New insights concerning the occurrence of fungi in water sources and their potential pathogenicity.

Fungi are known to occur ubiquitously in the environment. In the past years, the occurrence of filamentous fungi in the aquatic environment has been a subject of growing interest. This study describes the occurrence of various fungal genera in different drinking water sources being Penicillium and Trichoderma the most representative ones (30% and 17%, respectively). Also, 24 fungal species that have not been previously described in the aquatic environment are reported in this study, being once again the major species from the Penicillium genera. This study therefore contributes to the knowledge on the richness of fungi diversity in water. 68% of the described species were found to be able to grow at 30 °C but only Aspergillus fumigatus, Aspergillus viridinutans and Cunninghamella bertholletiae were able to grow at the higher temperature tested (42 °C). 66% of the species that were able to grow at 30 °C have spore sizes below 5 µm which enables them to cause breathing infections. These were therefore identified as potential pathogenic species.

Volatile compounds in samples of cork and also produced by selected fungi.

The production of volatile compounds by microbial communities of cork samples taken during the cork manufacturing process was investigated. The majority of volatiles were found in samples collected at two stages: resting after the first boiling and nontreated cork disks. Volatile profiles produced by microbiota in both stages are similar. The releasable volatile compounds and 2,4,6-trichloroanisole (TCA) produced in cork-based culture medium by five isolated fungal species in pure and mixed cultures were also analyzed by gas chromatography coupled with mass spectrometry (GC-MS).The results showed that 1-octen-3-ol and esters of fatty acids (medium chain length C8-C20) were the main volatile compounds produced by either pure fungal species or their mixture. Apparently, Penicillium glabrum is the main contributor to the overall volatile composition observed in the mixed culture. The production of releasable TCA on cork cannot be attributed to any of the assayed fungal isolates.

Assessment of the presence and dynamics of fungi in drinking water sources using cultural and molecular methods.

A comparison of different isolation techniques and culture media for detection of filamentous fungi and yeasts in the aquatic environment revealed that the use of membrane filtration with the media dichloran rose bengal chloramphenicol (DRBC) optimized fungi detection in terms of abundance and variety in three untreated water sources with very different characteristics (surface water, spring water, and groundwater). The diversity of the fungi population captured by direct DNA extraction of fungi collected by membrane filtration was compared with the isolates obtained after selective growth using different culture media through amplification of the internal transcribed spacer gene and denaturing gradient gel electrophoresis (DGGE). The Czapek-Dox agar, Sabouraud dextrose agar, and DRBC media showed closer similarities to those obtained by the uncultured biomass for the different water sources. Based on these data and the best enumeration results, DRBC is recommended for the assessment of fungi in water sources using culture-based methods. DGGE was also used to monitor temporal variations in the fungal population structure and showed that each water matrix possessed a distinct population profile as well as that changes in the fungal community can be expected in the different matrices throughout the year.

Weissella halotolerans W22 combines arginine deiminase and ornithine decarboxylation pathways and converts arginine to putrescine.

AIMS: To demonstrate that the meat food strain Weissella halotolerans combines an ornithine decarboxylation pathway and an arginine deiminase (ADI) pathway and is able to produce putrescine, a biogenic amine. Evidence is shown that these two pathways produce a proton motive force (PMF). METHODS AND RESULTS: Internal pH in W. halotolerans was measured with the sensitive probe 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein. Membrane potential was measured with the fluorescent probe 3,3'-dipropylthiocarbocyanine iodine. Arginine and ornithine transport studies were made under several conditions, using cells loaded or not loaded with the biogenic amine putrescine. ADI pathway caused an increase in DeltapH dependent on the activity of F(0)F(1)ATPase. Ornithine decarboxylation pathway generates both a DeltapH and a DeltaPsi. Both these pathways lead to the generation of a PMF. CONCLUSIONS: Weissella halotolerans W22 combines an ADI pathway and an ornithine decarboxylation pathway, conducing to the production of the biogenic amine putrescine and of a PMF. Transport studies suggest the existence of a unique antiporter arginine/putrescine in this lactic acid bacteria strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The coexistence of two different types of amino acid catabolic pathways, leading to the formation of a PMF, is shown for a Weissella strain for the first time. Moreover, a unique antiport arginine/putrescine is hypothesized to be present in this food strain.

Occurrence of filamentous fungi and yeasts in three different drinking water sources.

In order to determine the occurrence of fungi in different drinking water sources and capture variability in terms of matrix composition and seasonal effects, surface water, spring water, and groundwater samples were collected in numerous sampling events. The occurrence and significance of fungi detected in the different water sources are reported and discussed in terms of colony-forming units per millilitre and by the identification of the most frequently detected isolates, at the species level, based on morphology and other phenotypic characters. All the samples were also analyzed in terms of total coliforms and Escherichia coli that are widely monitored bacteria considered as microbiology indicators of water quality. All the groundwater samples showed significantly lower levels of total coliforms, E. coli, and fungi compared to the surface and spring water samples. No significant correlations were found between the levels of fungi detected in all the matrices and the physico-chemical parameters and bacteria regularly monitored by drinking water utilities. Fifty-two fungi isolates were identified in this study, most of which have never been described to occur in water sources. The results obtained show that fungi occur widely in drinking water sources and that further studies should be conducted to address their biodegradation potential as well as if the drinking water treatment processes currently used are effective in removing these organisms and the potential secondary metabolites produced.

