Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity.

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

Production and migration of ochratoxin A and citrinin in Comté cheese by an isolate of Penicillium verrucosum selected among Penicillium spp. mycotoxin producers in YES medium.

Moldy food products that are not subject to pathogenic bacterial contamination could be trimmed by consumers to remove fungal mycelium before consumption. However, prior to giving such recommendations to consumers, it is necessary to evaluate potential mycotoxin migration in these products. This study aimed at quantifying citrinin (CIT) and ochratoxin A (OTA) accumulation and migration in a French semi-hard Comté cheese after artificial inoculation with a CIT- and OTA-producing Penicillium verrucosum strain. At 8 °C, CIT and OTA production started after 14 days and 28 days incubation, respectively; while at 20 °C, both mycotoxins were produced from day 7. At 20 °C, maximum CIT concentration, about 50000 ng/g, was 20 fold that at 8 °C. Regardless of temperature, maximum OTA concentration was about 4000 ng/g cheese. Maximum concentrations were obtained in the upper part of the cheese, but depending on incubation time, mycotoxins were detected up to 1.6 cm in depth. As long as only white mycelium developed on the cheese surface, trimming can be acceptable, but a blue mold color (due to fungal sporulation) was associated with the accumulation of significant amounts of mycotoxins so the product should be discarded.

Modelling the effect of water activity reduction by sodium chloride or glycerol on conidial germination and radial growth of filamentous fungi encountered in dairy foods.

Water activity (aw) is one of the most influential abiotic factors affecting fungal development in foods. The effects of aw reduction on conidial germination and radial growth are generally studied by supplementing culture medium with the non-ionic solute glycerol despite food aw can also depend on the concentration of ionic solutes such as sodium chloride (NaCl). The present study aimed at modelling and comparing the effects of aw, either modified using NaCl or glycerol, on radial growth and/or conidial germination parameters for five fungal species occurring in the dairy environment. The estimated cardinal values were then used for growth prediction and compared to growth kinetics observed on commercial fresh cheese. Overall, as compared to glycerol, NaCl significantly increased the fungistatic effect resulting from aw reduction by extending latency and/or reducing radial growth rates of Paecilomyces niveus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti but not of Mucor lanceolatus. Besides, NaCl significantly reduced aw range for conidial germination and delayed median germination time of P. expansum but not of P. roqueforti. Despite these observations, cardinal aw values obtained on glycerol-medium yielded similar predictions of radial growth and germination time in commercial fresh cheese as those obtained with NaCl. Thus, it indicates that, for the studied species and aw range used for model validation, the use of NaCl instead of glycerol as a aw depressor had only limited impact for fungal behavior prediction.

Genetic basis for mycophenolic acid production and strain-dependent production variability in Penicillium roqueforti.

Mycophenolic acid (MPA) is a secondary metabolite produced by various Penicillium species including Penicillium roqueforti. The MPA biosynthetic pathway was recently described in Penicillium brevicompactum. In this study, an in silico analysis of the P. roqueforti FM164 genome sequence localized a 23.5-kb putative MPA gene cluster. The cluster contains seven genes putatively coding seven proteins (MpaA, MpaB, MpaC, MpaDE, MpaF, MpaG, MpaH) and is highly similar (i.e. gene synteny, sequence homology) to the P. brevicompactum cluster. To confirm the involvement of this gene cluster in MPA biosynthesis, gene silencing using RNA interference targeting mpaC, encoding a putative polyketide synthase, was performed in a high MPA-producing P. roqueforti strain (F43-1). In the obtained transformants, decreased MPA production (measured by LC-Q-TOF/MS) was correlated to reduced mpaC gene expression by Q-RT-PCR. In parallel, mycotoxin quantification on multiple P. roqueforti strains suggested strain-dependent MPA-production. Thus, the entire MPA cluster was sequenced for P. roqueforti strains with contrasted MPA production and a 174bp deletion in mpaC was observed in low MPA-producers. PCRs directed towards the deleted region among 55 strains showed an excellent correlation with MPA quantification. Our results indicated the clear involvement of mpaC gene as well as surrounding cluster in P. roqueforti MPA biosynthesis.

Yamadazyma barbieri f.a. sp. nov., an ascomycetous anamorphic yeast isolated from a Mid-Atlantic Ridge hydrothermal site (-2300 m) and marine coastal waters.

