cip
ro
flox
acin
e.
Kan
amyc
in;
Pur
om
om
yci
n.
An
tim
yci
nA
;F
usid
ate;
Spi
ram
ycin
.
Gra
mic
idin
S.
Spe
ctin
omyc
in.
Mit
hra
cin
.
Sul
fagu
anid
ine;
ison
iazi
de.
Cep
hal
ori
din
e;T
etra
cy
clin
e;M
ito
my
cin
C;
Pef
loxa
cine
.
Vio
myc
in;
Ble
omyc
in;
Cap
reom
ycin
;H
yg
ro
my
cm.
Eth
ambu
tol;
Nal
idix
icac
id;
Nim
araz
ole
;D
DS
.L
inco
myc
in;
Ny
stat
in.
Am
ph
ote
rici
nB
.
Lev
amis
ole;
thio
tep
a;C
hlo
ram
ph
enic
ol;
Cic
lo
hexi
mid
e;Q
uin
ine;
Min
o
cycl
in;
Dau
no
rub
icin
.E
ryth
rom
yci
n;
Do
xo
rub
ici
n;D
ista
my
cin
A.
N-a
cety
lsu
lfan
ilam
ide;
Sulf
an
ilam
ide;
Met
ron
ido
zole
;A
mpi
cill
in;
Met
hic
illi
n;
Pen
icil
lin
G;
Sul
mer
azin
e;S
ulfa
thia
zole
;S
ulfi
zoxa
zo
le;
To
bra
my
cin
.
Van
com
yci
n;
Bac
itra
cin
;C
olis
tin
met
han
esu
lfo
nat
e.
~ Il'
<: a- 3 c ~ Il'
:;0;
n> o ......
t:I a aa w 00
'-l
388 H. L. David, S. Clavel-Seres, F. Clement, and K.-S. Goh
Antibacterial agents: The following drugs were kindly supplied by their producers: ansamycin (Carlo Erba), clofazimine (Bayer), pristinamycin (Specia), thipeta (Specia),colistin(Roger Bellon), trobicin (Upjohn), daunorubicine (Specia), amikacin (Bristol), minocyc1in(Lederle), trimethroprim (Hoffman LaRoche), lividomycin (Roger Bellon), novobiocin(Theraplix), nimarozole (Carlo Erba), bleomycin (Roger Bellon), doxorubicine (Roger Bellon), and mithracin (Pfizer). The other drugs listed in Table 1 were purchased from SigmaChemical Company (USA).
Results and Discussion
The antibacterial action of 64 molecules against Mycobacterium avium ATCC15769 is depicted in Table 1. The most active drugs were rifampicin, ansamycin,clofazimine, and pristinamycin (Table 2 and Fig. 1). These drugs are all hydrophobicmolecules, however there was no clear correlation between the hydrophobicities andthe molecular weights of the various compounds tested. Nonetheless, it was apparentthat the water soluble drugs had little antibacterial action. Although ansamycin,clofazimine and pristinamycin were significantly active against M. aviumATCC 15769(Table 1, Fig. 1) they were not efficient antibacterial agents against all of the clinicalisolates, as shown in Table 2. These data showed that these drugs may not be usefulchemotherapeutic agents against all cases of disease, as discussed previously (1) inrespect to other chemotherapeutic agents.
A 8MINUTES DRUG IN J'fJ/ml
-15 0 60 120 180 1.0 5.0 1 .0100
161
- 1cl~....~
~ 103Ci 50
lu~
~
s~ a:e ~ 10
4::la:Cl
105
0 106
Fig. 1. Uptake (A) and antibacterial action (B) of rifamycin SV (0-), rifampicin (0-),ansamycin (e-), and clofazimine (.-) in M. avium ATCC 15769. The data for doxorubicine, daunorubicine, and amphotericin B are not shown as there was no uptake norantibacterial action. For pristinamycin (+--) only the antimicrobial action is shown (seetext).
M. avium; Uptake of Drugs 389
Table 2. Number of strains with the indicated proportion of resistant bacteria at the indicat-ed concentrations of the drugs in Middlebrook 7H10 agar medium. The drug concentra-tions are in Itg/ml
Ansamycin Clofazimine Pristinamycin
Survivors 0.1 0.5 1.0 0.5 1.0 5.0 1.0 5.0 10.0
1.0 1 1 4 2 15 X 10-1 2 1 3 110-1 1 3 1 4 15 X 10-2 3 3 2 210-2 1 35 X 10-3 1 110-3 1 1 1 1 15 X 10-4 1 1 110-4 1 1 1 1 15 X 10-5 2 2 110-5 1 1 1 15 X 10-6 1 1 110--{; 1 5 8 1 6
To study the interaction of the drugs and the bacteria we measured their disappearance from the extracellular medium using an approach similar to that of Nikaido in hisstudies of transmembrane diffusion in Salmonella typhimurium (9). Rifamycin SV, andits rifampicin and ansamycin derivatives were taken up almost instantaneously (Fig. 1).Clofazimine had very low solubility in aqueous solutions and it had high affinity tofats, however at the concentration of 10.0 ug/ml used it remained in solution uponcentrifugation. The uptake of pristinamycin could not be determined because bacterialsubstances absorbing in the ultraviolet part of the spectrum were released from thebacteria treated with this antibiotics. The drugs used as controls (doxorubicin,daunorubicin, and amphotericin B) were not taken up by the bacteria.
