FEMS Microbiology Letters 123 (1994) 11-18
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07 00
Published by Elsevier
11
FEMSLE 06211
MlnlRevlew
Mycobacterial cell wall: Structure and role in natural
resistance to antibiotics
Vincent Jarher
a
and Hlroshl Nlkaldo
b,*
a Laboratotre de Bactdrtologte-Virologte, Facultd de Mddecme Ptttd-Salp~trtOre, F-75634 Parts, France, and b Department of Molecular
and Cell Biology, 229 Stanley Hall 3206, Umverstty of Cahforma, Berkeley, CA 94720-3206, USA
(Received 20 June 1994, revision received 12 August 1994, accepted 22 August 1994)
Abstract Mycobacterla show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents The low
permeability of the mycobacterial cell wall, with its unusual structure, IS now known to be a major factor in this resistance Thus
hydrophilic agents cross the cell wall slowly because the mycobacterial porin is inefficient in allowing the permeation of solutes and
exists in low concentration Lipophlhc agents are presumably slowed down by the lipid bilayer which is of unusually low flmdity and
abnormal thickness Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires
synergistic contribution from a second factor, such as the enzymatic inactwatlon of drugs
Key words Mycobactertum, Cell wall, Mycohc acid
Introduction
(M avtum, M xenopt, M kansastt, M chelonae,
M fortuttum)
There are nearly 60 commonly recognized
species of Mycobactenum, but most are saprophytic inhabitants of sod A few are major human
pathogens that cause tuberculosis (M tuberculosis, M afncanum, M boris) and leprosy (M leprae), two well-documented diseases Several socalled 'atypical mycobacterla' are essentially
saprophytic, yet can cause opportumstlc refections, especially in lmmunocomprommsed patients
Sulfonamtdes and pentctlhn, which drastically
modified the outcome of many hfe-threatenlng
bacterial refections, were meffectwe against tuberculosis Slmdarly, pemcllhn was xneffectwe and
sulfonamldes showed only bacterlostatlc effects
against leprosy [1] Among the major antibiotics,
only a few showed good activity against M tuberculosts complex, and almost none were actwe
against the atypical species (Table 1) This disappointing situation encouraged studies on mechamsms of mtrtnslc drug resistance m mycobactena
In the present review, we will concentrate on the
structure and barrier properties of the mycobacterial cell wall In promoting antibiotic resistance
* Corresponding author Tel (510) 642 2027, Fax (510) 643
9290
SSD1 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 3 6 3 - 7
12
Table 1
Activity of major chemotherapeutic agents against mycobacte-
c o p e p t t d o h p t d s [3,5] T h e cell wall also c o n t a i n s
several p r o t e i n s [5,6]
rla
Agent (year introduced)
Sulfonamldes (1935)
Pemcflhn G (1944)
Streptomycin (1944)
Chloramphemcol (1947)
Tetracychnes (1948)
Erythromycln (1952)
Isonlazld (1952)
Novoblocln (1955)
Vancomycm (1956)
Broad-spectrum
fl-lactams (1961)
Qumolones (1962)
Fusldlc aod (1962)
Rffamplcm (1966)
Fluoroqumolones (1979)
M tuberculosls
M leprae Atypical
mycobacteria
+
.
+
.
.
.