Dual role for the tyrosine decarboxylation pathway in Enterococcus faecium E17: response to an acid challenge and generation of a proton motive force.

In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.

Penicillium glabrum cork-colonising isolates - preliminary analysis of their genomic similarity

The cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are not completely understood although they are considered to be involved in the so-called "cork taint" of wine. It is essential to (a) identify environmental constraints which define the appearance of the colonising fungal species and (b) trace their origin to the forest and/or the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was confirmed, followed by a high diversity of Penicillium species. Penicillium glabrum, which was found in all samples, was the most frequently isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork specific fungi can contribute(AU)

Cork stoppers industry: defining appropriate mould colonization.

AIMS: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. METHODS AND RESULTS: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. CONCLUSIONS: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Lactobacillic acid accumulation in the plasma membrane of Oenococcus oeni: a response to ethanol stress?

It is known that ethanol strongly interferes with the development and activity of lactic acid bacteria in wine. In this work, it was observed that membrane composition was dependent of ethanol concentration and cell physiological state. The protein electrophoretic profile was modified in the membranes of Oenococcus oeni cultured in presence of 8 and 10% ethanol. Concerning the membrane lipid composition, it was observed that O. oeni maintained a high level of phospholipid biosynthesis via the relative increased biosynthesis of phosphoethanolamine and sphingomyelin in presence of ethanol. On the other hand, ethanol induced an increase in the membrane lactobacillic acid percentage at the expense of cis-vaccenic acid. This increased synthesis of lactobacillic acid appears as the more significant change induced by ethanol in O. oeni membrane. The increase of lactobacillic acid in the membrane of O. oeni clearly appears as a factor that provides protection against the toxic effect of ethanol, balancing the increase of membrane fluidity normally attributed to ethanol. The results presented in this paper constitute evidence that lactobacillic acid may have a part in the survival and or adaptive mechanisms developed by O. oeni under culture adverse conditions, allowing these bacteria to maintain their activity in the presence of ethanol, namely performing malolactic fermentation in wine.

Cork taint in wine: scientific knowledge and public perception: a critical review.

The manufacturing process of cork stoppers includes a stabilization period of the cork slabs, following boiling, during which mold growth completely covers the cork slabs. This process has been used traditionally for several decades; however, due to the possibility of certain molds isolated from cork to produce off flavor compounds, especially 2,4,6-trichloroanisole and 2,3,4,6-tetrachloroanisole, recently cork stoppers are being unsoundly targeted with the accusation of inducing cork taint in wine. This article reviews the manufacturing process of cork stoppers, the diversity of microorganisms associated with cork, and finally the diversity and origins of the compounds associated with cork taint in wine, focusing on those currently considered as more important. Some important results recently obtained by the authors are also included. The current idea of suppressing mold growth during cork stopper manufacturing is discussed, as well as the erroneous idea of imputing, directly and exclusively, to cork the responsibility of the so-called cork taint in wine.

Biogenic amines occurrence in wine. Amino acid decarboxylase and proteolytic activities expression by Oenococcus oeni.

This work deals with the study of the proteolytic and amino acid decarboxylase activities of selected Oenococcus oeni isolates and the effect of yeast autolysis on biogenic amines production in wine. A total of 220 isolates of O. oeni were tested for decarboxylase and proteolytic activity. Only six isolates showed both activities, but only after a period of adaptation in a growth medium containing wine. The results reported on this paper show that proteolytic activity was dependent on medium composition and bacterial growth phase. It can be assumed that the ability of O. oeni to use wine peptides and to produce biogenic amines is not a constant characteristic of this species, and enzymatic system expression appears to be closely dependent on nutritional and energetical composition of the medium. It also seems to be strain dependent and not widespread among this bacterial community.

Mycobiota in Portuguese 'normal' and 'green' cork throughout the manufacturing process of stoppers.

The compounds responsible for the so-called 'cork taint' include, among others, some microbial metabolites which can be produced by the microbial population colonizing the unprocessed cork and stoppers. This study was intended to obtain information on the mycobiota associated with Portuguese cork throughout the manufacturing process of stoppers. Samples of barks and stoppers of both 'normal' and 'green' cork were examined. Moulds were isolated from 'normal' and 'green' cork throughout the entire cork stopper manufacturing process. Yeasts were rarely detected in the corks. Fungal contamination was not detected in finished stoppers from the company under study.

In vitro reassembly of the malolactic fermentation pathway of Leuconostoc oenos (Oenococcus oeni).

The mechanism of metabolic energy generation by malolactic fermentation was studied with artificial membrane vesicles of Leuconostoc oenos (Oenococcus oeni). (Note that although L. oenos was recently reclassified as O. oeni [L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995], the old designation was kept in the present work.) Purified malolactic enzyme was entrapped in artificial membrane vesicles prepared from L. oenos cells able to transport L-malate. We show that the in vitro reconstituted system, including an electrogenic L-malate carrier and the decarboxylating malolactic enzyme, generated a proton motive force that was able to drive intravesicular accumulation of leucine.

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.