Synergic activity of D-cycloserine and ß-chloro-D-alanine against Mycobacterium tuberculosis

Suzana David,*

Unit of Mycobacteriology and Centro de Malária e outras Doenças Tropicais /IHMT/Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisbon, Portugal


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
D-Cycloserine (DCS) is a peptidoglycan inhibitor. Although very effective against Mycobacterium tuberculosis, it is seldom employed in the management of this infection due to its high toxicity. ß-Chloro-D-alanine (another peptidoglycan inhibitor) reduces the MIC of DCS from 50 to 2.5 mg/L at a concentration significantly below its MIC for this organism. A reduction in bacterial viability and significant growth inhibition (as quantified by the X/Y quotient for the Bactec radiometric procedure) were observed with subinhibitory concentrations of both drugs. It is suggested that this powerful synergic effect should be the object of in vivo and eventually clinical trials.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
D-Cycloserine (DCS) is an important drug in the treatment of tuberculosis. However, it is seldom used in the management of this disease owing to toxicity associated with effective dosages. Nevertheless, the worldwide resurgence of tuberculosis, the emergence of multiply drug-resistant tuberculosis and the problematic use of available drugs required to treat these infections have resulted in the proposal that DCS now be considered as an alternative therapy.1

Compounds that increase the permeability of the mycobacterial cell wall by inhibiting the synthesis of cell wall components enhance the activity of conventional drugs as a result of increased penetration of these latter agents to susceptible internal targets.2 This enhancement of antimicrobial activity theoretically affords the use of lower concentrations of antibiotics associated with toxicity.

In Mycobacterium tuberculosis as in other bacteria, DCS is primarily an inhibitor of peptidoglycan synthesis.3 DCS is an analogue of D-alanine known to inhibit M. tuberculosis cell wall synthesis by competing with D-alanine for the essential enzymes D-alanyl-D-alanine synthetase and D-alanine racemase. The primary site of action seems to be D-alanyl-D-alanine synthetase. DCS also inhibits the synthesis of mycoside-C, a D-alanine-containing peptidoglycolipid of the cell wall in Mycobacterium avium.2

O-carbamyl-D-serine and DCS when employed in combination exerted a synergic effect against M. tuberculosis. These drugs are known to inhibit distinct enzymes involved in the sequential synthesis of the peptidoglycan. The potentiation of DCS activity against mycobacterial growth by O-carbamyl-D-serine is effective in part through its inhibition of D-alanine racemase.4 ß-Chloro-D-alanine (ßCDA), another alanine analogue, is an inhibitor of D-glutamate-D-alanine transaminase and alanine racemase.5,6 It has been shown to act synergically to inhibit bacterial growth in the presence of ß-lactam antibiotics.7,8 However, the combination has not achieved clinical application.

Since ßCDA is known to affect enzymes involved in peptidoglycan synthesis, we investigated the combined effect of this compound on the antituberculous activity of DCS.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Strains

The M. tuberculosis clinical isolate employed in this study (38396) was susceptible to first-line antibiotics as determined using the Bactec 460TB radiometric criteria, SIRE (Becton Dickinson, Sparks, MD, USA). It has been tested by us previously in drug combination studies.9 Work was not carried out on the M. tuberculosis type strain H37Rv as this strain is particularly sensitive to DCS, with Bactec MICs of 5–10 mg/L. The selected strain was subcultured in Bactec 12B vials before the experiments.

Drugs

DCS and ßCDA were purchased from Sigma–Aldrich Quimica, S.A. (Madrid, Spain). Stock solutions were prepared in sterile distilled water, sterilized by filtration using 0.2 µm Filtropur filters (Sarstedt, Nümbrecht, Germany) and frozen at –20°C until use.

In vitro drug combination studies

In vitro activities of D-cycloserine and ßCDA used alone or in combination were carried out using subinhibitory concentrations of these drugs. For this, MICs were determined using procedures recommended for the Bactec 460 radiometric system.9,10 Briefly, a control was prepared containing a 1 x 102 dilution of the initial innoculum used in the drug-containing vials, termed the 1:100 control. When the growth index (GI) of the 1:100 control vial reached 30, the GI was read on one additional day. The difference in the GI values ({triangleup}GI) was calculated for these 2 days. The MIC corresponding to the drug concentration resulting in more than 99% inhibition of the bacterial population, was defined as the lowest concentration for which the {triangleup}GI of the drug-containing vial was less than the {triangleup}GI of the 1:100 control, obtained from the reading after the GI had reached 30.

Viability was determined by plating the bacterial suspensions from individual Bactec vials at the beginning and at the end of the experiments.10 For this purpose, 0.1 mL of the culture from Bactec vials was taken and serially diluted 10-fold to provide successive dilutions ranging from 10–1 to 10–5. Bactericidal activity was determined by plating a 0.1 mL aliquot from each of the 10-fold dilutions on Mycobacteria 7H11 agar (Difco Laboratories, Detroit, MI, USA). Colony forming units (cfu) were assessed after 21 days' incubation at 37°C. The successive dilutions and the minimal plating volume used under our experimental conditions avoided any artefactual decrease in bacterial viable counts because of drug carry-over.

Inhibition studies were carried out comparing the bacterial population (cfu/mL), from individual Bactec vials, after 10 days' incubation in the absence or the presence of the drugs.