Two yeast strains that are members of the same species were isolated from different marine habitats, i.e. one from Mid-Atlantic Ridge ocean water samples located in the direct vicinity of black smokers near the Rainbow deep-sea hydrothermal vent and one from Brazilian marine water samples off the Ipanema beach. Strains CLIB 1964T and CLIB 1965 are anamorphic ascomycetous yeasts affiliated to the Yamadazyma clade of Saccharomycetales. Interestingly, these strains were phylogenetically and distinctly positioned into a group of species comprising all species of the genus Yamadazyma isolated from marine habitats including deep-sea hydrothermal vents, i.e.Candida atmosphaerica,C. spencermartinsiae,C. atlantica,C. oceani and C. taylorii. These strains differed significantly in their D1/D2 domain sequences of the LSU rRNA gene from the closely related species mentioned above, by 2.6, 3.0, 3.4, 3.8 and 6.0 %, respectively. Internal transcribed spacer region sequence divergence was also significant and corresponded to 4.6, 4.7, 4.7, 12.0 and 24.7 % with C. atlantica,C. atmosphaerica, C. spencermartinsiae,C. oceani and C. taylorii, respectively. Phenotypically, strains CLIB 1964T and CLIB 1965 could be distinguished from closely related species by their inability to assimilate l-sorbose. CLIB 1964T (=CBS 14301T=UBOCC-A-214001T) is the designated type strain for Yamadazyma barbieri sp. nov. The MycoBank number is MB 815884.

Deciphering the Microbiota and Volatile Profiles of Algerian Smen, a Traditional Fermented Butter.

In Algeria, Smen is a fermented butter produced in households using empirical methods. Smen fermentation is driven by autochthonous microorganisms; it improves butter shelf-life and yields highly fragrant products used as ingredients in traditional dishes as well as in traditional medicine. The present study is aimed at investigating microbial diversity and dynamics during Algerian Smen fermentation using both culture-dependent and culture-independent approaches, as well as by monitoring volatile organic compound production. To reach this goal, fifteen Smen samples (final products) produced in households from different regions in Algeria were collected and analyzed. In addition, microbial and volatile compound dynamics at the different stages of Smen manufacturing were investigated for one Smen preparation. The results showed that Smen is a microbiologically safe product, as all hygiene and safety criteria were respected. The dominant microorganisms identified by both techniques were LAB and yeasts. Lactococcus spp. and Streptococcus thermophilus were the main bacterial species involved in spontaneous raw milk fermentation preceding butter-making, while lactobacilli and enterococci were the only bacteria found to be viable during Smen maturation. Regarding fungal diversity, yeast species were only recovered from two mature Smen samples by culturing, while different species (e.g., Geotrichum candidum, Moniliella sp.) were identified in all samples by the culture-independent approach. Using microbial analysis of a single batch, many of these were found viable during manufacturing. Concerning the volatile profiles, they were highly diverse and characterized by a high prevalence of short chain fatty acids, methylketones, and esters. Correlation analysis between microbial diversity and volatile profiles showed that several yeast (Moniliella sp., K. marxianus) and LAB (e.g., Lactococcus spp., S. thermophilus) species were strongly correlated with one or more volatile organic compound families, including several ethyl esters and methyl ketones that can be linked to pleasant, sweetly floral, fruity, buttery, and creamy odors. This study clearly identified key microorganisms involved in Smen fermentation and maturation that could be used in the future for better fermentation control and improvement of quality attributes.

Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations.

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had ß-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

Production and migration of patulin in Penicillium expansum molded apples during cold and ambient storage.

The ability of three Penicillium expansum isolates to produce patulin was first evaluated in YES medium after incubation at 25 °C to select a high patulin producer. Then, a spore suspension of the selected P. expansum 3.78 strain was inoculated onto the surface of Golden delicious apples and incubated at 8 or 20 °C until the mold lesion reached a diameter of 1, 2 or 3 cm. For each lesion size, patulin was quantified from apple samples cut into 1 cm depthwise fractions and widthwise sized cylinders. Maximum patulin concentration, about 80,000 ng/g apple, was obtained at 8 °C for the center and surface sample of the 3 cm diameter lesion. Patulin was systematically found at the highest concentration in the lesions, but still quantified up to one centimeter next to the lesion. Patulin concentrations were not significantly different between the 8 and 20 °C incubation temperature, except for the 3 cm large lesions. Based on these findings, and for lesions less than or equal to 3 cm in diameter, we recommend to consumers to cut off at least 1 cm around and below the mold spot to limit patulin exposure. Apples should also be stored at cool temperatures, below 8 °C, to delay lesion development.

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk.

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Functional diversity within the Penicillium roqueforti species.

