Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two-phase systems.

An extractive fermentation technique was developed using a thermoseparating reagent to form a two-phase system for simultaneous cell cultivation and downstream processing of extracellular Burkholderia cepacia lipase. A 10% (w/w) solution of ethylene oxide-propylene oxide (EOPO) with a molecular mass of 3900 g/mol and pH 8.5, a 200 rpm speed, and 30 °C were selected as the optimal conditions for lipase production (55 U/ml). Repetitive batch fermentation was performed by continuous replacement of the top phase every 24h, which resulted in an average cell growth mass of 4.7 g/L for 10 extractive batches over 240 h. In scaling-up the process, a bench-scale bioreactor was tested under the conditions that had been optimized in flasks. The production rate and recovery yield were higher in the bioreactor compared to fermentation performed in flasks.

Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by bacteria of the Burkholderia cepacia complex.

The repeating unit of cepacian, the exopolysaccharide produced by the majority of the microorganisms belonging to the Burkholderia cepacia complex, was isolated from inner bacterial membranes and investigated by mass spectrometry, with and without prior derivatisation. Interpretation of the mass spectra led to the determination of the biological repeating unit primary structure, thus disclosing the nature of the oligosaccharide produced in vivo. Moreover, mass spectra recorded on the native sample revealed that acetyl substitution was very variable, producing a mixture of repeating units containing zero to four acyl groups. At the same time, finding acetylated oligosaccharides showed that binding of these substituents occurred in the cellular periplasmic space, before the polymerisation process took place. In the chromatographic peak containing the repeating unit, oligosaccharides shorter than the repeating unit co-eluted. Mass spectrometric analysis showed that they were biosynthetic intermediates of the repeating unit and further investigation revealed the biosynthetic sequence of cepacian building block.

Colloidal and bacterial deposition: role of gravity.

The role of gravitational force on the deposition of 0.5, 1.1, and 1.8 mum carboxylate-modified polystyrene latex (CML) microspheres and bacterium Burkholderia cepacia G4g has been evaluated using a parallel plate flow chamber system. This experimental system utilized an inverted and an upright optical microscope attached with image-capturing devices to directly observe and determine the deposition kinetics onto glass surfaces located at the top and bottom of the flow chamber. Deposition kinetics was quantified at 10 mM KCl under electrostatically unfavorable and favorable attachment conditions and at two flow rates (0.06 and 3 mL/min), simulating the range of flow velocities from groundwater to rapid granular filtration. Comparing the particle deposition kinetics on the top and bottom surfaces under identical flowing exposure time, fluid chemistries, and hydrodynamic conditions, results showed that significant differences were observed between the two surfaces, suggesting that gravity was a significant driving force for the initial stages of deposition of particles that were larger than 1 mum size. Simulation results utilizing a particle trajectory model confirmed these experimental observations. This was further supported by additional deposition experiments with 1.1 mum microspheres suspended in a deuterium oxide (D(2)O)/water mixture (heavy water) where the density of colloid and the suspending heavy water were effectively the same. Under this condition, deposition rates were observed to be identical between the top and bottom surfaces. Results from normal and heavy water solutions indicated that the greater deposition of colloidal particles larger than 1 mum on the bottom in normal water solutions is due to gravity. Finally, the experimental results were compared with deposition studies using smaller 0.5 mum colloids as well as some theoretical calculations of expected rates of particle deposition.

Isolation and characterization of a novel Burkholderia cepacia with strong antifungal activity against Rhizoctonia solani.

Strain CF-66 with strong antifungal activity against Rhizoctonia solani was isolated from compost samples. It is clearly demonstrated that strain CF-66 is belonging to Burkholderia cepacia complex by the morphological and biochemical tests and 16S rDNA sequence. The B. cepacia complex consists of a group of bacteria currently organized into nine genomovars, among them genomovar II and genomovar III, contain the highly epidemic strains. However, it was known that strain CF-66 is not a member of genomovar II or III of the B. cepacia complex by species-specific polymerase chain reaction assay. In this study, the antifungal compound CF66I produced by strain CF-66 was purified and characterized. Based on the nuclear magnetic resonance, GC-MS spectral and infrared spectral data, CF66I was confirmed to have amide bonds, alpha-methyl fatty acid, bromine, and some structural units such as CH(2)CH(2)O. CF66I is stable to high temperature, proteolytic enzymes, and organic solvents. CF66I inhibit the growth of a variety of plant pathogenic fungi and pathogenic yeast, whereas bacterial cells are unaffected. CF66I mainly reduced hyphal extension rates in a dose-dependent manner and induced severe change in cell morphology that resulted in swelled and formed very short hyphae with multiple branches.