The visual observation of the bacterial mass in the above experiments showed thatthey were colored by rifamycin SV, rifampicin, ansamycin and clofazimine but not bydoxorubicin, daunorubicin and amphotericin B (pristinamycin being almost colorlessits attachement to the bacterial mass was not vizualized by direct observation). Following these observations we treated the bacterial mass with the above drugs for 4 hours.The cell mass was washed once with distilled water and the sediments were washedwith chloroform-methanol (2:1, v/v). The chloroform extracts were washed, weredried and were then applied onto TLC plates. As shown in Fig. 2 the drugs interactedwith the mycobacterial lipids but not in an indiscriminate fashion. In the TLC systemused the mycobacterial polar lipids were retained at the origin and they were notstained by the antibiotics. The lipids that interacted with the antibiotics were of the lowpolarity kind. Rifamycin SV and rifampicin formed lipid-antibiotic complexes with thesame relative polarity; the lipid-ansamycin complex had a different relative mobility;pristinamycin reacted with at least two distinct lipids; and clofazimine interacted withat least four distinct lipids. In the extract of the clofazimine treated bacteria a largeproportion of the drug was apparently free, while in the extracts of the bacteria treated
25 Zbl. Bakt. Hyg. A 265/3-4
390 H. L. David, S. Clavel-Seres, F. Clement, and K.-S. Goh
with the other drugs the free antibiotics were not detected. Consequently we concludedthat two factors may have contributed to the uptake and subsequent antibacterialefficacy of these drugs. One factor was their solubility into fats which was perhapsnonspecific and related to their hydrophobicities and low polarities; the other factorwas the binding of the drug to the structural lipids in the wall which may be specificbetween sites in the antibiotics and sites in the amphiphatic lipids of the wall.
In the Gram-negative bacteria that are surrounded by a membrane bilayer it hasbeen shown that this outermost membrane acted as a penetration barrier (9, and for arecent review see reference 6). The mycobacterial cells are not limited by a membranebilayer but instead by a polysaccharide outerlayer (4, 11). It was suggested that themycolic acids esterifying the arabinogalactanmucopeptide basal layer formed a coherent monolayer into which the loosely bound complex mycobacterial lipids inserted toform a functional bilayer (2, 4, 8). Thus, like in the Gram-negative bacteria, themycobacterial outerlayer may also act as a penetration barrier (4, 10, 11, 12). As wehad hypothesized that the penetration barrier was located in the cell wall outermostlayer we thought that there were two possible approaches to circumvent natural multi-
A 8
. ....· :.-
:0:· ...... :· ...'
---
8 1
m~:....
..... .
....
RSV RIF ANS PRleLO1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Fig. 2. TLC of rifamycin SV (RSV, lane 1), rifampicin (RIF, Lane 2), ansamycin (ANS, lane3), pristinamycin (PRI, lane 4), and clofazimine (CLOF, lane 5). A. TLC of the free antibiotics; B. TLC of the chloroform-methanol extracts of the bacteria treated with the indicatedantibiotics; B' is the same run as B after spraying the plates using the Bial spraying reagentfor glycolipids (Sigma Chemical Company, USA).In A and B the spots were the natural color of the antibiotics, except for pristinamycin thatwas revealed from its quenching (dotted spots) under ultraviolet light observation. In B' thespots labeled m had the typical golden-brown color of the mycosides C, and the dotted spotsin lane 4 quenched under ultraviolet light examination and gave a golden-brown color uponspraying with the Bial spray reagent.
M. avium; Uptake of Drugs 391
Table 3. Relationships between the minimal inhibitory concentrations of the indicated drugsand their relative polarities determined on TLC using silica gel plates and chloroformmethanol 90: 10, v/v, as the solvent
M.Le. in ug/ml
Antibiotic 7H9 medium 7H10 medium Relative polarities (Rf'S).
