Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi.

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl.

Debaryomyces hansenii showed an increased growth in the presence of either 1 M, KCl or 1 M NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86Rb+ and 22Na+. 86Rb+ transport was saturable, with K(m) and Vmax values higher for cells grown in 1 M NaCl. 22Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1 M KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.

Drug effects on intracellular mycobacteria determined by mass spectrometric analysis of the Na(+)-to-K+ ratios of individual bacterial organisms.

The successful establishment of a drug screening system for intracellular cultivable and noncultivable mycobacteria based on the mass spectrometric determination of bacterial viability is described. To compare drug efficacies on intra- and extracellular mycobacteria, the mycobacteria were subjected to drug treatment either after phagocytosis by the mouse macrophage cell line RAW 264.7 or in cell-free medium. After reisolation, their viability was monitored by analyzing the intrabacterial sodium-to-potassium ratios for a limited number of individual organisms. This approach offers a reliable and quick tool for monitoring the influence of intracellular growth and of additional permeation barriers on intracellular drug efficacy and will thus provide useful information for the rational development and testing of optimized antimycobacterial drugs. In particular, the methodology is applicable to the noncultivable species Mycobacterium leprae, because the mass spectrometric analysis of the intrabacterial sodium-to-potassium ratio allows the determination of bacterial viability independent from their ability to multiply in vitro. Because of the improved metabolic activity of intracellularly growing M. leprae compared with that of extracellularly growing M. leprae, the spectrum of antileprosy drugs that can be tested in vitro could even be extended to those interfering with DNA replication and cell division.

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.