Antibiotic treatment options for Burkholderia cepacia infection are limited because of high intrinsic resistance. The problem is complicated by development of cross-resistance between antibiotics of different classes. We isolated antibiotic-resistant mutants by stepwise exposure to chloramphenicol (Chlor) and to trimethoprim/sulphamethoxazole (T/S) for four B. cepacia strains: ATCC13945, Per (clinical isolate), Cas and D4 (environmental isolates). Chlor(r) mutants did not produce chloramphenicol acetyl-transferase. Cross-resistance, defined as greater than four-fold increase in MIC by microtitre dilution method, was consistently seen in both types of mutants. For chloramphenicol-resistant (Chlor[r]) and trimethoprim/sulphamethoxazole-resistant (Tr/Sr) mutants of B. cepacia ATCC13945 and Cas, no MIC change was seen for piperacillin, ceftazidime, rifampicin, gentamicin, tobramycin, polymyxin B or azithromycin. B. cepacia-Per and -D4 mutants showed cross-resistance to ceftazidime and to piperacillin. Comparison of outer membrane protein (OMP) profiles of B. cepacia and their mutants by SDS-PAGE revealed Tr/Sr) mutants to be deficient in a major OMP (molecular weight 39-47 kDa). Tr/Sr mutants also expressed additional OMPs not found in wild type strains at 75-77 kDa for B. cepacia-ATCC13945 and -Cas, and 20-21 kDa in B. cepacia-D4 and -Per. No OMP changes occurred in Chlor(r) mutants. Lipopolysaccharide (LPS) profiles of each type of mutant showed new high and low molecular weight LPS bands. Cross-resistance seems to be mediated by alterations in porin and LPS for Tr/Sr mutants, but only by LPS in Chlor(r) mutants.

Infections in cystic fibrosis (CF) due to Burkholderia cepacia are challenging due to their resistance to antibiotics. We explored a new strategy for increasing the permeability of B. cepacia using cationic agents, including amino compounds, to reduce the MICs of standard antibiotics. Twenty-eight B. cepacia isolates from four CF centres in North America and four non-CF B. cepacia were examined by standard microtitre broth dilution methods for susceptibility to a variety of antibiotics in the presence of non-inhibitory concentrations of diaminoacetone (DAA), methylglyoxal bis-guanylhydrazone (MGBH), chlorpromazine (CPZ) and prochlorperazine (PCPZ). The proportion of isolates with greater than four-fold reductions in MIC in the presence of 0.3 mM CPZ or 0.4 mM PCPZ were 90% and 94% for gentamicin, 80% and 83% for tobramycin, 45% and 17% for ceftazidime, and 35% and 17% for amifloxacin. CPZ showed the same degree of reduction in the MIC of azithromycin in 79% strains (MIC50 reduced to 16 from > or = 256 mg/L). Non-CF B. cepacia showed a greater than four-fold reduction in MIC with CPZ for gentamicin, tobramycin and azithromycin and two-fold reduction for ceftazidime. Little or no reduction in MIC was seen with DAA or MGBH for any antibiotic. Addition of magnesium ions to the medium competitively inhibited any MIC reduction effect seen with the cationic agents. CPZ and PCPZ appeared to enhance the permeability of B. cepacia to antibiotics based upon ionic charge characteristics of the antibiotic. No significant differences were seen in outer membrane protein and lipopolysaccharide profiles between the culture treated with CPZ and the respective control culture of strain B. cepacia ATCC 13945. The fluorescent probe 1N-phenylnaphthylamine had no increased access across the outer membrane in the presence of CPZ for B. cepacia ATCC 13945. However, thin-section electron microscopy revealed separation between the outer membrane and the rest of the cytoplasm accompanied by a widening of the periplasmic space. These data provide a rationale for investigating amino compounds as potential permeability-increasing agents against B. cepacia.

Chlorpromazine and prochlorperazine have previously been shown to enhance the susceptibility of Burkholderia cepacia to aminoglycosides. To screen other non-antibiotic drugs containing similar amine (-N-CH3) groups, we examined a range of such agents that are in current clinical use for the treatment of non-infectious diseases, in combination with antibiotics that are ineffective against B. cepacia. At a concentration of 0.2 mM, theobromine, theophylline, trifluoperazine, fluophenazine and coumarin-152 significantly reduced (by four-fold) the MICs of gentamicin and ceftazidime. Theobromine and theophylline also reduced the MICs of amikacin and azithromycin.

Electron microscopy is still the most frequently used method for visualization of subcellular structures in spite of limitations due to the preparation required to visualize the specimen. High resolution X-ray microscopy is a relatively new technique, still under development and restricted to a f e w large synchrotron X-ray sources. We utilized a single-shot laser (nanosecond) plasma to generate X-rays similar to synchrotron facilities to image live cells of Candida albicans.The emission spectrum was tuned for optimal absorption by carbon-rich material. The photoresist was then scanned by an atomic force microscope to give a differential X-ray absorption pattern. Using this technique, with a sample image time of 90 min, w e have visualized a distinct 152.24 nm thick consistent ring structure around cells of C. albicans representing the cell wall, and distinct 'craters' inside, one of 57090 nm diameter and three smaller ones, each 400 nm in diameter. This technique deserves further exploration concerning its application in the ultrastructural study of live, hydrated microbiological samples and of macromolecules.Keywords : Candida albicans, X-ray micrography, high resolution, ultrastructure, laser plasmas INTRODUCTIONThe detection and study of microbial clells is performed by low magnification optical microscopy, and direct and indirect labelling techniques. In bacteriology, cationic (methylene blue, crystal violet, safranin) or anionic (eosin, acid fuchsin, congo red) colour dyes are used for resolution at the 1-100 pm scale and radioactively labelled substrate uptake is measured for very slow growing mycobacteria (Chapin-Robertson & Edberg, 1991). In virology, for nm-scale detection, secondaryconjugated fluorescent antibodies are applied. Visual ultrastructural studies on subcellular organelles are possible with variations of electron microscopy (thin section, scanning and freeze fracture) a1 though specimen preparation steps such as fixation, dehydration, resin embedding, ultra-thin sectioning, coating and staining are very technical, extensive and may introduce artefacts in the original sample. Although electron microscopy can be used at the nm resolution level, the sample Abbreviations: AFM, atomic force microscopy; PMMA, poly(methy1 methacrylate).preparation steps involved limit its use for routine studies of microbial cells (Kay, 1976 ; Lichfeld, 1976).X-ray microscopy is a relatively new technique that has not been applied to any significant extent for biological specimens (DeMeis, 1996). It eliminates specimen preparatipn which may alter the target, has resolution at the 100 A level (Ohnesorge & Binning, 1993) and is able to probe the internal structures of in vivo assemblies, enabling the observation of complex features in their natural, live state (Feder et al., 1985;Hoh et al., 1992).Most X-ray microscope development has been made so far using large synchrotron sources (Neiman, 1992), thus limiting X-ray microscopy as a research tool to where such facilities are available. The use of a laser plasma X-r...

A compact, high-resolution, laser-plasma, x-ray contact microscopy method using a table-top Nd:glass laser system has been developed. This x-ray microscopy system was applied for the observation of macrophage ultrastructures. These images were produced using proximity imaging in which a ?-ns pulse of soft x-rays with