Culture conditions and sample preparation methods affect spectrum quality and reproducibility during profiling of Staphylococcus aureus with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

UNLABELLED: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI-TOF MS to characterize a model system consisting of five methicillin-sensitive (MSSA) and five methicillin-resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI-TOF MS to characterize S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results indicated that MALDI-enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction-based approach. These findings should enhance future efforts to maximize the performance of this approach to characterize strains of S. aureus.

Microbial fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications and challenges.

Recent threats posed by pathogenic microorganisms in food, recreational waters, and as agents of bioterror have underscored the need for the development of more rapid, accurate, and cost-effective methods of microbial characterization and identification. This chapter focuses on the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapidly characterize and identify microorganisms through generation of characteristic fingerprints of intact cells. While most efforts have focused on bacteria, this technology has also been applied to fungi and viruses. Results of most studies suggest that MALDI-TOF MS can be used to rapidly and accurately characterize microorganisms. A variety of quantitative approaches have been employed in the analysis of MALDI-TOF MS fingerprints of microorganisms. The reproducibility of fingerprints of intact cells remains a primary concern and limitation associated with this approach. Protocols and instrumentation used have varied considerably and likely account for much of the variability in reproducibility reported. Key first steps to overcoming this limitation will be the development of standard approaches to quantifying reproducibility and the development of standard protocols for sample preparation and analysis.

Influence of a nonaqueous phase liquid (NAPL) on biodegradation of phenanthrene.

A series of batch reactor experiments was carried out to examine the effect of a nonaqueous phase liquid (NAPL) on the biodegradation of a hydrophobic solute. A mathematical program model that describes physical processes of solute solubilization and partitioning between the NAPL and aqueous phases as well as microbial degradation and oxygen utilization was used to analyze the test data. The model calculates the cumulative changes in concentration of substrate, cell mass, carbon dioxide, and dissolved oxygen as a function of time. The equations incorporate the effects of solute solubilization, partitioning, biodegradation, as well as oxygen availability. Hexadecane was used as the model NAPL and was not biodegraded in the timeframe of the experiments performed. The model solute was the polyaromatic hydrocarbon, phenanthrene. In agreement with several previous studies, experimental measurements showed that hexadecane increased rates of mineralization of 15 mg phenanthrene when present at low mass but decreased rates at high mass. Model results suggest that partitioning of the phenanthrene into the hexadecane phase limits bioavailability at high NAPL mass. Further the model suggests that mineralization rates were higher with the low NAPL mass because aqueous phenanthrene concentrations were higher in those treatments from ca. 20 to 40 h than in other treatments. Finally, experiments showed that the presence of hexadecane, at all masses tested, resulted in a lower cell yield, effectively increasing the amount of CO(2) produced during the experiment. Model results suggest that this is due to changes in phenanthrene metabolism that are induced by the presence of the hexadecane phase. Model studies aimed at increasing rates of biodegradation by modifying operating conditions are described along with practical approaches to implementing these modifications.

A rhamnolipid biosurfactant reduces cadmium toxicity during naphthalene biodegradation.

A model cocontaminated system was developed to determine whether a metal-complexing biosurfactant, rhamnolipid, could reduce metal toxicity to allow enhanced organic biodegradation by a Burkholderia sp. isolated from soil. Rhamnolipid eliminated cadmium toxicity when added at a 10-fold greater concentration than cadmium (890 microM), reduced toxicity when added at an equimolar concentration (89 microM), and had no effect at a 10-fold smaller concentration (8.9 microM). The mechanism by which rhamnolipid reduces metal toxicity may involve a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface to alter cadmium uptake.

Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates.

Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.