Effect of pH on the toxicity of nickel and other divalent metals to Burkholderia cepacia PR1(301).

Nickel (Ni) is a common cocontaminant at many waste sites where the soils and sediments often are acidic, thereby influencing metal availability. Growth of Burkholderia cepacia PR1(301) was not affected at 3.41 mM Ni at pH 5, but was inhibited by 73.2% at pH 6 and inhibited completely at pH 7 compared to growth without Ni. This pH effect was not observed in the Ni-resistant strains, Ralstonia metallidurans CH34 and 31A. Predicted Ni speciation did not explain the observed toxicity trends. Sorption of Ni to PR1 increased with increasing pH (1.49, 1.12, and 3.88 mg Ni/g dry weight at pH 5, 6, and 7, respectively), but was low at all three pH values, and most likely does not explain the observed pH effect. Growth inhibition of PRI with increasing pH also was observed for other divalent cations, with growth observed at 4.24 mM Co, 2.22 mM Cd, and 3.82 mM Zn at pH 5 and 6, but totally inhibited at pH 7. These studies suggest that, at circumneutral pH, PRI would be considered sensitive to Ni and other divalent cations, in spite of the ability to grow in higher concentrations at lower pH values.

Efficacy of disinfectants and hot water against biofilm cells of Burkholderia cepacia.

The effects of various disinfectants and hot water on planktonic cells and biofilm cells of Burkholderia cepacia were investigated. The survival rate of viable B. cepacia cells in suspension decreased to 0.001% or lower within 15 s of exposure to 0.5% benzalkonium chloride, within 30 s of exposure to 0.5% alkyldiaminoethyl glycine, or within 1 min of exposure to 0.1% alkyldiaminoethyl glycine, and decreased to about 0.1% with 60 min of exposure to 0.1% benzalkonium chloride or 0.5% chlorhexidine gluconate, but did not decrease to 1% or less with 60 min of exposure to 0.1% chlorhexidine gluconate. There were no effects of 0.1% and 0.2% chlorhexidine gluconate and 0.1% benzalkonium chloride against biofilm cells of B. cepacia, and 0.5% chlorhexidine gluconate, 0.5% benzalkonium chloride and 0.1% alkyldiaminoethyl glycine were barely effective against biofilm cells even after 60-min exposure. On the other hand, both planktonic cells and biofilm cells of B. cepacia were eradicated within 15 s by sodium hypochlorite, povidone-iodine, 80% v/v ethanol, and hot water at 65 degrees C or higher.

Epifluorescence microscope methods for bacterial enumeration in a 4-chlorophenol degrading consortium.

Epifluorescence microscope methods, namely BacLight, direct epifluorescence filter technique and Rhodamine 123, consistently underestimated plate bacterial counts in a 4-chlorophenol degrading consortium. Cells capable of passing through 0.2 microm filters, referred as 'ultramicrocells', were found. Although cell counts were higher when traditional methods were used, BacLight and direct epifluorescence filter technique were convenient techniques for the systematic monitoring of bacteria involved in biodegradation processes, as results were consistent and available within a short time.

Growth and survival of Pseudomonas pseudomallei in acidic environments.

A study was made on the growth and survival of Pseudomonas pseudomallei in culture environments differing in nutrients, initial pH, and aeration, in comparison with Pseudomonas cepacia and Pseudomonas aeruginosa. The observations led us to a view that P. pseudomallei has the highest adaptability to acidic environments among the three species. Unlike the other species, it grew in heart infusion broth of initial pH 4.5 under aeration and survived keeping a high level (10(9) per ml) of viable counts for as long as 30 days. This sort of adaptation was found to be more evident in the media of poor nutrition and under limited aeration.