448
7th FORUM IN MICROBIOLOGY
Takashima, T., Uela, C., Tsuyuguchi, 1. & Kishimoto, S. (1990), Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis. Infect. lmmun., 58, 3286-3292. Takayama, K. & Dana, A.K. {1989), Isolation and characlerizalion ol" novel myeolate-eontaining $1yeolipids from Cor),nebactetium diphtheriae. Proceedings 24th US-Japan Tuberculosis Conference, San Diego, CA, Aug. 23-25, 1989, pp. 108-111. Takayama, K., Dalta, A.K., Sievert, T., Randall, R.B., Wan,,, R. & Cotter, R.J. (1990), Characterization of a novel mycolate-
eontaininj glycolipid from Mycobacterium smegmatis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 13-17. Takayama. K. & Kilburn, J.O. (1989), Inhibition of syntlae~is of arabinogalactan by ethambutol in Mycobacterium smegmarls. A,~i,microb, Agents a. Chemother., 33, 1493-1499. Takayama, K. & Qureshi, N. (1984), Structure and synthesis of iipids, in "The mycobacteria. A sourcebook" (G.P. Kubica & L.G. Wayne) (pp. 315-344). Marcel Dekker, Inc., New York. Takayama, K., Wang, L. & David,
H.L. (1972), Effect of isoniazid on the in vtvo myeolie acid synthesis, cell growth, and viability of Mycobacterium tuberculosis. Antimicrob. Agents a. Chemother., 2, 29-35. Wallis, R.S., Amir-'l'ahmasseb, M. & EIIner, J.J. (t990), Identification by 2-D gel electrophoresis of monocytes-activating proteins of Mycobacterium tuberculosis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 117-121. Winder, F.G. & Collins, P.B. (1970), Inhibition by isoniazid of synthesis of myeolic acids in Mycobacrerium tuberculosis. J. gen. Microbiok, 63, 41-4g.
Stt dies on the role of the mycobacterial cell envelope in the multiple drug resistance of atypical myeobacteria S.E. Hoffner and S.B. Svenson Division for Mycobacteriology and Department c f Vaccine Development and P, oduclion, The National Bacteriological Laboratory, S-105 21 Stockholm
INTRODUCTION A broad resistance to various antibacterial agents is a common feature of myeobaeteria. The structure and function of the mycobacterial cell envelope is of key importance in this drug resistance. In particular, the captivity of the cell envelope to z¢clude drug entrance is thought t: be one major factor in the resistance o f m y c o b a c t e r i a against several drugs (David 1981; Rastogi et al., 1981). Hence, drugs a f f e c t i n g the biosynthesis and integrity of the mycobacterial cell envelope are of special interest as, by their mode of action, they are likely to enhance the penetration of other drugs which have intrace!!u!ar
targets such as ribosomes, RNA potymerase, etc. A m o n g the most drugresistant of all mycobacteria are those belonging to the Mycobacterium avium complex. Bacteria of this complex are also becoming increasingly clinically important, as they cause severe opportunistic infections in immunesuppressed patients, e.g. in HIVinfected patients, The relevance and the clinical usefulness of resistance/susceptibility data on atypical mycobacteria obtained by conventional in vitro susceptibility tests is doubtful. We feel that the use of conventional test methods such as the resistance ratio method on LdwensteinJensen medium with a susceptible M. tuberculosis strain as
reference should be avoided in the evaluation of drug susceptibility of atypical mycobacteria. A pronounced resistance of e.g. M. avium to each of the commonly used antimycobacterial drugs is often seen when the drugs are tested one by one. Therefore, such drugs are frequently considered to be " n o n effective" and are not recommended for use. However, the oppos[ze result (a high susceptibility) is frequently found when two-drug combinations of such "non-effective" drugs are tested. Tests of combinations of drugs also seem to be more relevant, as in the ciinicel setting combinations of drugs are always used in the treatment of M. avium infections.
