Debaryomyces is enriched in Crohn's disease intestinal tissue and impairs healing in mice.

Alterations of the mycobiota composition associated with Crohn's disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch's postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell-specific type 1 interferon-CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing.

Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Early gut mycobiota and mother-offspring transfer.

BACKGROUND: The fungi in the gastrointestinal tract, the gut mycobiota, are now recognised as a significant part of the gut microbiota, and they may be important to human health. In contrast to the adult gut mycobiota, the establishment of the early gut mycobiota has never been described, and there is little knowledge about the fungal transfer from mother to offspring. METHODS: In a prospective cohort, we followed 298 pairs of healthy mothers and offspring from 36 weeks of gestation until 2 years of age (1516 samples) and explored the gut mycobiota in maternal and offspring samples. Half of the pregnant mothers were randomised into drinking probiotic milk during and after pregnancy. The probiotic bacteria included Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis Bb-12 and Lactobacillus acidophilus La-5. We quantified the fungal abundance of all the samples using qPCR of the fungal internal transcribed spacer (ITS)1 segment, and we sequenced the 18S rRNA gene ITS1 region of 90 high-quantity samples using the MiSeq platform (Illumina). RESULTS: The gut mycobiota was detected in most of the mothers and the majority of the offspring. The offspring showed increased odds of having detectable faecal fungal DNA if the mother had detectable fungal DNA as well (OR = 1.54, p = 0.04). The fungal alpha diversity in the offspring gut increased from its lowest at 10 days after birth, which was the earliest sampling point. The fungal diversity and fungal species showed a succession towards the maternal mycobiota as the child aged, with Debaryomyces hansenii being the most abundant species during breast-feeding and Saccharomyces cerevisiae as the most abundant after weaning. Probiotic consumption increased the gut mycobiota abundance in pregnant mothers (p = 0.01). CONCLUSION: This study provides the first insight into the early fungal establishment and the succession of fungal species in the gut mycobiota. The results support the idea that the fungal host phenotype is transferred from mother to offspring. TRIAL REGISTRATION: Clinicaltrials.gov NCT00159523.

PH EFFECT ON ANTAGONISTIC ACTIVITY TOWARDS BACTERIA OF YEASTS ISOLATED FROM HUCUL DAIRY PRODUCTS AND GASTROINTESTINAL TRACT OF HUMAN.

The aim of this work was to study the influence of pH of medium on antagonistic ac- tivity of isolated from authentic Hucul dairy products and gastrointestinal tract (GIT) of Hucul long-livers yeasts towards potentially harmful for humans and animals bacteria. Among 52 tested yeast isolates 14 % yeasts showed considerable antagonistic activity towards Gram-positive bacteria Staphylococcus aureus and only 6 % of them inhibited growth of Gram negative bacteria belonging to genera Escherichia and Citrobacter Most ofyeasts with antagonistic activity (over 70 %) were isolatedfriom long-livers GIT There were identifed two optimal for antagonism areas of pH values of nutrient medium for tested yeasts being around 5.5 and 6.0 for Gram-positive bacteria and around 6.0 and 6.5 for Gram negative bacteria. It appeared that isolated fiom Hucul yogurt Saccharomyces pasterianus yeasts manifested their antagonistic activity in more acidic conditions com- pared to isolates fiom GIT.

Selection and evaluation of Debaryomyces hansenii isolates as potential bioprotective agents against toxigenic penicillia in dry-fermented sausages.

Biocontrol using autochthonous Debaryomyces hansenii isolates is a potentially suitable strategy for inhibiting toxigenic moulds in dry-cured meat products. The antifungal activity of 280 D. hansenii isolated from dry-cured meat products as well as the mode of action of the most active isolates against toxigenic penicillia were evaluated in this work. A 13.9% of the D. hansenii isolates showed inhibitory activity in a radial inhibition assay. The effects on penicillia growth of both the cell-free culture filtrate and volatile compounds from active yeast isolates were analysed. Penicillia growth inhibition by D. hansenii was probably based on additive or synergistic effects of several inhibiting factors such as competition for nutrient and space, and production of soluble or volatile compounds. When four D. hansenii isolates were tested on dry-fermented sausage, two of them produced a significantly growth reduction of the ochratoxigenic Penicillium verrucosum, keeping its counts under the level considered as hazardous for the mycotoxin presence. Therefore, the use of these two D. hansenii isolates during the processing of dry-fermented meat product could be a promising tool to control toxigenic moulds in the meat industry.

Draft genome sequence of salt-tolerant yeast Debaryomyces hansenii var. hansenii MTCC 234.

Debaryomyces hansenii is one of the most halotolerant species of yeast, and the genome sequence of D. hansenii strain CBS767 is already available. Here we report the 11.46-Mb draft genome of D. hansenii strain MTCC 234, which is even more halotolerant than strain CBS767. Comparative analysis of these sequences would definitely provide further insight into the halotolerance of this yeast.

Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

UNLABELLED: Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. PRACTICAL APPLICATION: Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes.

Extracellular lipolytic enzyme activity of a newly isolated Debaryomyces hansenii.

A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.

Fungal diversity in composting process of pig manure and mushroom cultural waste based on partial sequence of large subunit rRNA.

Fungal diversity during composting was investigated by culture-independent rDNA sequence analysis. Composting was carried out with pig manure and mushroom cultural waste using a field-scale composter (Hazaka system), and samples were collected at various stages. Based on partial sequence analysis of large subunit (LSU) ribosomal RNA (rRNA) and sequence identity values, a total of 12 different fungal species were found at six sampling sites; Geotrichum sp., Debaryomyces hansenii, Monographella nivalis, Acremonium strictum, Acremonium alternatum, Cladosporium sphaerospermum, Myriangium durosai, Pleurotus eryngii, Malassezia globosa, Malassezia restricta, Rhodotorula glutinis, and Fusarium sporotrichioides. Geotrichum sp. of the class Saccharomycetes was the most predominant fungal species throughout the composting process (185 out of a total of 236 identified clones, or 78.4%), followed by Acremonium strictum (7.6%), Monographella nivalis (5.1%), and Pleurotus eryngii (3.8%). The prevalence of Geotrichum sp. was the lowest (61.1%) at the beginning of composting, and then gradually increased to 92.5% after 10 days of composting.

"Pecorino di Filiano" cheese as a selective habitat for the yeast species, Debaryomyces hansenii.

The composition of yeast microflora in artisanal "Pecorino di Filiano" cheese, a typical product of the Basilicata region of Southern Italy, was studied during ripening. The isolates were identified by restriction analysis of the 18S rDNA amplified region with the combined use of Hinf I and Cfo I enzymes. The majority of the isolates were identified as Debaryomyces hansenii, whereas two yeasts were identified as Kluyveromyces lactis and one as Dekkera anomala. To evaluate natural biodiversity, D. hansenii "Pecorino di Filiano" isolates were submitted to genetic and technological characterization. RAPD-PCR analysis with P80 (5CGCGTGCCCA3) primer revealed significant polymorphism among D. hansenii isolates. About 30% of the isolates showed single molecular profiles, whereas the other D. hansenii yeasts were separated into three main patterns, differing for both the ripening time and the isolation source. Furthermore, the yeasts showed significant variability in their, "proteolytic activity". This work demonstrated the high predominance of D. hansenii among the yeast population of "Pecorino di Filiano" cheese, probably in consequence of the traditional salting process, which was selected for this salt tolerant species. This preliminary study allowed us to isolate autochthonous D. hansenii yeasts potentially useful as starters for the production of this artisanal cheese.

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.