Changes in the gut mycobiome in pediatric patients in relation to the clinical activity of Crohn's disease.

BACKGROUND: Numerous studies have shown that in Crohn's disease (CD), the gut microbiota is of great importance in the induction and maintenance of inflammation in the gastrointestinal tract. Until recently, studies have focused almost exclusively on bacteria in the gut. Lately, more attention has been paid to the role of intestinal fungi. AIM: To study the gut mycobiome analysis of pediatric patients with CD (in different stages of disease activity) compared to healthy children. METHODS: Fecal samples were collected from patients: With active, newly diagnosed CD (n = 50); active but previously diagnosed and treated CD (n = 16); non-active CD and who were in clinical remission (n = 39) and from healthy volunteers (n = 40). Fungal DNA was isolated from the samples. Next, next generation sequencing (MiSeq, Illumina) was performed. The composition of mycobiota was correlated with clinical and blood parameters. RESULTS: Candida spp. were overrepresented in CD patients, while in the control group, the most abundant genus was Saccharomyces. In CD patients, the percentage of Malassezia was almost twice that of the control (P < 0.05). In active CD patients, we documented a higher abundance of Debaryomyces hansenii (D. hansenii) compared to the non-active CD and control (P < 0.05) groups. Moreover, statistically significant changes in the abundance of Mycosphaerella, Rhodotorula, and Microidium were observed. The analyses at the species level and linear discriminant analysis showed that in each group it was possible to distinguish a specific species characteristic of a given patient population. Moreover, we have documented statistically significant correlations between: D. hansenii and patient age (negative); C. zeylanoides and patient age (positive); C. dubliniensis and calprotectin (positive); C. sake and calprotectin (positive); and C. tropicalis and pediatric CD activity index (PCDAI) (positive). CONCLUSION: Mycobiome changes in CD patients, and the positive correlation of some species with calprotectin or PCDAI, give strong evidence that fungi may be of key importance in the development of CD.

Exploring fungal diversity in Antarctic wildlife: isolation and molecular identification of culturable fungi from penguins and pinnipeds.

AIMS: To survey the diversity of fungal species that may be cultured from Antarctic penguins and pinnipeds, and to test the in vitro susceptibility to triazole drugs of any medically important Aspergillus spp. isolates. METHODS: During an expedition to Argentinean Antarctic research stations at Potter Peninsula (South Shetland Islands) and Primavera Cape (Antarctic Peninsula) in February 2019, samples (n = 212) were collected from fur seals (Arctocephalus gazella), elephant seals (Mirounga leonine), leopard seals (Hydrurga leptonyx), Weddell seals (Leptonychotes weddellii) and crabeater seals (Lobodon carcinophaga) and gentoo penguins (Pygoscelis papua). Oral, nasal and rectal swabs and skin/hair brushings were collected from pinnipeds, and skin/feather brushings, cloacal swabs and moulted feathers from penguins. Samples were cultured on Sabouraud dextrose agar and/or potato dextrose agar plates and fungal isolates identified by morphological criteria followed by PCR amplification and DNA sequencing. Antifungal susceptibility of Aspergillus spp. isolates to triazoles was tested. RESULTS: Fungi from 21 genera were isolated from 121/212 (57.1%) samples obtained from pinnipeds and penguins. Among pinnipeds from Potter Peninsula (fur seals and elephant seals), the most frequent fungal species were Debaryomyces hansenii and Rhodotorula mucilaginosa, isolated from the oral, nasal and/or rectal mucosa, and Antarctomyces psychrotrophicus isolated from the skin/hair of all sampled individuals. Among pinnipeds from Primavera Cape (leopard seals, Weddell seals and crabeater seals), the most frequent fungal species were Naganishia adeliensis and Cryptococcus neoformans var. uniguttulatus, isolated from the nasal/oral mucosa of 4/33 (15.2%) and 5/33 (12.1%) animals, respectively. The most frequently isolated fungal species from gentoo penguins (Potter Peninsula), were Pseudogymnoascus pannorum and A. pyschrotrophicus, which both were isolated from skin/feathers of 7/15 (46.7%) birds, and Thelebolus microsporus, isolated from the cloacal mucosa and skin/feathers of 5/15 (33.3%) and 2/15 (13.3%) birds, respectively. Fungi that are potentially pathogenic to both humans and animals (Aspergillus fumigatus, Asp. flavus, Asp. versicolor, Candida parapsilosis and Microsporum canis) were isolated from 4/38 (10.5%), 1/38 (2.6%), 2/38 (5.3%), 4/38 (10.5%) and 2/38 (5.3%) sampled pinnipeds, respectively. Only non-azole-resistant isolates of Asp. fumigatus and Asp. flavus were identified. CONCLUSIONS: The fungal biodiversity in Antarctic pinnipeds and gentoo penguins was explored using standard mycological culture followed by PCR and DNA sequencing. The frequency of fungal carriage varied among animal species, sample type and location. This study constitutes an epidemiologic approach to monitoring of these marine animals for emerging fungal pathogens.