STRUCTURE AND FUNCTIONS OF THE M¥COBACTERIAL CELL ENVELOPE
The role of the mycobacterial cell envelope, and especially its capacity to exclude drugs, and also the mechanisms through which drugs may act synergisticglly need better understanding. In this paper, we will briefly discuss some of our in vitro studies of the thermodynamics of the physieochemical interactions betv,'~¢ii drags and the cell envelope of M. avium and also some radio-respirometric studies of drug-induced inhibition of the mycobacterial metabolism. Microcalorimeirie stadies of
drug/cell envflope inletaetions T o investigate the i~itiat interactions between antimycobacterial drugs and the M. avium call envelope, a batch reaction microcalorimeter (LKB, Bromma, Sweden) was used. This instrument is equipped with twin-glass reaction vessel allowing direct estimation of differences in enthalpy changes between the two vessels, erie o f which is carrying the control sample. The method used has been reported earlier (F/offner et e L , 1990). The change in enthaipy during the primary interactions between the celt surface o f clinical isolates o f M, a v i u m and each o f the antibacterial drugs streptomycin, cthambutol, isoniazid and rifampiCin, as well as some of their combinations, was examined. When tested separately, e t h a m b u t o l gave the most pronounced calorimetric response. A strong and rapid endothermic reaction following a doseresponse pattern aver a wide range of ethambutoI concentrations was obtained. This reflects a strong immediate interaction of ethambutol with the outer envelope, which we interpret as a rapid binding of the drug to target structures of the outer cell envelope. Rifam~icin and isoniazid yielded only minor rapid
thermal effects, indicating that these drugs interact with the cell envelope in a non-binding fashion, WI-..m the M. avium cells were exposed to combinations of ethambutol and streptomycin (a combination which is bactericidal; see below), the thermal responses were similar to that seen for ethambutol alone, showing that the initial strong interaction with ethambutol is not affected by streptomycin. To investigate whether prior exposure of cells to ethambutol affects the subsequent interaction of a second drug, a series of experiments in which M. avium cells were first exposed to sublethal c o n c e n t r a t i o n s of ethambutol and subsequently exposed to streptomycin were carried out. A completely different thermal r e s p o n s e was obtained to streptomycin as compared to bacteria not earlier exposed to ett~ambutoL The reverse was not the case: proexposure of M. a v i . m to streptomycin did not a!tcr the initial chemical interaction of ethambutol. This suggests a change in the nature of the initial interaction of streptomycin aiter treatment with e t b a m b u t o i . Our hypothesis is that this change allows better penetration of the aminoglycoside, allowing it to act on its intracellular targets (i.e. the rib0so~es) and thereby further potentiate the antibacterial activity. The initial interactions between ethambatol and the M. ttvi~,,,n cell envelope of live and killed 1t4. aviu;n cells were compared in anothe~ se~ of experiments. The thermodynamics of the interaction between ethambutol and live or OV-killed M. avium ceils were very much the same. By contrast, heat- or ethanol-killed cells showed completely different thermal response curves, These data lead us to propose that some he::~t-labile and ethanol-
449
sensitive structure(s) (most likely of protein nature) serves as a receptor{s) for ethambutoi on the M. avium cell envelope (Hoffner et el., 1989a).