The combined effects of subinhibitory concentrations of the drugs were estimated using the X/Y quotient as described previously.9 In these calculations, X refers to the Bactec GI (see above) obtained with the drug combination, and Y to the lowest GI obtained at the same time with either drug used alone. The final quotient was expressed as the average value obtained from various Bactec 460 readings during exponential growth. An X/Y value of 1 indicated that there was no interaction between the two, an X/Y of <0.5 indicated an enhanced drug action and an X/Y of >2.0 indicated the presence of antagonism.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The potentiation of DCS activity by ßCDA was well demonstrated. Bactec MICs of DCS and ßCDA were 50 mg/L and 100 mg/L, respectively, for the clinical isolate used in this study. In the presence of low concentrations of ßCDA (5–10 mg/L) the MIC of DCS was reduced to as low as 2.5 mg/L. This represented a 20-fold reduction in the DCS MIC.

Results from the viability studies are shown in the FigureGo. Maximal reductions in viability of over three log units were observed in combinations of ßCDA with 0.5 x MIC of DCS for this strain. Significant drops were also obtained using combinations with lower concentrations of DCS, such as 2.5 mg/L (one-twentieth the MIC).



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Figure. Combined effect of various concentrations of DCS and ßCDA on M. tuberculosis viability expressed as the difference in the logarithms of the number of cfu/mL on the 10th day of incubation (t10) and that of cfu/mL on the day of inoculation (t0).

 
The increase in DCS activity in the presence of ßCDA was confirmed by inhibition studies (TableGo). Over 99% inhibition was obtained with five of the drug combinations tested each using concentrations of DCS far below the MIC of the drug. This was further confirmed from quantified X/Y calculations yielding values of <0.5 for all the combinations tested, indicating synergic interaction.


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Table. Results of the combined action of subinhibitory concentrations of DCS and ßCDA against M. tuberculosis, expressed as percentage inhibition or quantified by the X/Y quotient
 
In conclusion, these investigations showed that DCS and ßCDA act synergically against M. tuberculosis. This may occur by inhibiting sequential enzymic reactions necessary for peptidoglycan synthesis, similarly to the way in which O-carbamyl-D-serine acted synergically with DCS on mycobacterial growth inhibition.4 Further investigations would elucidate the biochemical basis of this synergic effect.

The powerful synergy that has been observed between DCS and ßCDA is a potentially important finding for the development of new effective drug regimens against tuberculosis. This is especially important when standard regimens cannot be recommended such as in the case of multiply drug-resistant tuberculosis. We suggest that the extent of this observation should be confirmed in other clinical isolates of M. tuberculosis, including multiply drug-resistant isolates, and should eventually be considered for clinical trials.


    Acknowledgments
 
I would like to thank Professor Leonard Amaral and Professor Hugo L. David, Instituto de Higiéne e Medicina Tropical and Centro de Malaria e Outras Doenças Tropicais (CMDT/IHMT), Lisbon, Portugal, for fruitful discussions and help in preparing the manuscript. I also thank Josefina Almeida and Rosário Bettencourt of the Mycobacteriology Unit for technical assistance. This investigation received financial support from the Comissão Nacional de Luta Contra a SIDA (CNLS), Portugal. Suzana David was a recipient of a post-doctoral fellowship from PRAXIS XXI BPD/9921/96 Fundação para a Ciência e Tecnologia (FCT), Portugal.


    Notes
 
* Tel/Fax: +351-1-362-24-58; E-mail: suzanadavid{at}ihmt.unl.pt Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Wolinsky, E. (1993). Statement of the Tuberculosis Committee of the Infectious Diseases Society of America. Clinical Infectious Diseases 16, 627–8.[ISI][Medline]

2 . David, H. L., Rastogi, N., Clavel-Seres, S. & Clement, F. (1988). Alterations on the outer wall architecture caused by the inhibition of mycoside C biosynthesis in Mycobacterium avium. Current Microbiology 17, 61–8.[ISI]

3 . David, H. L., Takayama, K. & Goldman, D. S. (1969). Susceptibility of mycobacterial D-alanyl-D-alanine synthetase to D-cycloserine. American Review of Respiratory Diseases 100, 579–81.

4 . David, H. L. (1970). Effect of O-carbamyl-D-serine on the growth of Mycobacterium tuberculosis. American Review of Respiratory Disease 102, 68–74.[ISI][Medline]

5 . Henderson, L. L. & Johnston, R. B. (1976). Inhibition studies of the enantiomers of beta chloroalanine on purified alanine racemase from B. subtilis. Biochemical and Biophysical Research Communications 68, 793–8.[ISI][Medline]

6 . Manning, J. M., Merrifield, N. E., Jones, W. M. & Gotschlich, E. C. (1974). Inhibition of bacterial growth by ß-chloro-D-alanine. Proceedings of the National Academy of Sciences, USA 71, 417–21.[Abstract]

7 . Soper, T. S. & Manning, J. M. (1976). Synergy in the antimicrobial action of penicillin and ß-chloro-D-alanine in vitro. Antimicrobial Agents and Chemotherapy 9, 347–9.[ISI][Medline]

8 . Sieradzki, K. & Tomasz, A. (1997). Suppression of ß-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureusthrough synergic action of early cell wall inhibitors and some other antibiotics. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 47–51.[Abstract/Free Full Text]

9 . Bosne-David, S., Barros, V., Cabo Verde, S., Portugal, C. & David, H. L. (2000). Intrinsic resistance of Mycobacterium tuberculosis to clarithromycin is effectively reversed by subinhibitory concentrations of cell wall inhibitors. Journal of Antimicrobial Chemotherapy 46, 391–5.[Abstract/Free Full Text]

10 . Rastogi, N., Goh, K. S., Bryskier, A. & Devallois, A. (1996). In vitro activities of levofloxacin used alone and in combination with first- and second-line antituberculous drugs against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 40, 1610–6.[Abstract]

Received 29 June 2000; returned 12 September 2000; revised 30 October 2000; accepted 31 October 2000