+
-
--
_ b
-
+
+
+
+
_+ c
-
+ and - indicate the presence and absence, respectwely, of
slgmflcant actwlty
a Mmocychne (1961) is active against M leprae
b Clanthromycm (1984) ts actwe against some atypical species
c Variable depending on speoes and particular compound
M y c o b a c t e r l a p r o d u c e cell walls of u n u s u a l
s t r u c t u r e T h e p e p t i d o g l y c a n c o n t a i n s N-glycol y l m u r a m l c acid i n s t e a d of the u s u a l N a c e t y l m u r a m i c acid [2], b u t a far m o r e dtstmctwe
f e a t u r e ts that u p to 60% of the wetght of the
m y c o b a c t e r l a l cell wall ts o c c u p i e d by hpids that
consist m a i n l y of u n u s u a l l y l o n g - c h a m fatty acids
c o n t a l n m g 60 to 90 carbons, the mycohc acids [3]
T h e chemtstry a n d i m m u n o l o g y of the various
c o m p o n e n t s of the m y c o b a c t e r l a l cell wall have
b e e n s t u d i e d extenswely, m a i n l y wtth the perspectwe of dlscovermg a n t t g e n s that could be
used for i m m u n i z a t i o n a n d diagnosis T h e covalently c o n n e c t e d s t r u c t u r e of the cell wall ts m a d e
of peptldoglycan, to which a r a b l n o g a l a c t a n is
l m k e d w a a p h o s p h o d l e s t e r bridge A b o u t 10% of
the a r a b m o s e restdues tn a r a b m o g a l a c t a n are m
t u r n s u b s t t t u t e d by mycohc a o d [4] T h e cell wall
also c o n t a i n s several types of ' e x t r a c t a b l e hpIds'
that are n o t covalently h n k e d to this basal skeleton, these i n c l u d e t r e h a l o s e - c o n t a l n i n g glycolIptds, p h e n o l - p h t h i o c e r o l glycostdes, a n d gly-
Physical organlzatmn of the lipids in the mycobacterial cell wall
K n o w l e d g e of the chemistry of mycobacterlal
hplds 1s u n f o r t u n a t e l y insufficient to u n d e r s t a n d
the b a r r i e r p r o p e r t i e s of the cell wall, b e c a u s e
these p r o p e r t i e s d e p e n d o n the physical organization of the hplds M o r e t h a n t e n years ago, M m n l k m p r o p o s e d a m o d e l for this physical organization, in which mycohc acid chains are p a c k e d
side by side m a direction p e r p e n d i c u l a r to the
p l a n e of the cell surface [3] It was also p r o p o s e d
that this mycohc a c l d - c o n t a m m g i n n e r leaflet is
covered by a n o u t e r leaflet c o m p o s e d of extractable llptds, the whole s t r u c t u r e thus p r o d u c mg a n a s y m m e t r i c lipid bilayer (Ftg 1) This
m o d e l has b e e n recently u p d a t e d [4,7], a n d has
received s u p p o r t from several r e c e n t studies Xray diffraction d a t a of purified M chelonae cell
wall showed that a large part of the h y d r o c a r b o n
chains In the cell wall are tightly p a c k e d in a
parallel array in a direction p e r p e n d i c u l a r to the
cell surface [8] T h e s e c o n d line of s u p p o r t came
from i m p r o v e d u n d e r s t a n d i n g of the a r a b l n o galactan structure I n this polysaccharlde, b o t h
the galactan m a i n c h a i n a n d the a r a b l n a n stde
b r a n c h e s are c o n s t r u c t e d in a m a n n e r that would
e n s u r e m a x t m u m f r e e d o m of m o v e m e n t b e t w e e n
--
Extractable hpld with C14 C18 acids
Mycohc acid
- Extractable hpld wrth intermediate
chamqength hydrocarbons Porln
11111111
AP~BINO~I.ACTAN
I
PEPTIDOGLYCAN
Fig 1 Modified Mlnnikm model for the structure of mycobactenal cell wall
13
sugar residues Thus all of the sugars are in
furanose form, and arabmose and galactose
residues are connected through 1,5 and 1,6 linkages, respectwely [4] Finally, mycohc acid
residues are linked to the tips of branches of this
highly branched polysaccharide [4] This structure
suggests that the parallel packing of the mycohc
acid chains may be achieved by the flexible movement of arabInogalactan Such packing would
otherwise be difficult since all mycohc acid
residues are covalently connected to this macromolecule The third line of evidence was generated by a quantltatwe reevaluation of exastIng
results [8] Data on the amount of mycohc acid