Radiorespirometric studies of the ar~ilmycobacterial effects o| eombiaed drags, and its relation with the mycobacterial cell envelope In contrast to most strains of M. tuberculosis, which generally show a primary susceptibility to all or most antimy¢obacteriaf drugs, many other clinically relevant myeobacteria are inherently muhiresistant. The most important example of lhi~ is the M. avium complex, in which the ceil ~'~all ztruetar¢ and its function as an exclusion barrier for many drugs has been shown to be a major resistance mechanism (David, 19gl, Rastogi et at., 1981). We have used the "Barter" r~diometric system {BectonDJckinson, MD, USA) for in vitro investigations of the antimycobacterial effects of various drug combinations. In this assay, the respirometric activity, registered ks the production o f 14co2, is compared in culturing vials with ~C-labelled substrate (Middlebrook et el., 1977) with or without the addition of a drug/drug-combination- In the first study, we investigated the antimyeobacterial interactions of all combinations of the drugs ethambutol, streptomycin, rifampicin and isoniazid against cl~rficol isolates of the M. aviurn complex. To make possible a irecise quantiticatior~ of the synergistic antimycebactcfial effect of a drug combination, we defined an interaction quotient based on the registered reduction of metabolic activity. Using this quotient, the level of inhibitory effect was calculated, and the antimycobacterial interactions of any drug combination could be classed as
450
7th FORUM IN MICROBIOLOGY
syncrgisik, additive, independeal or antagonistic (Hoffner et al., 1987). In this study, drug combinations of ethambutol with either streptomycin or rifampiein were shown to achieve a very pronounced antibacterial effect against all tested M. avium strains. Later, similar drug synergistic effects were demonstrated for combinations of ethambutol with fluorinated quinoline drugs (Hoffner et al. 1989b, Olsson-Liljequist et al., 1989), erytbromycin, and for some of the tested M. avium strains aiso with ceftazidime or fusidic acid (Hoffner et aL, 1989a). From these studies, we suggested that ethambutol is a key substance in potentiating the antimycohacterial aetivily of a wide spectrum of other antibacterial drugs, and that this is mediated through a reduction in the exclusion barrier of the mycohacteriaI cell envelope. M,~c¢ recently, we have investigated the drug susccptihiliW of ~ n.~,rwnberof clinical isolates of atypical mycobaeteria after pre-exposure to various a,itimycobacteria[ drugs. In these experiments, the bacteria were first exposed to a drug and thereafter subcultured for several generations in a drug.free broth. These cultures were sttbsequently exposed to another drug, All isolates appeared to be significantly more susceptible after pre-exposure to ethambutoi as compared to the corresponding contmi cultures. This effect of ethambutol was more pronounced when pre-exposurc were done at the higher concentrations (i0 compared to 2.5 rag/l) and also when longer periods of pre-exposure (7 compared to 3 days) were used. Thus, also in these experiments, ethambutol was shown to he the key to potentiate the effect of tile other drugs. This finding was consistent for all species tested (submitted).
Discussion and conclusions By combining studies of enthalpy changes by microcalorimetry and the more longlasting effects on the metabolism by radio-respirometry, information on both the initial physicochemical interactions and the subsequent biological effects on myeobacteria can be obtained. We have shown that ethambutol strongly interacts with the myeobacterial outer envelope and that it also affects the initial chemical interaction of other drugs. This subsequently leads te a strong reduction in the drug resistance of M. avium (K~illea[us el aL, 1989). We conclude that there must be at least two different mechanisms involved in the ethambutoldePendent modification of the cell envelope of M. avium: one which acts very rapidly and another of a more long-lasting character, which reduces the mycobaeterial resistance and impairs the ability of the cell enveiope to exclude drugs. This latter effect lasts for at least several generations after the withdrawal of ethambutol. We envisage two major possibilities for how ethambutol interferes with the permeability barrier of the mycobacterial cell. First, the initial rapid interaction between ethambutol and the mycobacterial cell envelope in itself could interfere with the permeability barrier. Secondly, and more likely, the breakdown of the permeability barrier may be a secondary event caused by an et hambutoI-induced disturbance of biosynthesis artd hence the integrity of the cell wall. Experiments to study this it1 more detail are in progress. Radiometric respir(~rnetry enables investigations of the antimycobacterial activity of drugs/drug combinations and also of modifications in drugs designed to overcome the ceil envelope permeability barrier of