Specific fungi associated with response to capsulized fecal microbiota transplantation in patients with active ulcerative colitis.

Objective: Fecal microbiota transplantation (FMT) is a novel microbial treatment for patients with ulcerative colitis (UC). In this study, we performed a clinical trial of capsulized FMT in UC patients to determine the association between the gut fungal community and capsulized FMT outcomes. Design: This study recruited patients with active UC (N = 22) and healthy individuals (donor, N = 9) according to the criteria. The patients received capsulized FMT three times a week. Patient stool samples were collected before (week 0) and after FMT follow-up visits at weeks 1, 4, and 12. Fungal communities were analysed using shotgun metagenomic sequencing. Results: According to metagenomic analysis, fungal community evenness index was greater in samples collected from patients, and the overall fungal community was clustered among the samples collected from donors. The dominant fungi in fecal samples collected from donors and patients were Ascomycota and Basidiomycota. However, capsulized FMT ameliorated microbial fungal diversity and altered fungal composition, based on metagenomic analysis of fecal samples collected before and during follow-up visits after capsulized FMT. Fungal diversity decreased in samples collected from patients who achieved remission after capsulized FMT, similar to samples collected from donors. Patients achieving remission after capsulized FMT had specific enrichment of Kazachstania naganishii, Pyricularia grisea, Lachancea thermotolerans, and Schizosaccharomyces pombe compared with patients who did not achieve remission. In addition, the relative abundance of P. grisea was higher in remission fecal samples during the follow-up visit. Meanwhile, decreased levels of pathobionts, such as Candida and Debaryomyces hansenii, were associated with remission in patients receiving capsulized FMT. Conclusion: In the metagenomic analysis of fecal samples from donors and patients with UC receiving capsulized FMT, shifts in gut fungal diversity and composition were associated with capsulized FMT and validated in patients with active UC. We also identified the specific fungi associated with the induction of remission. ClinicalTrails.gov (NCT03426683).

Autochthonous fungi are central components in microbial community structure in raw fermented sausages.

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.

Mycotoxigenic and phylogenetic perspective to the yeasts and filamentous moulds in mould-matured Turkish cheese.

This study was conducted to determine the diversity of yeasts and filamentous moulds in mould-matured cheese (MMC) consumed in Turkey. Overall, 120 samples were collected from 12 different geographical locations between March 2016 and April 2017. The morphological observation was applied in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular analyses to determine yeasts and filamentous moulds in the cheeses. High-performance liquid chromatography (HPLC) technique was used to evaluate the ability of mycotoxins production of fungal isolates and the presence of mycotoxins in cheese samples. A total of 241 fungi (81 filamentous moulds and 160 yeast) were recovered, and Penicillium roqueforti and Debaryomyces hansenii were the most frequently isolated species in all cheese samples. The rep-PCR results indicated a high level of genetic diversity among fungal isolates, regardless of isolation source or geographical origin. Filamentous mould strains isolated from MMC were found to synthesize at least one mycotoxin (Aflatoxin B1, B2, G1 and G2, citrinine, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid and roquefortine C). Although mycotoxin producing ability was observed from all isolates, none of the cheese samples were found positive for these mycotoxins. AFM1 was detected in 8 (6.6%) MMC samples from which 2 (1.6%) were above the legal limits (0.05 µg/kg) set by the Turkish Food Codex (TFC) and European Commission (EC). In conclusion, Turkish MMCs were found to be contaminated with toxigenic fungi, so a potential public health risk, while low, exists. Therefore, the selection of nontoxigenic filamentous mould strains for cheese manufacturing and control of the ripening conditions is a critical need to ensure the quality and safety of Turkish MMC.