present in a known amount of Bacillus CalmetteGu6rin (BCG) cells indicated that a monolayer of
mycohc acid, the presumptwe inner leaflet of the
bIlayer structure, can indeed cover the whole
surface of the bacterial cells Finally, although it
has been often argued that atypical species did
not contain much extractable lipid, analysis of M
chelonae cell wall showed the existence of sufficient amounts to produce the outer leaflet coverlng the entire cell surface (E Y Rosenberg and
H Nikaido, unpublished results) Moreover, various lines of ewdence indicate that extractable
hpids are located close to the outer surface for
example, glycopeptldohpids (mycosldes C) function as phage receptors [9]
All these pieces of evidence favour the bilayer
model of Minnlkln Mycolic acid is a branched
fatty acid with a long branch (40 to 64 carbons)
and a short branch (22 to 24 carbons) In addition
to their extraordinary lengths, mycohc acids contaIn very few double bonds or cyclopropane
groups (none in the shorter branch and at most
two in the longer branch) It is thus predicted
that the tuner leaflet will have very low fluidity,
indeed a nearly crystalline structure Since the
diffusion of hpophilIc solutes through a lipid btlayer requires a fluid interior, this construction is
expected to act as an effective barrier for the
penetration of lipophdm antibiotics This asymmetric bllayer structure is reminiscent of that of
the Gram-negative outer membrane, where the
low fluidity of the lipopolysaccharide leaflet is
known to impede the influx of hpophihc solutes
Ira]
Permeability of the mycobacterial cell wall
Mycobacterial cell wall has long been suspected to act as a permeation barrier for antibiotics Some redirect data were available For example, the antibiotic efficacy increased when detergents such as Tween were added to culture
media [1l], or when the cell wall structure became defective due to mutations [12] Furthermore, ammoglycosldes were shown to be more
actwe on ribosomes in cell extracts than on intact
mycobactenal cells, suggesting the role of a cell
surface barrier [11] In specms producing intrinsic
fl-lactamase, the enzyme is cryptic, and the hydrolysis of /3-1actams measured with disrupted
cells is far faster than that measured in intact
cells [13] Recent data also show a synergistic
effect between various agents and those agents
that are known to inhibit the synthesis of cell wall
components, such as ethambutol [14]
In spite of these largely quahtatwe results,
there was no quantitative measurement of the
permeability of mycobacterIal cell wall, until our
study [15] We used the method introduced for
estimation of outer membrane permeability in
Gram-negative bacteria [16] We measured the
rate of hydrolysis of/3-1actams by intact bacterial
cells, and calculated the cell wall permeability by
assuming that drug molecules first diffuse through
the cell wall (following Flck's first law of diffusion) and then are hydrolyzed by perlplasmlc
/3-1actamase (following the Mlchaehs-Menten kinetics) For this method, cells should exist as
unicellular dispersions in contact with the
medium A strain of M chelonae was chosen
because ~t produced homogeneous suspensions
without the use of detergents, and because it
produced a/3-1actamase of sufficmntly high activity which did not leak out into the medium The
cell wall permeability to cephalosporins measured
in this strain was indeed very low, and was about
three orders of magnitude lower than that of E
cob outer membrane, and ten times lower than
the permeability of the notoriously impermeable
P aerugtnosa outer membrane (Fig 2)
Another approach was that of studying the
kinetics of nutrient uptake If we assume that
nutrient molecules are taken up by high-affinity
14
10 1
10 3
m o r e slowly t h a n t h r o u g h t h e o u t e r m e m b r a n e o f
10 2
lo2® i~10 3
!
E
i~=
-
~101
glucose
cepha|ondme
~10-4 --=_-cephacetnle
: n-
~10 °
o10 5
10 ~
E cob [15] ( F i g 2)
M chelonae, c h o s e n as a m o d e l b e c a u s e its
glycerol
glycerol
fructose
nlffocephln
L~
cephalondme
cephacetnle
nltrocephln
• ~ 10 ~
rlOZ L ~ 7
:o_10
103 ~10-8
E colt
P aerugtnosa
glocose
gly,~rgl .