M. avium. Recently, Rastogi and
coworkers examined the antimycobaeterial activity of a derivative of isoniazid which was made amphiphatic by the introduction of a palmitoyl moiety. This derivative was found to be more active against M , avium than the corresponding native hydrophilie isoniazid itself, but an even better effect was seen when the modified drag was combined with mfluorophenylalanine, an inhibitor of mycoside C biosynthesis (Rastogi and Goh, 1990). This substance also increased the antimyeobaeterial effect of other drugs, such as rifampicin and fluorinated quinolones (Rastogi et.al., 1990), An important conclusion from our work is that when susceptibility tests are performed on atypical mycobacteria, they should include studies of the effects of combinations of drugs. This is also supported hy the fzcr. that patients with myeobacterial infections are not treated with single-drug therapy. Indeed, the clinical finding i ~-that such multidrug regimens often show good therapeutic effects. This, albeit the drugs included in such regimens, when tested one by one, show no antimycobacterial activity in vitro, This agrees with and is further supported by our findings that ethambutol, even at very low concentrations, strongly potentiates the antimycobacterial effects of several other drugs. We feel that such synergistic drug interactions are of clinical importance and that they have practical consequences for routine laboratory susceptibility testing of atypical mycobacteria. We also suggest that the earlier disregard for several "old" drugs (based on their lack of in vitro activity when tested separately) should be re-evaluated. The growing understanding of the architecture and function of the mycobacterial cell enve-
STRUCTURE AND FUNCTIONS OF THE MYCOBACTERIAL CELL ENVELOPE lope allows the emergence of new and more rational routes for designing new drugs and evaluating new drug combinations in the effort to combat mycobacterial infections.
Our thanks to Drs. Gunilla K~.llenius, Anthony E. Boozer and Ulrika Hjelm for important contributions. Financial support by The Swedish National Association against Heart and Lung Disease and King Oscar It's Jubilee Foundation is gratefully acknowledged.
References
David, H.L. l198t), Basis for lack of drug susceptibility of atypical myeobaeteria. Roy. infect. Dis., 3, 878-884. Hoffner, S.E., Svenson, S.B. & Kallenius, G. (1987), Synergistic of feels of antimycobacterial drugs on Myeobacterium avium complex determined radiometrically in liquid medium. Europ. J. Clin. MicrobioL, 6, 530-535.
Hoffner, S.E. K~itlenius, O., Beezer, A.E., & Svenson, S.B. (1989a), Studies on the mechanisms of the synergistie effects of ethambutol and other antibacterial drugs on Mycobacterium aviura complex. Aeta Leprol., 7 (Suppl i), 195-199. Hoffner, S.E., Kratz, M., OlssonLiljequist, B., Svenson, S.B. & K~illenius, G. (1989b), In vitro synergistic activity between ethambutol and fluorinated quinolones against Mycobacterium avium cornpiex, d. Antimicrob. Chemother., 24, 317-324. Hoffner, S.E., Svenson, S.B. & Beezer, A.E. (1990), Microcalorimetric studies of the initial interaction between antimyeobacterial drugs and Mycobacterium avium. J. Antimicrob. Chemotker., 25, 353-359. K~illenius, G., Svenson, S.B. & Hoffner, S.E. (1999), Ethambutol: a key for Mycobaeterium avium complex chemotherapy? Amer. Roy. Respir. Dis., 140, 264. Middlebrook, G., Reggiardo, Z. & Tigertt, W.D. (1977), Automatable radiometric detection of growth of Mycobacterium
451
tuberculosis in selective media. Amer. Roy. Respir. Dis., ltS, 1066-1069. Olsmn-Liljequist, B., Kallenius, G., Svenson, S.B. & Hoffner, S.E. 0989), Activity of ciprofloxacin and other quinoiones alone and in combination with
ethambutol against Mycobacterium avium complex. Roy, Jnfect. Dis., II, S1044-S1045. Rastogi, N., F#'hel, C., Ryter, A., Ohayon, H., Lesourd, M. & David, H.L. (1981), Multiple drug resistance in Mycobaeterium avium: is the wall architecture responsible for exclusion of antimicrobial agents? Antimicrob. Agents a. Chemother., 20, 666-677. Rastogi, N., Gob K.S. & David, H.L. (1990), Enhancement of ~2rug susceptibility of Mycobacterium avium by inhibitors of ceil envelope synthesis. Anlimierob. Agents a. Chemother., 34, 759-764. Rastogi, N. & Ooh K.S. (1990), Action of l-isonicotinyl-2-palmitoyl hydrazine against the Mycobacrerium avium complex and enhancement of its activity by m- fluorophenylalanine. Antimierob. Agents a. Chemother., 34, 2061-2064.
