Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms.

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na(+), K(+) ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na(+), K(+) ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na(+), K(+) ratios within a bacterial population. For this, the limiting value of the Na(+), K(+) ratio (up to which bacteria are viable) -which had been determined as 0.45 for cultivable bacteria - has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and - probably due to the restricted metabolism of M. leprae in a cell-free medium - ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.

Comparison of the intrabacterial Na+,K(+)-ratio and multiplication in the mouse foot pad as measures of the proportion of viable Myobacterium lepraemurium.

Drug are generally screened for activity against Mycobacterium leprae by administration to M. leprae-infected mice, and the efficacy of a chemotherapeutic regimen is assessed by inoculating mice with M. leprae recovered from the skin-biopsy specimens obtained at intervals during treatment. Both methods are expensive and time consuming. Although a number of methods has been proposed for the rapid distinction between viable and non viable M. leprae, none has found wide acceptance. Earlier work had shown that the ratios of the intrabacterial concentrations of Na(+) and K(+) (Na(+),K(+)-ratio) of individual bacterial cells, measured by means of laser microprobe mass analysis, are a sensitive indicator of the viability of cultivable organisms. Assuming that the maximal value (the "limiting value") of the Na(+),K(+)-ratio of viable cultivable organisms is valid for non-cultivable species, the degree of correspondence between intrabacterial Na(+),K(+)-ratios of M. lepraemurium after treatment in vivo with isoniazid, streptomycin and clofazimine, and the ability of these organisms to multiply in mice were examined. A linear relationship between the proportion of viable organisms, calculated from the limiting value of the Na(+),K(+)-ratio, and that calculated from ID50 was found, suggesting that, at least for M. lepraemurium, the intrabacterial Na(+),K(+)-ratio predicts the effect of drugs measured by the much more demanding technique of mouse inoculation.

Intrabacterial sodium-to-potassium ratios and ATP contents of Mycobacterium leprae from ofloxacin-treated patients.

In a clinical trial including 17 multibacillary leprosy patients the in vivo effectiveness of ofloxacin on Mycobacterium leprae was tested via mass spectrometric determination of intrabacterial ratios of the concentrations of the sodium and potassium ions of individual organisms and of the ATP content per 10(6) bacteria isolated from skin biopsies. After 3 months of treatment, the in vivo drug effect could be determined with at least one of the two methods in 14 cases. Both methods revealed that in two cases the bacteria definitely did not respond to a 3-month ofloxacin monotherapy (200 mg twice daily). In three further cases a nonresponse of the M. leprae organisms was suspected from the mass spectrometric measurements. In the responder cases, the M. leprae were severely impaired. From the intrabacterial cation ratios the percentage of viable organisms averaged over all untreated biopsies was determined to be 58% and the percentage-killing during the first 3 months of treatment was 72%.

Determination of in vivo and in vitro drug effects of mycobacteria from the mass spectrometric analysis of single organisms.

Laser microprobe mass analysis of single bacterial organisms allows the determination of their intrabacterial ratio of sodium-to-potassium ions and the registration of fragment ions originating from their organic bacterial cell matrices as mass fingerprint spectra. It has been established previously that the intrabacterial cation ratio provides information on the physiological state of an individual bacterial cell. In the present experiments it is also shown, with different cultivable mycobacterial species and strains (drug sensitive and resistant) exposed to various drugs, that data derived from the evaluation of the mass fingerprint spectra reflect changes in the degree of impairment. The analysis of Mycobacterium leprae derived from a limited number of skin biopsies of lepromatous/borderline lepromatous leprosy patients under World Health Organization-recommended multiple-drug therapy (WHO/MDT) showed impairment of the organisms with both of the methods of measurement in proportion to the duration of treatment except in one case. In one M. leprae population from a patient who had been treated for 19 months, the fingerprint evaluation gave the first evidence for an insufficient response to treatment. This was further confirmed by the unusual frequency distribution of the Na+,K+ ratios which revealed the existence of two subpopulations, one impaired and one unimpaired.

Monitoring of bacterial drug response by mass spectrometry of single cells.

The application of the laser microprobe mass analyser LAMMA 500 to the solution of problems in the field of microbiology is reported. The special features of this instrument allow the analysis of single bacterial cells, and questions can be answered which are not accessible to the normally applied integral methods. Thus it is possible to establish distributions of, for instance, elemental concentrations within a bacterial population and of correlations between measured characteristics of a bacterium with its morphology. The mass spectrum of a single bacterial cell comprises information on its intracellular cation contents as well as on the organic matrix. The relation between the sodium and potassium contents can serve as a criterion of the physiological state of a cell and of its viability. The information from the organic matrix can be extracted from the complex spectra of fragment ions produced by the interaction of the laser with the cell by applying multivariate data analysis, thus rendering additional information. Examples will be given for in vitro drug screening and in vivo therapy control in leprosy, an infection which is caused by a mycobacterial species, Mycobacterium leprae; this organism does not multiply in artificial growth media so that only limited numbers of organisms are available for microbiological investigations.

Results from cation and mass fingerprint analysis of single cells and from ATP measurements of M. leprae for drug sensitivity testing: a comparison.

The physiologic states of Mycobacterium leprae isolated from patient biopsies were studied using single cell mass spectrometry by laser microprobe mass analysis (LAM-MA) and ATP bioluminescence assay. The changes in the physiologic state of M. leprae after the patients had been treated with dapsone (DDS) monotherapy were also studied. The shift of the low intracellular Na+, K+-ratio of untreated M. leprae cells to higher values under DDS therapy, as measured from a limited number of single bacteria, correlates with a decrease in the ATP content. Further information on the influence of the drug could be drawn from the multivariate analysis of mass fingerprints of the organic matrix of the cells. Evidence is provided that the combination of the measurement of the intracellular cation ratios and of the mass fingerprint analysis could give fast answers to the question of drug resistance and to the persister hypothesis. The ATP bioluminescence assay and the single cell mass analysis should be alternatives to the mouse foot pad test.

Detection of externally induced impairments in single bacterial cells by laser microbe mass analysis.

Applying the laser microbe mass analyzer method (LAMMA), mass spectra (fingerprints) were taken from single bacterial cells not treated or treated with high temperature, X-irradiation, isonicotinic acid hydrazide (INH), or diaminodiphenylsulfone (DDS). Spectra of treated cells ("M. lufu," M. tuberculosis H37Ra, E. coli) differ from those of controls in that the K+/Na+ ratio was smaller and in that the intensities of peaks with m/e greater than 100 were lower. From the results with M. leprae the possible application of this new method for monitoring the effectiveness of leprosy therapy is proposed.