cepR~llOnolne
nrtrocephm
M chelonae
Fig 2 Permeablhty coefficients and half-eqmhbratlon hmes
of nutrients and cephalosporms The permeabdJty coefficients
are those across the outer membrane or the cell wall, taken
from [15] Half-eqmhbratlon nmes indicate the rime needed
for lntracellular concentration to reach one-half of the external concentrahon, in this calculation we have disregarded the
presence of a second permeabdlty barrier (cytoplasmic membrane) and of degradatwe enzymes
active t r a n s p o r t systems as soon as they cross t h e
cell wall, we c a n e v a l u a t e cell wall p e r m e a b i l i t y
by c o m b i n i n g t h e diffusion e q u a t i o n with t h e
M ] c h a e h s - M e n t e n kinetics T h e a b o v e a s s u m p n o n is p r o b a b l y valid, as a m i n o acids a r e k n o w n
to b e t r a n s p o r t e d across t h e m y c o b a c t e n a l cytop l a s m i c m e m b r a n e by high affinity systems (for
r e f e r e n c e , see [15]) This a p p r o a c h a g a i n sugg e s t e d t h a t small n u t r i e n t s p e n e t r a t e t h r o u g h t h e
M chelonae cell wall m o r e t h a n 10000 t i m e s
1,000
glutamate
leuclne
500
g 2oo
~
1O0
~
50
prohne
tryptopl~an
glyclne
glucose
~5
10
~
5
2
1
glutamate
p r o p e m e s w e r e c o n v e n i e n t for o u r assay, h a p p e n s to b e o n e of t h e m o s t d r u g - r e s ] s t a n t species
a m o n g m y c o b a c t e r ] a T h u s ]t b e c o m e s I m p o r t a n t
to study the cell wall p e r m e a b d ] t y o f o t h e r myc o b a c t e n a l s p e c i e s M smegmatts, widely u s e d m
studies of m o l e c u l a r biology, was f o u n d to show
a b o u t t e n - f o l d h i g h e r p e r m e a b d ] t y to /3-1actams,
by us]ng an assay s l m d a r to t h a t u s e d for M
chelonae [17] T h e assay h a d to b e m o d i f i e d for
M tuberculosts, which has a s t r o n g e r t e n d e n c y to
a g g r e g a t e U s e of th~s m o d i f i e d assay s h o w e d t h a t
M tuberculosts H 3 7 R a was m o r e p e r m e a b l e , by a
factor o f n e a r l y ten, t h a n the M chelonae s t r a m
s t u d i e d e a r h e r ( E Y R o s e n b e r g a n d H N]ka]do,
u n p u b h s h e d results)
T h e s e results are c o n s i s t e n t with o u r knowle d g e t h a t atypical species, such as M chelonae,
are usually m o r e r e s i s t a n t to a n u m b e r of a g e n t s
t h a n M tuberculosts ( T a b l e 1) It is also consist e n t with t h e e a r l i e r o b s e r v a t i o n t h a t n u t r i e n t s
a r e a c c u m u l a t e d m u c h m o r e r a p i d l y by M smegmatts a n d M phlet t h a n by M chelonae ( F i g 3)
It a p p e a r s t h a t t h e cell wall o f the f o r m e r species
]s a b o u t o n e o r two m a g n i t u d e s m o r e p e r m e a b l e
t h a n t h a t of M chelonae F o r a m i n o acids, M
tuberculosts a p p e a r e d to b e i n t e r m e d i a t e b e t w e e n
the highly i m p e r m e a b l e M chelonae a n d the m o r e
p e r m e a b l e M phlet [19]
T h e s e results i n d i c a t e that, a l t h o u g h m y c o b a c t e n a ]n g e n e r a l have low p e r m e a b d l t y cell wall,
t h e p r e c i s e values o f p e r m e a b i l i t y m a y differ
a m o n g d i f f e r e n t species by a f a c t o r o f p e r h a p s u p
to 100 A s s e e n a m o n g G r a m - n e g a t w e rods, t h o s e
s p e c i e s t h a t a r e o b h g a t e p a r a s i t e s such as M
tuberculosts a p p e a r to have a relatively high p e r m e a b d l t y , w h e r e a s s o m e sod i n h a b i t a n t s such as
M chelonae s e e m to p r o t e c t t h e m s e l v e s by p r o d u c m g a very low p e r m e a b i l i t y wall
glucose
leucme
glycerol
M ehelonae M smegmatts
M phlot
F~g 3 Estimated permeabd~ty coefficients for nutrients m M
phleg, M smegmatts, and M chelonae Calculation was performed as m [15], utlhzmg the transport data from [19] and
from papers cited in [18]
Mechanism
of diffusion across cell wall
Hydrophthc pathway
The Gram-negatwe bacteria, whose outer
m e m b r a n e acts as a p e r m e a b d l t y b a r n e r , have
15
developed porto channels that allow the diffusion
of small, hydrophfllc nutrient molecules [1(3] The