Structure, function and biogenesis of the cell envelope of mycobacteria in relation to bacterial physiology, pathogenesis and drug resistance; some thoughts and possibilities arising from recent structural information M.R.
McNeil and P.J. Brennan
Department o f Microbiology, Colorado State University, Fort Collins, C O 80523 (USA)
1. - - I n t r o d u c t i o n a n d hislorical c o n s i d e r a t i o n s
The standard schematic molecular models o f the cell
envelope o f Escherichia coli, those that appear in countless textbooks and review articles (Davis e t a l . , 1980; DiRienzo et aL, 1978; Freer, 1985; Luften-
berg and van Alpen, 1983 ; Raetz and Dawhan, 1990), the origins o f which are seemingly lost ( N i k a i d o a n d N a k a e , 1979; Osborn a n d Wu, 1980; Schnait-
7th FORUM IN MICROBIOLOGY
Takashima, T., Uela, C., Tsuyuguchi, 1. & Kishimoto, S. (1990), Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis. Infect. lmmun., 58, 3286-3292. Takayama, K. & Dana, A.K. {1989), Isolation and characlerizalion ol" novel myeolate-eontaining $1yeolipids from Cor),nebactetium diphtheriae. Proceedings 24th US-Japan Tuberculosis Conference, San Diego, CA, Aug. 23-25, 1989, pp. 108-111. Takayama, K., Dalta, A.K., Sievert, T., Randall, R.B., Wan,,, R. & Cotter, R.J. (1990), Characterization of a novel mycolate-
eontaininj glycolipid from Mycobacterium smegmatis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 13-17. Takayama. K. & Kilburn, J.O. (1989), Inhibition of syntlae~is of arabinogalactan by ethambutol in Mycobacterium smegmarls. A,~i,microb, Agents a. Chemother., 33, 1493-1499. Takayama, K. & Qureshi, N. (1984), Structure and synthesis of iipids, in "The mycobacteria. A sourcebook" (G.P. Kubica & L.G. Wayne) (pp. 315-344). Marcel Dekker, Inc., New York. Takayama, K., Wang, L. & David,
H.L. (1972), Effect of isoniazid on the in vtvo myeolie acid synthesis, cell growth, and viability of Mycobacterium tuberculosis. Antimicrob. Agents a. Chemother., 2, 29-35. Wallis, R.S., Amir-'l'ahmasseb, M. & EIIner, J.J. (t990), Identification by 2-D gel electrophoresis of monocytes-activating proteins of Mycobacterium tuberculosis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 117-121. Winder, F.G. & Collins, P.B. (1970), Inhibition by isoniazid of synthesis of myeolic acids in Mycobacrerium tuberculosis. J. gen. Microbiok, 63, 41-4g.