PristinamycinAnsamycinRifampicinRifamycin SVClofazimineDaunorubicineDoxorubicineAmphotericin B1
1.00.10.51.00.5
100.0100.0100.0
10.01.00.51.00.1
ND2
NDND
0.900.860.690.680.510.000.000.00
Amphotericin B is not an antibacterial agent, and it was included as a control.ND = not done.
pie drug-resistance in these bacteria: one approach would be to determine the nature ofthe possible interactions of the rare active drugs with the surface amphiphils uponwhich to design new chemotherapeutic agents; and the other approach would be tospecifically inhibit the synthesis of the outer layer thus rendering the bacteria permeable to current chemotherapeutic agents. The present report dealt with the first of thesetwo approaches.
Judging from our data the majority of the 64 compounds tested had little antibacterial action against M. avium, even though many of them were hydrophobic molecules.The most active drugs (ansamycin, rifampicin, clofazimine, and pristinamycin) werehydrophobic compounds of low polarity. Although appreciated retrospectively thedevelopment of the Rifamycin SV derivatives appeared as an example of drug-targetting because their antibacterial activities correlated with their hydrophobicities andpolarities, as well as their interactions with the mycobacterial surface lipids. The othertwo active drugs (pristinamycin and clofazimine) also were of low polarity and interacted with surface lipids. These findings indicated that further investigations on theinteractions of these drugs with purified mycobacterial lipids are necessary for thedesigning of molecular structures to target other antibacterial agents as it appeared thatdiffusion across the wall may require their specific attachment to specific sites in thesurface amphiphils.
Acknowledgements. We thank the Chemical and Drug Companies for supplying variousdrugs used in our study.
References
1. David, H. 1.: Basis for lack of drug susceptibility of atypical mycobacteria. Rev. infect.Dis. 3 (1981) 878-884
2. David, H. 1. and N. Rastogi: Antibacterial action of colistin (polymixin E) againstMycobacterium aurum. Antimicrob. Agents Chemother. 27 (1985) 701-707
392 H. L. David, S. Clavel-Seres, F. Clement, and K.-S. Goh
3. David, H. L., V. Uvy-Frebault et F. Papa: Methodes de Laboratoire pour Mycobacteriologie Clinique. Institut Pasteur (1986)
4. David, H. L., N. Rastogi, S. Claoel-Seres, F. Clement, and F. Thorel: Structure of the cellenvelope of Mycobacterium avium. ZbI. Bakt. Hyg. A 264 (1987) 49-66
5. Kent, P. I. and G. P. Kubica: Public Health Mycobacteriology: A Guide for the LevelIIILaboratory. United States Department of Health and Human Services, USPHS, CDC(1985)
6. Lambert, P. A.: The bacterial surface and drug resistance. In: Role of the Envelope inthe Survival of Bacteria in Infection, ed. by C. S. F. Easmon, ]. [eliaszeioicz, M. R. W.Brown, and P. A. Lambert, Medical Microbiology, Vol. 3, pp. 1-19. Academic Press,London (1983)
7. Luft, ]. H.: Ruthenium red and violet. I. Chemistry, purification, methods of use forelectron microscopy and mechanisms of action. Anat. Rec. 171 (1971) 347-368
8. Minnikin, D. E.: Lipids: complex lipids, their chemistry, biosynthesis and roles. In: TheBiology of the Mycobacteria, ed. by C. Ratledge and]. Stanford, pp. 95-184. Academic
. Press, London (1982)9. Nikaido, H.: Outer membrane of Salmonella typhimurium. Transmembrane diffusion
of some hydrophobic molecules. Biochem. Biophys. Acta 433 (1976) 118-13210. Rastogi, N. and H. L. David: Ultrastructural and chemical studies on wall deficient
forms, spheroplasts, and membrane vesicles from Mycobacterium aurum. J. gen. Microbiol. 124 (1981) 71-79
11. Rastogi, N., C. Frehel, A. Ryter, H. Oyon, M. Lesourd, and H. L. David: Multiple drugresistance in Mycobacterium avium: Is the wall architecture responsible for the exclusion of chemotherapeutic agents? Antimicrob. Agents Chemother. 20 (1981) 666-f,77
12. Rastogi, N., V. Levy-Prebault, and H. L. David: Spheroplast format ion from ninerapidly growing mycobacteria. Curro Microb iol. 9 (1983) 201-204
13. Woodley, C. L. and H. L. David: Effect of temperature on the rate of the transparent toopaque colony type transition in Mycobacterium avium. Antimicrob. AgentsChemother. 9(1974) 113-119
Dr. Hugo L. David, Service de la Tuberculose et des Mycobacteries, Institut Pasteur, 25Rue du Dr. Roux, F-75724 Paris 15, France