Fungal community succession and volatile compound dynamics in Harbin dry sausage during fermentation.

This study investigated the fungal community succession and volatile compound dynamics of Harbin dry sausage during a twelve-day fermentation using high-throughput internal transcribed spacer amplicon sequencing and headspace solid-phase microextraction gas chromatography-mass spectrometry. Aspergillus pseudoglaucus was found to be the primary species in the sausages during fermentation, whereas Lasiodiplodia theobromae, Alternaria alternata, Aspergillus caesiellus, and Trichosporon asahii were also prevalent. Additionally, a total of 72 volatile compounds were identified in the dry sausages, of which 24 key compounds (odor activity value > 1) dominated flavor development, including 3 aldehydes, 1 ketone, 4 alcohols, 9 esters, 4 alkenes, and 3 other compounds. Furthermore, correlation analysis suggested that most of the core fungi were positively correlated with the key volatile compounds, particularly A. pseudoglaucus, Aspergillus gracilis, Trichosporon caseorum, Debaryomyces hansenii, and T. asahii. Our findings provide novel insights into the fungal ecology and flavor development of Harbin dry sausages.

Use of High-Throughput Sequencing to Identify Fungal Communities on the Surface of Citri Reticulatae Pericarpium During the 3-Year Aging Process.

Citri Reticulatae Pericarpium (CRP) is a natural product that is used widely in food and is an ingredient in traditional Chinese medicine. CRP improves gradually with aging; this process typically takes 3 years or more. During the aging process, CRP can be colonized with fungi and mildew. Molds and mildew may result in an increased flavonoid content; however, this has been observed only in response to fungi of the genera Penicillium and Aspergillus. As fungal colonization may alter the quality and properties of CRP, it is critical to have an understanding of the fungal communities detected on the surface of CRP during the aging process. We used a high-throughput sequencing (HiSeq) platform to sequence internal transcribed spacers (ITS) region to identify the contaminants associated with CRP during the 3-year aging process. We also evaluated the distribution of the dominant fungi of the genera Aspergillus and Penicillium over time. At the phylum level, we identified Ascomycota (36.26%) and Basidiomycota (18.98%), along with smaller populations of Mucoromycota, Glomeromycota, and Mortierellomycota. At the genus level, the fungi detected include Wallemia (12.40%), Cystofilobasidium (4.62%), Zasmidium (4.52%), Cladosporium (3.72%), Hanseniaspora (3.55%), Fusarium (3.49%), Kurtzmaniella (2.03%), Candida (1.74%), Passalora (1.47%), Ceramothyrium (1.33%), Mucor (1.07%), and Aspergillus (1.03%). Fungi of the genus Penicillium were detected primarily during the first year of storage. By contrast, fungi of the genus Aspergillus were not detected during the early stages (fresh peel-8 months), but appeared gradually at later stages of the aging process. Taken together, our results indicate that HiSeq is an effective method to study the changes in fungal communities that develop on the CRP surface over time. These findings provide a basis for further research into the correlation between dominant fungi and the mechanisms underlying the successful aging of CRP.

Biochemical changes and microbial community dynamics during spontaneous fermentation of Zhacai, a traditional pickled mustard tuber from China.