Penicillium roqueforti is used as a ripening culture for blue cheeses and largely contributes to their organoleptic quality and typical characteristics. Different types of blue cheeses are manufactured and consumed worldwide and have distinct aspects, textures, flavors and colors. These features are well accepted to be due to the different manufacturing methods but also to the specific P. roqueforti strains used. Indeed, inoculated P. roqueforti strains, via their proteolytic and lipolytic activities, have an effect on both blue cheese texture and flavor. In particular, P. roqueforti produces a wide range of flavor compounds and variations in their proportions influence the flavor profiles of this type of cheese. Moreover, P. roqueforti is also characterized by substantial morphological and genetic diversity thus raising the question about the functional diversity of this species. In this context, 55 representative strains were screened for key metabolic properties including proteolytic activity (by determining free NH2 amino groups) and secondary metabolite production (aroma compounds using HS-Trap GC-MS and mycotoxins via LC-MS/Q-TOF). Mini model cheeses were used for aroma production and proteolysis analyses, whereas Yeast Extract Sucrose (YES) agar medium was used for mycotoxin production. Overall, this study highlighted high functional diversity among isolates. Noteworthy, when only P. roqueforti strains isolated from Protected Designation of Origin (PDO) or Protected Geographical Indication (PGI) blue cheeses were considered, a clear relationship between genetic diversity, population structure and the assessed functional traits was shown.

Insights into Penicillium roqueforti Morphological and Genetic Diversity.

Fungi exhibit substantial morphological and genetic diversity, often associated with cryptic species differing in ecological niches. Penicillium roqueforti is used as a starter culture for blue-veined cheeses, being responsible for their flavor and color, but is also a common spoilage organism in various foods. Different types of blue-veined cheeses are manufactured and consumed worldwide, displaying specific organoleptic properties. These features may be due to the different manufacturing methods and/or to the specific P. roqueforti strains used. Substantial morphological diversity exists within P. roqueforti and, although not taxonomically valid, several technological names have been used for strains on different cheeses (e.g., P. gorgonzolae, P. stilton). A worldwide P. roqueforti collection from 120 individual blue-veined cheeses and 21 other substrates was analyzed here to determine (i) whether P. roqueforti is a complex of cryptic species, by applying the Genealogical Concordance Phylogenetic Species Recognition criterion (GC-PSR), (ii) whether the population structure assessed using microsatellite markers correspond to blue cheese types, and (iii) whether the genetic clusters display different morphologies. GC-PSR multi-locus sequence analyses showed no evidence of cryptic species. The population structure analysis using microsatellites revealed the existence of highly differentiated populations, corresponding to blue cheese types and with contrasted morphologies. This suggests that the population structure has been shaped by different cheese-making processes or that different populations were recruited for different cheese types. Cheese-making fungi thus constitute good models for studying fungal diversification under recent selection.

Use of denaturing high-performance liquid chromatography (DHPLC) to characterize the bacterial and fungal airway microbiota of cystic fibrosis patients.

The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

Use of response surface methodology to optimise environmental stress conditions on Penicillium glabrum, a food spoilage mould.

Fungi are ubiquitous microorganisms often associated with spoilage and biodeterioration of a large variety of foods and feedstuffs. Their growth may be influenced by temporary changes in intrinsic or environmental factors such as temperature, water activity, pH, preservatives, atmosphere composition, all of which may represent potential sources of stress. Molecular-based analyses of their physiological responses to environmental conditions would help to better manage the risk of alteration and potential toxicity of food products. However, before investigating molecular stress responses, appropriate experimental stress conditions must be precisely defined. Penicillium glabrum is a filamentous fungus widely present in the environment and frequently isolated in the food processing industry as a contaminant of numerous products. Using response surface methodology, the present study evaluated the influence of two environmental factors (temperature and pH) on P. glabrum growth to determine 'optimised' environmental stress conditions. For thermal and pH shocks, a large range of conditions was applied by varying factor intensity and exposure time according to a two-factorial central composite design. Temperature and exposure duration varied from 30 to 50 °C and from 10 min to 230 min, respectively. The effects of interaction between both variables were observed on fungal growth. For pH, the duration of exposure, from 10 to 230 min, had no significant effect on fungal growth. Experiments were thus carried out on a range of pH from 0.15 to 12.50 for a single exposure time of 240 min. Based on fungal growth results, a thermal shock of 120 min at 40 °C or a pH shock of 240 min at 1.50 or 9.00 may therefore be useful to investigate stress responses to non-optimal conditions.