Stt dies on the role of the mycobacterial cell envelope in the multiple drug resistance of atypical myeobacteria S.E. Hoffner and S.B. Svenson Division for Mycobacteriology and Department c f Vaccine Development and P, oduclion, The National Bacteriological Laboratory, S-105 21 Stockholm
INTRODUCTION A broad resistance to various antibacterial agents is a common feature of myeobaeteria. The structure and function of the mycobacterial cell envelope is of key importance in this drug resistance. In particular, the captivity of the cell envelope to z¢clude drug entrance is thought t: be one major factor in the resistance o f m y c o b a c t e r i a against several drugs (David 1981; Rastogi et al., 1981). Hence, drugs a f f e c t i n g the biosynthesis and integrity of the mycobacterial cell envelope are of special interest as, by their mode of action, they are likely to enhance the penetration of other drugs which have intrace!!u!ar
targets such as ribosomes, RNA potymerase, etc. A m o n g the most drugresistant of all mycobacteria are those belonging to the Mycobacterium avium complex. Bacteria of this complex are also becoming increasingly clinically important, as they cause severe opportunistic infections in immunesuppressed patients, e.g. in HIVinfected patients, The relevance and the clinical usefulness of resistance/susceptibility data on atypical mycobacteria obtained by conventional in vitro susceptibility tests is doubtful. We feel that the use of conventional test methods such as the resistance ratio method on LdwensteinJensen medium with a susceptible M. tuberculosis strain as
reference should be avoided in the evaluation of drug susceptibility of atypical mycobacteria. A pronounced resistance of e.g. M. avium to each of the commonly used antimycobacterial drugs is often seen when the drugs are tested one by one. Therefore, such drugs are frequently considered to be " n o n effective" and are not recommended for use. However, the oppos[ze result (a high susceptibility) is frequently found when two-drug combinations of such "non-effective" drugs are tested. Tests of combinations of drugs also seem to be more relevant, as in the ciinicel setting combinations of drugs are always used in the treatment of M. avium infections.
STRUCTURE AND FUNCTIONS OF THE M¥COBACTERIAL CELL ENVELOPE
The role of the mycobacterial cell envelope, and especially its capacity to exclude drugs, and also the mechanisms through which drugs may act synergisticglly need better understanding. In this paper, we will briefly discuss some of our in vitro studies of the thermodynamics of the physieochemical interactions betv,'~¢ii drags and the cell envelope of M. avium and also some radio-respirometric studies of drug-induced inhibition of the mycobacterial metabolism. Microcalorimeirie stadies of
drug/cell envflope inletaetions T o investigate the i~itiat interactions between antimycobacterial drugs and the M. avium call envelope, a batch reaction microcalorimeter (LKB, Bromma, Sweden) was used. This instrument is equipped with twin-glass reaction vessel allowing direct estimation of differences in enthalpy changes between the two vessels, erie o f which is carrying the control sample. The method used has been reported earlier (F/offner et e L , 1990). The change in enthaipy during the primary interactions between the celt surface o f clinical isolates o f M, a v i u m and each o f the antibacterial drugs streptomycin, cthambutol, isoniazid and rifampiCin, as well as some of their combinations, was examined. When tested separately, e t h a m b u t o l gave the most pronounced calorimetric response. A strong and rapid endothermic reaction following a doseresponse pattern aver a wide range of ethambutoI concentrations was obtained. This reflects a strong immediate interaction of ethambutol with the outer envelope, which we interpret as a rapid binding of the drug to target structures of the outer cell envelope. Rifam~icin and isoniazid yielded only minor rapid
thermal effects, indicating that these drugs interact with the cell envelope in a non-binding fashion, WI-..m the M. avium cells were exposed to combinations of ethambutol and streptomycin (a combination which is bactericidal; see below), the thermal responses were similar to that seen for ethambutol alone, showing that the initial strong interaction with ethambutol is not affected by streptomycin. To investigate whether prior exposure of cells to ethambutol affects the subsequent interaction of a second drug, a series of experiments in which M. avium cells were first exposed to sublethal c o n c e n t r a t i o n s of ethambutol and subsequently exposed to streptomycin were carried out. A completely different thermal r e s p o n s e was obtained to streptomycin as compared to bacteria not earlier exposed to ett~ambutoL The reverse was not the case: proexposure of M. a v i . m to streptomycin did not a!tcr the initial chemical interaction of ethambutol. This suggests a change in the nature of the initial interaction of streptomycin aiter treatment with e t b a m b u t o i . Our hypothesis is that this change allows better penetration of the aminoglycoside, allowing it to act on its intracellular targets (i.e. the rib0so~es) and thereby further potentiate the antibacterial activity. The initial interactions between ethambatol and the M. ttvi~,,,n cell envelope of live and killed 1t4. aviu;n cells were compared in anothe~ se~ of experiments. The thermodynamics of the interaction between ethambutol and live or OV-killed M. avium ceils were very much the same. By contrast, heat- or ethanol-killed cells showed completely different thermal response curves, These data lead us to propose that some he::~t-labile and ethanol-
449
sensitive structure(s) (most likely of protein nature) serves as a receptor{s) for ethambutoi on the M. avium cell envelope (Hoffner et el., 1989a).