Zhacai is a traditional fermented vegetable that has been consumed in China for centuries. It is currently manufactured by spontaneous fermentation and therefore mostly relies on the activities of autochthonous microorganisms. Here, we characterized microbial community dynamics and associated biochemical changes in 12% salted Zhacai during a 90-day spontaneous fermentation process using high-throughput sequencing and chromatography-based approaches to identify associations between microorganisms and fermentation characteristics. Amplicon sequencing targeting bacterial 16S rRNA genes revealed that bacterial communities were dominated by halophilic bacteria (HAB, i.e., Halomonas and Idiomarina) and lactic acid bacteria (LAB, i.e., Lactobacillus-related genera and Weissella) after 30 days of fermentation. In addition, the relative abundances of the fungal genera Debaryomyces, Sterigmatomyces, and Sporidiobolus increased as fermentation progressed. Concomitantly, pH decreased while titratable acidity increased during fermentation, along with associated variation in biochemical profiles. Overall, the levels of organic acids (i.e., lactic and acetic acid), free amino acids (i.e., alanine, lysine, and glutamic acid), and volatiles (i.e., alcohols, esters, aldehydes, and ketones) increased in mature Zhacai. In addition, the abundances of Lactobacillus-related species, Halomonas spp., Idiomarina loihiensis, as well as that of the yeast Debaryomyces hansenii, were strongly correlated with increased concentrations of organic acids, amino acids, biogenic amines, and volatiles. This study provides new detailed insights into the succession of microbial communities and their potential roles in Zhacai fermentation.

The potential correlations between the fungal communities and volatile compounds of traditional dry sausages from Northeast China.

The fungal communities and volatile compounds of traditional dry sausages collected from five different regions in Northeast China, including Harbin (HRB), Daqing (DQ), Suihua (SH), Hegang (HG) and Mudanjiang (MDJ) were investigated in this study. The results revealed clear differences among the fungal community structures of the sausages. Aspergillus pseudoglaucus, Debaryomyces hansenii, and Trichosporon asahii were found to be the predominant species in the sausages from HRB, HG, and MDJ, respectively. Candida zeylanoides was the predominant species in the sausage from DQ and SH. Additionally, 88 volatile compounds were identified in all sausages, of which 31 volatile compounds were the most important flavor contributors (odor activity value > 1). Potential correlation analysis revealed that 8 fungi (D. hansenii, C. zeylanoides, T. asahii, A. pseudoglaucus, Aspergillus sydowii, Penicillium expansum, A. alternata, and Alternaria tenuissima) showed significant positive correlations with ≥3 key volatile compounds. Among these fungi, D. hansenii was regarded as a core functional fungus responsible for the formation of the volatile compounds, given its strong connection with the highest number of key volatile compounds. These results provide detailed insight into the fungal communities of traditional dry sausages and a deeper understanding of the contribution of these fungi to sausage flavor.

Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population.

BACKGROUND: A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. RESULTS: Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. CONCLUSION: A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

DNA metabarcoding of fungal diversity in air and snow of Livingston Island, South Shetland Islands, Antarctica.

We assessed fungal diversity present in air and freshly deposited snow samples obtained from Livingston Island, Antarctica, using DNA metabarcoding through high throughput sequencing (HTS). A total of 740 m3 of air were pumped through a 0.22 µm membrane. Snow obtained shortly after deposition was kept at room temperature and yielded 3.760 L of water, which was filtered using Sterivex membranes of 0.22 µm mesh size. The total DNA present was extracted and sequenced. We detected 171 fungal amplicon sequence variants (ASVs), 70 from the air and 142 from the snow. They were dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. Pseudogymnoascus, Cladosporium, Mortierella and Penicillium sp. were the most dominant ASVs detected in the air in rank order. In snow, Cladosporium, Pseudogymnoascus, Penicillium, Meyerozyma, Lecidea, Malassezia, Hanseniaspora, Austroplaca, Mortierella, Rhodotorula, Penicillium, Thelebolus, Aspergillus, Poaceicola, Glarea and Lecanora were the dominant ASVs present. In general, the two fungal assemblages displayed high diversity, richness, and dominance indices, with the assemblage found in snow having the highest diversity indices. Of the total fungal ASVs detected, 29 were only present in the air sample and 101 in the snow sample, with only 41 present in both samples; however, when only the dominant taxa from both samples were compared none occurred only in the air and, among the rare portion, 26 taxa occurred in both air and snow. Application of HTS revealed the presence of a more diverse fungal community in the air and snow of Livingston Island in comparison with studies using traditional isolation methods. The assemblages were dominated by cold-adapted and cosmopolitan fungal taxa, including members of the genera Pseudogymnoascus, Malassezia and Rhodotorula, which include some taxa reported as opportunistic. Our results support the hypothesis that the presence of microbiota in the airspora indicates the possibility of dispersal around Antarctica in the air column. However, further aeromycology studies are required to understand the dynamics of fungal dispersal within and beyond Antarctica.

