Influence of cell geometry and number of replicas in the reproducibility of whole cell FTIR analysis.

Fourier Transform InfraRed (FTIR) spectroscopy is an increasingly used technique in biology, especially for whole cell metabolomic fingerprint. The reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. Evidence exists suggesting that the cell shape and dimension can be further elements to consider in whole cell FTIR analysis. In this study we aimed to address the effect of cell geometry on the FTIR spectra and to define the extent of variability occurring between machine and biological replicas with a standardized protocol. The yeast species Saccharomyces cerevisiae (large oval-shaped cells) and Debaryomyces hansenii (small round shaped cells) were employed for their different morphology. Thirty machine replicas of each were analyzed separately and after averaging in groups of three, showing a three to four-fold reduction of the variability. Similarly, a two-fold reduction of variability was observed when thirty biological replicas of the two yeast species were analyzed. The optimal number of replicas to average was then estimated with a bootstrap-like procedure in which biological and machine replicas were randomly resampled 2000 times and averaged in groups spanning from 2 to 12 replicas. This simulation has shown that little if any advantage can be obtained by increasing the number of replicas over five and that the variability exhibited by the small regular cells of D. hansenii was always roughly half of that displayed by the large S. cerevisiae cells, confirming the results obtained with standard non-bootstrapped averages.

Development of a novel, FTIR (Fourier transform infrared spectroscopy) based, yeast bioassay for toxicity testing and stress response study.

Fourier transform infrared spectroscopy (FTIR) was used to analyze the metabolomic alterations caused to yeast cells by four chemical compounds: ethanol, sodium hypochlorite, sodium chloride and sulfur dioxide, each tested at five different concentrations. The complex of four stressing agents at different concentrations, inducing cell mortalities ranging from 1% to 100%, has given the opportunity to prove that FTIR can individuate the presence of a stress before the cells start dying. A series of "Stress Indexes" was calculated with an expressly designed "R" script, to estimate the level of stress induced by the chemical agents at different concentrations. These estimation procedures allowed the direct comparison of the stress induced by the four agents at different concentrations. The response spectra, calculated as difference between the spectrum of the cells under stress and that of the cells maintained in water, showed different shapes in the diverse experimental conditions, suggesting a specificity of the response and the possibility to classify it. The contribution of five different spectral regions (fatty acids, amides, mixed zone, carbohydrates and typing region) could be calculated separately, gaining additional information on the stressing effects. Spectral alterations were detected at concentrations as low as 10% ethanol, 20 ppm bleach, 1 M NaCl and 100 mg L(-1) SO(2). These preliminary findings suggest that FTIR technology and a series of simple algorithms can be employed to study response of cells to various stressing situations, not limited to chemical agents. The ease and rapidity of the FTIR analysis suggest that this approach could be used as a bioassay in several applications and particularly in ecotoxicology and in environmental microbiology.

Comparison of molecular and metabolomic methods as characterization tools of Debaryomyces hansenii cheese isolates.

Debaryomyces hansenii is one of the yeast species most frequently isolated from cheese and salty foods, however little is known about the phenotypic and molecular variability of its strains. In order to explore the possibilities of a large study on its biodiversity, some D. hansenii strains were selectively isolated from pecorino cheese sampled in ten different Italian regions. All isolates were identified as D. hansenii on the basis of conventional and molecular taxonomic analysis. The D1/D2 domain sequences of the 26S-rDNA did not show any variation, confirming the extreme homogeneity of this species. PCR-duplex-RAPD banding patterns analyzed with PCoA showed interesting clustering related to the geographic areas of isolation, although some overlapping between strains derived from different districts could be observed. A FTIR (Fourier Transform Infrared Spectroscopy) metabolomic fingerprint produced groupings weakly related to those observed with RAPD and less associated with the isolation locales. The discriminatory power of metabolomic fingerprint was able to discriminate strains otherwise considered identical. This preliminary study showed that, in spite of the homogeneity at the 26S-rDNA level, the D. hansenii strains exhibit high molecular and metabolomic variability somehow linked to the places of isolation. Further studies will be necessary to better investigate on the link between terroir and strain variability, as well as on the relation between genotypic and metabolomic fingerprints.

Direct spectroscopic (FTIR) detection of intraspecific binary contaminations in yeast cultures.

Fourier transform infrared spectroscopy has proved to be a good method to identify and characterize microorganisms. This technique has been proposed as a tool to determine the level of contamination in binary mixtures of strains belonging to different species and even to diverse kingdoms, showing a good linear relationship between spectral outputs and contamination levels. The monitoring of intraspecific contamination is a critical point in both laboratory practice and industrial monitoring, but it is challenged by the difficulty to discriminate between very similar cultures belonging to the same species. In this paper we considered binary intraspecific mixtures of strains belonging to three species (Saccharomyces cerevisiae, Debaryomyces hansenii and Rhodotorula minuta). Results showed that contaminated and pure cultures can be discriminated on the basis of their infrared spectra and that different spectral areas respond to the contamination according to the species under test. Moreover, some spectral areas change linearly with the increase of contaminants, giving the possibility of using this procedure for preliminary estimations of the contamination in addition to the even more important opportunity to indicate the presence of contaminants of the same species at low levels in fermentation cultures.

General evidence supporting the hypothesis that Saccharomyces cerevisiae vaginal isolates originate from food industrial environments.

Saccharomyces cerevisiae strains isolated from pregnant women were identified and characterized by molecular techniques which disclosed a wide chromosomal variability and possible segregations due to sporulation. The morphological analysis showed that very few strains were able to sporulate and generate pseudohyphae, whereas none produced proteases, raising some doubts on the importance of these characters in strain pathogenicity. The analysis of ethanol production revealed that these strains are quite similar to those found in fermentative plants, suggesting a possible derivation from the food industrial environment. Since the absence of relevant amounts of sugar does not confer selective advantage to strong fermentative metabolisms, these findings suggest that a metabolic adaptation to the vaginal environment did not occur yet.