Radiorespirometric studies of the ar~ilmycobacterial effects o| eombiaed drags, and its relation with the mycobacterial cell envelope In contrast to most strains of M. tuberculosis, which generally show a primary susceptibility to all or most antimy¢obacteriaf drugs, many other clinically relevant myeobacteria are inherently muhiresistant. The most important example of lhi~ is the M. avium complex, in which the ceil ~'~all ztruetar¢ and its function as an exclusion barrier for many drugs has been shown to be a major resistance mechanism (David, 19gl, Rastogi et at., 1981). We have used the "Barter" r~diometric system {BectonDJckinson, MD, USA) for in vitro investigations of the antimycobacterial effects of various drug combinations. In this assay, the respirometric activity, registered ks the production o f 14co2, is compared in culturing vials with ~C-labelled substrate (Middlebrook et el., 1977) with or without the addition of a drug/drug-combination- In the first study, we investigated the antimyeobacterial interactions of all combinations of the drugs ethambutol, streptomycin, rifampicin and isoniazid against cl~rficol isolates of the M. aviurn complex. To make possible a irecise quantiticatior~ of the synergistic antimycebactcfial effect of a drug combination, we defined an interaction quotient based on the registered reduction of metabolic activity. Using this quotient, the level of inhibitory effect was calculated, and the antimycobacterial interactions of any drug combination could be classed as
450
7th FORUM IN MICROBIOLOGY
syncrgisik, additive, independeal or antagonistic (Hoffner et al., 1987). In this study, drug combinations of ethambutol with either streptomycin or rifampiein were shown to achieve a very pronounced antibacterial effect against all tested M. avium strains. Later, similar drug synergistic effects were demonstrated for combinations of ethambutol with fluorinated quinoline drugs (Hoffner et al. 1989b, Olsson-Liljequist et al., 1989), erytbromycin, and for some of the tested M. avium strains aiso with ceftazidime or fusidic acid (Hoffner et aL, 1989a). From these studies, we suggested that ethambutol is a key substance in potentiating the antimycohacterial aetivily of a wide spectrum of other antibacterial drugs, and that this is mediated through a reduction in the exclusion barrier of the mycohacteriaI cell envelope. M,~c¢ recently, we have investigated the drug susccptihiliW of ~ n.~,rwnberof clinical isolates of atypical mycobaeteria after pre-exposure to various a,itimycobacteria[ drugs. In these experiments, the bacteria were first exposed to a drug and thereafter subcultured for several generations in a drug.free broth. These cultures were sttbsequently exposed to another drug, All isolates appeared to be significantly more susceptible after pre-exposure to ethambutoi as compared to the corresponding contmi cultures. This effect of ethambutol was more pronounced when pre-exposurc were done at the higher concentrations (i0 compared to 2.5 rag/l) and also when longer periods of pre-exposure (7 compared to 3 days) were used. Thus, also in these experiments, ethambutol was shown to he the key to potentiate the effect of tile other drugs. This finding was consistent for all species tested (submitted).