Lactobacillus delbrueckii subsp. bulgaricus F17 and Leuconostoc lactis H52 supernatants delay the decay of strawberry fruits: a microbiome perspective.

Strawberries are vulnerable to physical injuries and microbial invasion. To explore if beneficial lactic acid bacteria can improve the shelf life and edible quality of postharvest strawberry fruits, the effects of Lactobacillus delbrueckii subsp. bulgaricus (ital.) F17 (F17) and Leuconostoc lactis (ital.) H52 (H52) inoculation on the strawberry microbial community structure and saleable characteristics were examined by bacterial 16S rRNA and fungal ITS sequencing techniques. Lactobacillus (ital.) F17 and Leuconostoc lactis (ital.) H52 isolated from the traditional fermented yak milk in the Qinghai-Tibetan Plateau were used as the potential probiotic inocula. Samples from treated strawberries stored at 25 °C for 0, 12, 24, 48, and 72 hours were analyzed for their pH, weight loss percentage, decay percentage, total soluble solid content (SSC) and microbial counts, and for microbiome community diversity and canonical correspondence analysis. The results showed that F17 and H52 did not only significantly reduce the weight loss and decay percentage of strawberry fruits, but also delayed the decrease of the total SSC and pH (P < 0.05). In addition, F17 and H52 significantly inhibited the growth and colonization of aerobic mesophilic bacteria, yeast, mold and coliform bacteria. In particular, by comparing the microbiota composition of the samples, F17 significantly inhibited Pantoea, Mycospherella, unclassified_Pleosporales, Aureobasidium and Phoma at the genus level, whereas H52 inhibited Bacillus, Streptophyta, Mycospherella, Aureobasidium and Phoma. Moreover, analysis of alpha and beta diversity revealed that F17 and H52 had a significantly greater inhibitory effect on bacterial species compared to fungi. The results of canonical correspondence analysis revealed that the total SSC and pH were positively correlated with bacteria, whereas the decay percentage, weight loss percentage and total SSC were positively associated with fungi. Additionally, Podosphaera, Hanseniaspora, Botrytis and unclassified_Pleosporales were positively correlated with strawberry fruit decay and weight loss percentage. As a general result, Lactobacillus F17 and Leuconostoc lactis H52 have the potential to promote biological preservation, which is economically important to reduce the loss due to strawberry spoilage.

Identification of Microorganisms Associated with the Quality Improvement of Dry-Aged Beef Through Microbiome Analysis and DNA Sequencing, and Evaluation of Their Effects on Beef Quality.