penetration rate of cephalosporms across M chelonae cell wall is not very dependent on the
hpophdlclty of the drug or temperature [15], suggesting that these hydrophlhc solutes traverse an
aqueous pathway, perhaps through s~mdar poreforming proteins
When detergent extracts of M chelonae cell
wall were reconstituted into proteohposomes, a
channel-forming actlwty that was destroyed by
proteases was detected [20] Purification of this
channel-forming actw~ty resulted m the ~dentlficat~on of a 59 kDa cell wall protein that allows
the diffusion of small, hydrophdlc solutes The
channel diameter was estimated at about 2 nm
[20] The purified protein also produced shghtly
cat,on-selective channels of a defined s~ze on
reconstltutlon into planar bdayers [20] Most importantly, the M chelonae porln is a minor protern of the cell wall, unhke enterobacterml porms
that are the most abundant proteins in the cell
Furthermore, the M chelonae porto produced
permeabdlty far lower than that produced by an
equal weight of E cob porto [20] These observations, therefore, explain why the permeabdlty of
the M chelonae cell wall to hydrophlhc solutes is
so low
Ltpophthc pathway
In principle, hpophlhc solutes should be able
to traverse any biological membrane by dxssolvlng
into the hydrocarbon interior of the lipid bllayer
On the one hand, the low flm&ty of the mycohc
acid leaflet and the unusual thickness of the
bllayer would slow down such a process m mycobacterlal cell wall On the other hand, this
pathway may play a relatively prominent role m
solute transport because of the extreme inefficiency of the porto pathway m mycobactena
The ewdence suggesting the contribution of a
hpophlhc pathway to llpophdlc solute transport
comes from the observation that the more
llpophd~c denvatwes of chemotherapeutic agents
are often more actwe against mycobacterm Thus
the ad&t~on of a CI6 fatty acyl chain to ~somazld
led to higher actwlty against M avtum [21] Correlation w~th hpophfl~c~ty was seen also w~th te-
tracychnes, the more hydrophoblc derwatlves such
as doxycychne and mmocychne being more active
against M chelonae, M fortuttum and M marm u m [22,23] than the less hydrophobic forms
Moreover, mlnocychne IS efficacious in the treatment of leprosy [24] These comparisons are usually carried out by using many isolates, so that the
chnlcal usefulness of the drugs can be estimated
However, for our purpose, the heterogeneity of
the strains sometimes complicates the interpretation of data Thus one study of tetracychnes [22]
provided the mimmal inhibitory concentration
(MIC) values of individual strains, and it was
clear that among relatwely susceptible strains,
mmocychne was most actwe, followed by doxycychne with the least actwe form being tetracycline
But the tra&tlonal indicators such as MICs0 or
MIC90 do not give any suggestion of the differences in efficacy, because these values are biased
so strongly by the presence of a large number of
highly resistant strains (for example, see [23])
Clarlthromycln, which is slightly more hydrophoblc than erythromycm, is more actwe agamst
atypical species [25,26] and M tuberculosis [27]
Among nfamycms, more hydrophoblc derwatwes
are more actwe against M tuberculosis and M
avtum [28] Some of them were reported to be
mactwe against M fortuttum and M chelonae,
but again th~s ~s hkely to be due to the generally
high levels of resistance m these species and the
problem of heterogeneity mentioned above
Among fluoroqulnolones, the more hydrophoblc sparfloxacm is more actwe than the reference
fluoroqumolones against many mycobacterm mcludmg M avturn [29] Clprofloxacm, when made
more hydrophoblc by the addition of alkyl substltuents, becomes more active against M tuberculosts and M avtum [30] With M leprae, a good
positive correlation was seen between the
llpophfllClty of fluoroqumolone and its efficacy
[31, the data are analyzed m 18] However, In
comparison to various agents, other factors such
as the affinity to the target are expected to come
into play, and a perfect correlation w~th just one
parameter cannot be expected Indeed, PD127391, which should be as hydrophlhc as
clprofloxacm, showed a remarkably strong actwlty
[311
16
In view of the complexity of the correlation,