Discussion and conclusions By combining studies of enthalpy changes by microcalorimetry and the more longlasting effects on the metabolism by radio-respirometry, information on both the initial physicochemical interactions and the subsequent biological effects on myeobacteria can be obtained. We have shown that ethambutol strongly interacts with the myeobacterial outer envelope and that it also affects the initial chemical interaction of other drugs. This subsequently leads te a strong reduction in the drug resistance of M. avium (K~illea[us el aL, 1989). We conclude that there must be at least two different mechanisms involved in the ethambutoldePendent modification of the cell envelope of M. avium: one which acts very rapidly and another of a more long-lasting character, which reduces the mycobaeterial resistance and impairs the ability of the cell enveiope to exclude drugs. This latter effect lasts for at least several generations after the withdrawal of ethambutol. We envisage two major possibilities for how ethambutol interferes with the permeability barrier of the mycobacterial cell. First, the initial rapid interaction between ethambutol and the mycobacterial cell envelope in itself could interfere with the permeability barrier. Secondly, and more likely, the breakdown of the permeability barrier may be a secondary event caused by an et hambutoI-induced disturbance of biosynthesis artd hence the integrity of the cell wall. Experiments to study this it1 more detail are in progress. Radiometric respir(~rnetry enables investigations of the antimycobacterial activity of drugs/drug combinations and also of modifications in drugs designed to overcome the ceil envelope permeability barrier of
M. avium. Recently, Rastogi and
coworkers examined the antimycobaeterial activity of a derivative of isoniazid which was made amphiphatic by the introduction of a palmitoyl moiety. This derivative was found to be more active against M , avium than the corresponding native hydrophilie isoniazid itself, but an even better effect was seen when the modified drag was combined with mfluorophenylalanine, an inhibitor of mycoside C biosynthesis (Rastogi and Goh, 1990). This substance also increased the antimyeobaeterial effect of other drugs, such as rifampicin and fluorinated quinolones (Rastogi et.al., 1990), An important conclusion from our work is that when susceptibility tests are performed on atypical mycobacteria, they should include studies of the effects of combinations of drugs. This is also supported hy the fzcr. that patients with myeobacterial infections are not treated with single-drug therapy. Indeed, the clinical finding i ~-that such multidrug regimens often show good therapeutic effects. This, albeit the drugs included in such regimens, when tested one by one, show no antimycobacterial activity in vitro, This agrees with and is further supported by our findings that ethambutol, even at very low concentrations, strongly potentiates the antimycobacterial effects of several other drugs. We feel that such synergistic drug interactions are of clinical importance and that they have practical consequences for routine laboratory susceptibility testing of atypical mycobacteria. We also suggest that the earlier disregard for several "old" drugs (based on their lack of in vitro activity when tested separately) should be re-evaluated. The growing understanding of the architecture and function of the mycobacterial cell enve-
STRUCTURE AND FUNCTIONS OF THE MYCOBACTERIAL CELL ENVELOPE lope allows the emergence of new and more rational routes for designing new drugs and evaluating new drug combinations in the effort to combat mycobacterial infections.
Our thanks to Drs. Gunilla K~.llenius, Anthony E. Boozer and Ulrika Hjelm for important contributions. Financial support by The Swedish National Association against Heart and Lung Disease and King Oscar It's Jubilee Foundation is gratefully acknowledged.
References
David, H.L. l198t), Basis for lack of drug susceptibility of atypical myeobaeteria. Roy. infect. Dis., 3, 878-884. Hoffner, S.E., Svenson, S.B. & Kallenius, G. (1987), Synergistic of feels of antimycobacterial drugs on Myeobacterium avium complex determined radiometrically in liquid medium. Europ. J. Clin. MicrobioL, 6, 530-535.
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Structure, function and biogenesis of the cell envelope of mycobacteria in relation to bacterial physiology, pathogenesis and drug resistance; some thoughts and possibilities arising from recent structural information M.R.
McNeil and P.J. Brennan
Department o f Microbiology, Colorado State University, Fort Collins, C O 80523 (USA)
1. - - I n t r o d u c t i o n a n d hislorical c o n s i d e r a t i o n s
The standard schematic molecular models o f the cell
envelope o f Escherichia coli, those that appear in countless textbooks and review articles (Davis e t a l . , 1980; DiRienzo et aL, 1978; Freer, 1985; Luften-
berg and van Alpen, 1983 ; Raetz and Dawhan, 1990), the origins o f which are seemingly lost ( N i k a i d o a n d N a k a e , 1979; Osborn a n d Wu, 1980; Schnait-