The objective of this study was to isolate and identify the microorganisms, especially yeasts and molds, related to the improvement of beef quality during dry-aging of beef through microbiome analysis, and to examine the possibility of using them as starter culture strains to improve the efficiency of dry-aging beef production. Beef sirloins were dry-aged for 28 days using different wind speeds (0, 2.5, and 5 m/s) at 1 to 3 °C and 75% relative humidity, and microbial compositions were confirmed by microbiome analysis. Mold and yeast samples were plated on potato dextrose agar supplemented with 10% tartaric acid, and the isolated colonies were identified by DNA sequencing. The isolates were subjected to microbial characterization (morphological characterization, growth condition, and enzyme activity). Microbiome analysis showed that the dominant microorganisms were molds and yeasts identified as Pilaira anomala SMFM201611 and Debaryomyces hansenii SMFM201707. Pilaira anomala SMFM201611 and D. hansenii SMFM201707 were inoculated into 24 sirloins of the lowest grade. All samples were dry-aged for 0, 14, 21, and 28 days and analyzed for microbial growth, pH, shear force, ultrastructure, and flavor compounds (free amino acids and free fatty acids). Inoculation with P. anomala SMFM201611 and D. hansenii SMFM201707 improved tenderness and cause the breakdown of myofibrils by proteolysis. Both microorganisms also produced free amino acids and fatty acids through proteolytic and lipolytic activities. These results indicate that P. anomala SMFM201611 and D. hansenii SMFM201707 isolated and identified from dry-aged beef can improve the quality of low-grade beef during dry-aging. PRACTICAL APPLICATION: During dry-aging, mold and yeast improve the quality of dry-aged beef. Pilaira anomala SMFM201611 and Debaryomyces hansenii SMFM201707 isolated from dry-aged beef can improve tenderness by breaking down myofibrils. Both microorganisms improve flavor by producing free fatty acids and amino acids, and the taste and aroma characteristics of low-grade beef may be improved during the dry-aging process.

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica.

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.

Microbiota of Iberian dry-cured ham as influenced by chemical composition, high pressure processing and prolonged refrigerated storage.

The effect of high pressure processing (HPP) on the microbiota of ripened Iberian ham of different water activity, salt concentration and intramuscular fat content was investigated before and after a 5-month refrigeration period. At the beginning of the refrigeration period, the only significant effects of chemical composition were those of water activity on psychrotrophs and Micrococcaceae in untreated hams, and of the salt-in-lean ratio on lactic acid bacteria in HPP-treated hams. At the end of the refrigeration period, the only significant effect was that of intramuscular fat content on moulds and yeasts in HPP-treated samples. All microbial groups were significantly affected by HPP, with reductions ranging from 1.7 to 2.0 log cycles after treatment. A significant recovery of all microbial groups took place in HPP-treated hams during the refrigeration period, with increases ranging from 0.5 to 1.1 log cycles. In spite of this recovery, microbial levels in HPP-treated hams remained significantly lower than in untreated hams. Staphylococcus accounted for 93.4% of Iberian ham bacterial isolates, with S. equorum as the most abundant species. Representatives of the Tetragenococcus, Carnobacterium and Streptomyces genera, not previously reported in dry-cured ham, were also isolated. Most of the yeast isolates (75.0%) were identified as Debaryomyces hansenii.

Yeast-like fungi and yeasts in withered grape carposphere: Characterization of Aureobasidium pullulans population and species diversity.

Yeast-like fungi and yeasts residing on carposphere of withered grapes for Italian passito wine production have been scarcely investigated. In the present study, isolates from single berries, both sound and damaged, of Nosiola, Corvina and Garganega varieties were analyzed at the end of the withering process. Great variation of cell concentration among single berries was observed. In sound berries, yeast-like fungi were significantly more frequent than yeasts. Species identification of isolates was carried out by BLAST comparative analysis on gene databases and phylogenetic approach. All yeast-like fungi isolates belonged to Aureobasidium pullulans. They displayed different culture and physiological characteristics and inhibitory capacity against phytopathogenic fungi. Moreover, PCR profile analysis revealed high genotypic similarity among these strains. A total of 35 species were recognized among yeast isolates. Ascomycetes prevailed over basidiomycetes. To the best of our knowledge, Naganishia onofrii and Rhodosporidiobolus odoratus were identified for the first time among yeasts isolated from grapes, must or wine. Hanseniaspora uvarum and Starmerella bacillaris were the most frequent species. Most species were found only in one grape variety (nine in Nosiola, 10 in Corvina and five in Garganega). The sanitary state of withered grapes could have an important impact on the structure of these epiphytic populations.