we clearly need studies of drug penetration into
the cell, but few such studies exist Preliminary
data suggest that clarlthromycln indeed penetrates more rapidly into M awum cells than does
erythromycln (F Doucet-Populaire and V Jarher, unpublished results)
Cell wall barrier is a necessary, but not a sufficient, factor for resistance
Although mycobacterlal cell wall IS a formldable permeation barner, production of clinically significant levels of resistance usually requires the participation of an additional resistance mechanism A similar situation exists with
the outer m e m b r a n e barrier of Gram-negative
bacteria, where a second factor synergistically
and dramatically increases resistance, by removing the lnflowlng antibiotic molecules either by
chemical modification (notably by hydrolysis In
the case of/3-1actams [32]) or by active efflux into
the medium [33]
At least the first mechanism was shown to be
operating in mycobacterla, most species of which
produce /3-1actamase [13] In fact, quantitative
analysis with M chelonae showed that the synergism between the cell wall barrier and the
perlplasmlc fl-lactamase decreased the fl-lactam
concentration at the target precisely to the level
sufficient for inhibition of the most susceptible
PBP (2 mg m l - i for cephalothln, for example),
when the external concentration of the drug was
equal to M I C (about 3000 mg m1-1 for
cephalothln) [34] We emphasize that the low
permeability of the mycobacterlal cell wall is essential for this high degree of resistance Thus,
although the activity of fl-lactamase found in M
chelonae is only 20% of that m TEM-plasmldcontaining E coh, the synergistic effect of the
cell wall barrier is able to decrease the perlplasmlc concentration of cephaloridine to 0 2% of the
external concentration, m contrast, the T E M containing E coh with its high outer m e m b r a n e
permeability can lower the cephalorldlne concentration In the perlplasm only to about 80% of the
external concentration
Although the cell wall b a r n e r exerts a powerful influence in the presence of a synergistic
factor such as /3-1actamase, half-equilibration
across the cell wall takes place in several minutes
(see Fig 2), a much shorter time in comparison
with the generation time of these organisms The
barrier alone should not thus produce significant
resistance Mathematical analysis along the lines
presented in [35] shows that even the extremely
low level of cephalosporln permeability observed
in M chelonae would lower the periplasmlc drug
concentration by less than 0 5% In comparison
with the external concentration, if it acted alone
in the total absence of/3-1actamase actwity Thus
a second synergistic factor must obviously exist
for most other antibiotics to which mycobacterla
are resistant, this could be degradation or active
efflux (Amlnoglycoside-inactwatlng enzyme has
been reported in some mycobacteria [36] ) In any
case, this analysis gives us some hope that an
effectwe therapy may become possible if we could
eliminate the second synergistic factor Indeed,
M tuberculosts, which has a relatively high permeabihty cell wall (see above), becomes susceptible to ampicilhn if its /3-1actamase is inhibited by
clavulanate [37] Similar approaches are difficult
with atypical species, because even a very small
amount of residual /3-1actamase activity will be
able to cope with the few molecules of fl-lactam
that penetrate through their low permeability cell
wall [34] Nevertheless, cefamycins (cefoxitln and
cefmetazole), which are nearly completely stable
to the class A fl-lactamases, are actwe against M
fortuttum [38]
Finally, scattered evidence suggests that, in
mycobacterla, the targets of antibiotic action often have a lower affinity than in many common
bacteria This is true with PBPs [34] In wild-type
stratus of M tuberculosis, M at,rum, and M
smegmatts, the GyrA protein, the target for fluoroquinolones, harbours a characteristic alteration
found in quinolone-resistant mutants of E cob
[39,40] The ribosomes of M actum and M smegmatts also appear to have a lower affinity to
macrohdes than those of Staphylococcus aureus
(F Doucet-Populaire and V Jarller, unpublished