Diversity and Characteristics of the Meat Microbiological Community on Dry Aged Beef.

Beef was dry aged for 40-60 days under controlled environmental conditions in a refrigerated room with a relative humidity of 75%-80% and air-flow. To date, there is little information on the microbial diversity and characteristics of dry aged beef. In this study, we explored the effect of change in meat microorganisms on dry aged beef. Initially, the total bacteria and LAB were significantly increased for 50 days during all dry aging periods. There was an absence of representative foodborne pathogens as well as coliforms. Interestingly, fungi including yeast and mold that possess specific features were observed during the dry aging period. The 5.8S rRNA sequencing results showed that potentially harmful yeasts/molds (Candida sp., Cladosporium sp., Rhodotorula sp.) were present at the initial point of dry aging and they disappeared with increasing dry aging time. Interestingly, Penicillium camemberti and Debaryomyces hansenii used for cheese manufacturing were observed with an increase in the dry aging period. Taken together, our results showed that the change in microorganisms exerts an influence on the quality and safety of dry aged beef, and our study identified that fungi may play an important role in the palatability and flavor development of dry aged beef.

Distribution of apple and blackcurrant microbiota in Lithuania and the Czech Republic.

The microbial assemblies on the surface of plants correlate with specific climatic features, suggesting a direct link between environmental conditions and microbial inhabitation patterns. At the same time however, microbial communities demonstrate distinct profiles depending on the plant species and region of origin. In this study, we report Next Generation Sequencing-based metagenomic analysis of microbial communities associated with apple and blackcurrant fruits harvested from Lithuania and the Czech Republic. Differences in the taxonomic composition of eukaryotic and prokaryotic microorganisms were observed between plant types. Our results revealed limited geographic differentiation between the bacterial and fungal communities associated with apples. In contrast, blackcurrant berries harvested from different regions demonstrated high diversity in both bacterial and fungal microbiota structures. Among fungal and bacterial microorganisms, we identified both potentially beneficial (Cryptococcus, Hanseniaspora, Massilia, Rhodotorula, Sphingomonas) and phytopathogenic microorganisms (Cladosporium, Pantoea, Phoma, Pseudomonas, Septoria, Taphrina) indicating their important roles in ecological and evolutionary processes.

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.

Application of MALDI-TOF MS fingerprinting as a quick tool for identification and clustering of foodborne pathogens isolated from food products.

Foodborne pathogens can be associated with a wide variety of food products and it is very important to identify them to supply safe food and prevent foodborne infections. Since traditional techniques are timeconsuming and laborious, this study was designed for rapid identification and clustering of foodborne pathogens isolated from various restaurants in Al-Qassim region, Kingdom of Saudi Arabia (KSA) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sixty-nine bacterial and thirty-two fungal isolates isolated from 80 food samples were used in this study. Preliminary identification was carried out through culture and BD Phoenix™ methods. A confirmatory identification technique was then performed using MALDI-TOF MS. The BD Phoenix results revealed that 97% (67/69 isolates) of bacteria were correctly identified as 75% Enterobacter cloacae, 95.45% Campylobacter jejuni and 100% for Escherichia coli, Salmonella enterica, Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. While 94.44% (29/32 isolates) of fungi were correctly identified as 77.77% Alternaria alternate, 88.88% Aspergillus niger and 100% for Aspergillus flavus, Penicillium digitatum, Candida albicans and Debaryomyces hansenii. However, all bacterial and fungal isolates were 100% properly identified by MALDI-TOF MS fingerprinting with a score value ≥2.00. A gel view illustrated that the spectral peaks for the identified isolates fluctuate between 3,000 and 10,000 Da. The results of main spectra library (MSP) dendrogram showed that the bacterial and fungal isolates matched with 19 and 9 reference strains stored in the Bruker taxonomy, respectively. Our results indicated that MALDI-TOF MS is a promising technique for fast and accurate identification of foodborne pathogens.