results), and RpoB, the rlfampicin-sensltwe subunit of R N A polymerase appears to have a se-
17
quence found m resistant mutants of other bacterm [41] T h e lower suscepttblhty of targets described above may also contribute as a synerglsttc
factor to the drug resistance of mycobacterla,
espectally that of the atypical species It seems
that mycobacterla, as the ultimate sod bacteria,
have learned to combine all possible resistance
mechanisms This makes it difficult to treat mycobacterlal diseases m the age of antibiotics, once
we become infected by these well-protected bacterm
Acknowledgements
We apologize to the authors of many excellent
studies that could not be ctted because of space
Itmltatlon Research m the laboratory of H N has
been supported by grants from National Institutes of Health (AI-09644 and 33702) Research
in the laboratory of V J has been supported by
the Agence Natlonale de Recherche sur S I D A
and Association Francalse Raoul Follereau
References
1 Ji, B and Grosset, J (1990) Recent advances of
chemotherapy in leprosy Leprosy Rev 61, 313-322
2 Draper, P (1982) The anatomy of mycobacteria In The
Biology of Mycobactena (Ratledge, C and Stanford, J ,
Eds), vol 1, pp 9-52 Academic Press, London
3 Mmmkm, D (1982) Complex hplds their chemistry,
biosynthesis, and roles In The Biology of Mycobactena
(Ratledge, C and Stanford, J Eds), vol 1, pp 94-184
Academic Press, London
4 McNeil, M and Brennan, P J (1991) Structure, function
and biogenesis of the cell envelope of myeobacterm m
relation to bacterial phys,ology, pathogenes~s and drug
resistance Res Mlcrobiol 142, 451-463
5 Brennan, P J (1989) Structure of mycobacterla Recent
developments m defining cell wall carbohydrates and proterns Rev Inf Dis 11, $420-$430
6 Young, D , Garbe, T , Lathigra, R and Abou-Zeld, C
(1990) Protein antigens structure, funcuon, and regulation In Molecular Biology of Mycobactena (McFadden,
J , Ed ), pp 1-36 Academic Press, London
7 Mmmkm, D E (1991) Chemical principles m the orgamzatlon of hpld components m the mycobacterial cell envelope Res Mlcrobml 142, 423-427
8 Nlkaido, H , Kam, S -H and Rosenberg, E Y (1993) Physi-
cal orgamzatton of hplds m the cell wall of Myeobactenum
chelonae Mol Mlcroblol 8, 1025-1030
9 Assehneau, C and Assehneau, J (1984) Waxes, mycosldes
and related compounds In The Mycobactena, a Sourcebook (Kublca, G P and Wayne, L G , Eds), part A,
pp 345-359 Marcel Dekker, New York
10 Ntkaldo, H and Vaara, M (1985) Molecular basis of
bacterial outer membrane permeability Mlcroblol Rev
49, 1-32
11 Mlzuguchl, Y, Udou, T and Yamada, T (1983) Mechamsm of antibiotic resistance m Mycobactenum mtracellulare MlcroNol Immunol 27, 425-431
12 David, H L , Rastogl, N , Clavel-Seres, S, Clement, F and
Thorel, M F (1987) Structure of the cell envelope of
Myeobactertum at,turn Zbl Bakteriol Hyg A 264, 49-66
13 Kaslk, J E (1979) Mycobactenal /3-1actamases In Betalactamases (Hamdton-Mdler, J M T and Smith, J T , Eds ),
pp 339-350 Academic Press, London
14 Hoffner, S E , Kratz, M , Olsson-Liljeqmst, B, Svensson,
S B and Kallemus, G (1989) In-vttro synergtstlc activity
between ethambutol and fluorinated qumolones against
Mycobactenum a~'tum J Antimlcrob Chemother 24, 317324
15 Jarlier, V and Nlkaldo, H (1990) Permeabdlty barrier to
hydrophdlc solutes in Mycobaetenum chelonet J Bactenol 172, 1418-1423
16 Zlmmermann, W and Rosselet, A (1977) Function of the
outer membrane of Eschertchla coil as a permeabdlty
barrier to beta-lactam antibiotics Ant~mlcrob Agents
Chemother 12, 368-372
17 Tnas, J and Benz, R (1994) PermeaNhty of the cell wall
of Mycobactenum smegmatls Mol Mlcroblol in press
18 Connell, N D and Nikmdo, H (1994) Membrane permeabdlty and transport m Mvcobactertum tuberculosts In
Tuberculosis Pathogenesis, Protectmn, and Control
(Bloom, B R , Ed), pp 333-352 American Society for
MlcroNology, Washington, DC
19 Sundaram, K S and Venkltasubramanmn, T A (1978)
Tryptophan uptake by Mycobactenum tuberculosts H37Rv
effect of rlfampm and ethambutol Ant~microb Agents
Chemother 13, 726-730
20 Trms, J , Jarher, V and Benz, R (1992) Porms m the cell
wall of mycobacterm Science 258, 1479-1481
21 Rastogi, N and Goh, K S (1990)Action of lqsomcotmyl2-palmltoyl hydrazlde against the Mycobactertum avtum
complex and enhancement of its actwlty by m-fluorophenylalanlne Antlmlcrob Agents Chemother 34, 2061-2064
22 Wallace, R J J r , Dalovislo, J R and Pankey, G A (1979)
Disk dfffusmn testing of susceptlNhty of Mycobactertum
fortuttum and Mvcobactertum chelonet to anttbacterml
agents Anttmicrob Agents Chemother 16, 611-614
23 Swenson, J M , Thornsberry, C and Sdcox, V A (1982)
Rapidly growing mycobactena testing of susceptlbdxty to
34 anttmtcrobtal agents by broth dilution Antimlcrob
Agents Chemother 22, 186-192
24 Gelber, R H (1987) Acttvlty of mmocychne in Mycobactertum leprae-mfected mice J Infect Dis 156, 236-239
18
25 Fernandes, P B, Hardy, D J , McDamel, D , Hanson, C W
and Swanson, R N (1982) In vttro and m vwo activales of
clanthromycln against Mycobactenum awum Antimlcrob
Agents Chemother 33, 1531-1534
26 Brown, B A , Wallace, R J J r , Onyl, G O , De Rosas, V ,
Wallace, R J , III (1992) Actwttaes of four macrohdes,
including clanthromycm, against Mycobactenum fortutturn, Mycobactertum chelonae, and M chelonae-hke organisms Antamlcrob Agents Chemother 36, 180-184
27 Gorztnskl, E A , Gutman, S I and Allen, W (1989) Comparative antlmycobacterlal actwatles of dlfloxacm,
temafloxacln, enoxacm, pefloxacm, reference fluoroqumolones, and a new macrohde, clarlthromycm Antlmlcrob Agents Chemotber 33, 591-592
28 Heffets, L B , Lmdhom-Levy, P J and Flory, M A (1990)
Bactenodal activity m vitro of various rffamycms against
Mycobactenum at,turn and Mycobactenurn tubereulosts
Amer Rev Resplr Das 141,626-630
29 Yajko, D M , Sanders, C A , Nassos, P S and Hadley,
W K (1990) In vttro susceptibility of Mycobactenum avaurn
complex to the new fluoroqumolone sparfloxacm (CI-978,
AT-4140) and comparison with oprofloxacm Antlmacrob
Agents Chemother 34, 2442-2444
30 Haemers. A , Leysen, D C , Bollaert, W , Zhang, M and
Pattyn, S R (1990) Influence of N-substitution on antlmycobactenal actwlty of oprofloxacm Antamacrob Agents
Chemother 34, 496-497
31 Franzblau, S G and Whate, K E (1990) Comparative in
vitro activities of 20 fluoroqumolones against Mycobactenurn leprae Antlmacrob Agents Chemother 34, 229-231
32 Nlkaado, H and Normark, S (1987) Sensatway of Eschenchta cob to various /3-1actams is determined by the
interplay of outer Membrane permeabdaty and degradation
by perlplasmac/3-1actamases Mol Macrobtol 1, 29-36
33 Nakmdo, H (1994) Prevention of drug access to bacterial
targets permeablhty barriers and active efflux Scaence
264, 382-388
34 Jarher, V , Gutman, L and Nlkaldo, H (1991) Interplay of
cell wall barrier and /3-1actamase activaty determines hagh
resistance to fl-lactam antlbmtacs m Mycobactenum chelonae Antamacrob Agents Chemother 35 1937-1939
35 Nakaldo, H and Gehrmg, K (1988) Sagmficance o1 outer
membrane barrier m fl-lactam resistance In Antibiotic
Inhabltlon of Bactenal Cell Surface Assembly and Functmn (Actor, P , Daneo-Moore, L , Hlggms, M L , Salton,
M R J and Shockman, G D , Eds ), pp 419-430 Am Soc
Microblol, Washington, DC
36 Hull, S I, Wallace, R J , Jr, Bobey, D G , Price, K E ,
Goodhmes, R A , Swenson, J M and Sdcox, V A (1984)
Presence of ammoglycoslde acetyltransferase and plasmlds
m Mycobactenum fortuztum Am Rev Resp Das 129,
614-618
37 Sorg, T B and Cynamon, M H (1987) Comparison of four
fl-lactamase lnhabltors m combination with ampacdhn
against Mycobactenum tuberculosis J Antlmlcrob
Chemother 19, 59-64
38 Casal, M J , Rodrtguez, F C and Benavente, M (1985) In
vitro susceptlbdaty of Mycobactenum fortmtum and Mycobactenum chelonel to cefmetazole Anttmacrob Agents
Chemother 27, 282-283
39 Cambau, E , Sougakoff, W and Jarher, V (1994) Amphficataon and nucleotide sequence of the qumolone resistance-determmmg regmn tn the gyrA gene of mycobactena FEMS Macrobtol Lett 116, 49-54
40 Taklff, H E , Salazar, L , Guerrero, C , Phlhpp, W , Huang,
W M , Kreaswarth, B , Cole, S T , Jacobs, W R , Jr and
Telentl, A (1994) Cloning and nucleoUde sequence of
Mycobactenum tuberculosts gyrA and gyrB genes and detection of quinolone resistance mutatmns Antimlcrob
Agents Chemother 38, 773-780
41 Mdler, L P , Crawford, J T and Shmmck, T M (1994) The
rpoB gene of Mycobactenum tuberculosts Antamlcrob
Agents Chemother 38, 805-811