Microbial characterization of an artisanal production of Robiola di Roccaverano cheese.

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005.

Galactomyces geotrichum mold isolated from a traditional fried cottage cheese produced omega-3 fatty acids.

Thirty nine strains of Galactomyces geotrichum molds were isolated from a traditional fried cottage cheese and production of polyunsaturated fatty acids (PUFA) was assessed. Among them eleven strains produced an extracellular lipids enriched in n-6 and n-3 PUFA. The extracellular lipids produced by G. geotrichum strain 38 contained the highest amounts of total PUFA (24.3%), with the highest contribution of n-3 fatty acids (17.9%), where α-linolenic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids were the main contributors. To obtain maximal production of PUFA, composition of the medium consisted of 10 g/L rapeseed oil, 5 g/L yeast extract, 0.05 g/L K2HPO4, 0.17 g/L MgSO4, 0.015 g/L MnSO4, 0.015 g/L ZnSO4, 0.05 g/L FeSO4, and 10 mg/L vitamin B12. The optimal growth conditions at 30 °C involve: aeration at 1.5 vvm (volume of air per volume of broth per minute) at pH 6.5. The cheese produced under described conditions contained higher amount of n-3 PUFA (0.25 mg/g cheese) in comparison to control (0.01 mg/g). α-Linolenic acid predominated among n-3 fatty acids. Galactomyces geotrichum is a natural microflora of dairy products, and could be used to enrich food/cheese in deficient omega-3 lipids.

Invasive infections due to Saprochaete and Geotrichum species: Report of 23 cases from the FungiScope Registry.

Saprochaete and Geotrichum spp. are rare emerging fungi causing invasive fungal diseases in immunosuppressed patients and scarce evidence is available for treatment decisions. Among 505 cases of rare IFD from the FungiScope™ registry, we identified 23 cases of invasive infections caused by these fungi reported from 10 countries over a 12-year period. All cases were adults and previous chemotherapy with associated neutropenia was the most common co-morbidity. Fungaemia was confirmed in 14 (61%) cases and deep organ involvement included lungs, liver, spleen, central nervous system and kidneys. Fungi were S. capitata (n=14), S. clavata (n=5), G. candidum (n=2) and Geotrichum spp. (n=2). Susceptibility was tested in 16 (70%) isolates. All S. capitata and S. clavata isolates with the exception of one S. capitata (MIC 4 mg/L) isolate had MICs>32 mg/L for caspofungin. For micafungin and anidulafungin, MICs varied between 0.25 and >32 mg/L. One case was diagnosed postmortem, 22 patients received targeted treatment, with voriconazole as the most frequent first line drug. Overall mortality was 65% (n=15). Initial echinocandin treatment was associated with worse outcome at day 30 when compared to treatment with other antifungals (amphotericin B ± flucytosine, voriconazole, fluconazole and itraconazole) (P=.036). Echinocandins are not an option for these infections.

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.

Molecular study of Geotrichum strains isolated from Armada cheese.

The aim of this work was the genetic characterization at the strain level of 39 presumed Geotrichum candidum isolates isolated throughout the artisanal manufacturing and ripening of Armada cheese and tentatively identified at genus and/or species level by phenotypic characteristics. The molecular identification of the strains included among others the amplification and sequencing of the D1/D2 domains of the 26S rRNA gene. A restriction fragment length polymorphism (RFLP) analysis with the ITS1-5.8S-ITS2 PCR amplicons and a randomly amplified polymorphic DNA (RAPD) analysis with five different primers were carried out. The bands pattern profile obtained through RFLP by enzymatic restriction with HinfI was the same for all the strains studied, which confirmed the classification of the strains at species level. A RAPD-PCR analysis with three different primers was applied to assess the intraspecific diversity, in this way 16 band profiles were obtained for the 39 strains studied by the combined use of primers Ari1 and Omt1. This study contributes to know the occurrence and genotypic biodiversity of G. candidum in Armada cheese.

Genetic diversity of dairy Geotrichum candidum strains revealed by multilocus sequence typing.

The introduction of multilocus sequence typing (MLST) for strain characterization provided the first sequence-based approach for genotyping many fungi, leading to reproducible, reliable, and exchangeable data. A MLST scheme based on the analysis of six housekeeping genes was developed for genotyping Geotrichum candidum. The scheme was first developed using 18 isolates for which the complete sequences of the alanyl-tRNA synthetase (ALA1), pyruvate kinase (CDC19), acetyl-coA acetyltransferase (ERG10), glutaminyl-tRNA synthase (GLN4), phosphoglucoisomerase (PGI1), and phosphoglucomutase (PGM2) housekeeping genes were determined. Multiple sequence alignments of these genes were used to define a set of loci showing, as closely as possible, the same phylogenetic resolution level as complete gene sequences. This scheme was subsequently validated with 22 additional isolates from dairy and non-dairy sources. Overall, 58 polymorphic sites were indexed among 3,009 nucleotides analyzed. Depending on the loci, four to eight alleles were detected, generating 17 different sequence types, of which ten were represented by a single strain. MLST analysis suggested a predominantly clonal population for the 40 G. candidum isolates. Phylogenetic analysis of the concatenated sequences revealed a distantly related group of four isolates. Interestingly, this group diverged with respect to internal transcribed spacers 1 (ITS1), 5.8S, and ITS2 analysis. The reproducibility of the MLST approach was compared to random amplification of microsatellites by PCR (RAM-PCR), a gel profiling method previously proposed for G. candidum strain typing. Our results found MLST differentiation to be more efficient than RAM-PCR, and MLST also offered a non-ambiguous, unique language, permitting data exchange and evolutionary inference.

The use of FAME analyses to discriminate between different strains of Geotrichum klebahnii with different viabilities.

A considerable decline in viability of spray dried cells of Geotrichum klebahnii was observed and was attributed to an undefined alteration of the used strain. As common techniques were not able to distinguish the altered from the still viable strains, we used the fatty acid methyl ester (FAME) analysis. On the basis of FAME data we were able to discriminate the three strains under investigation. Especially the ratios of cis/trans fatty acid ratios and of saturated/unsaturated fatty acid were significantly reduced in the less viable strain, pointing to an increased stress level in this strain. These findings clearly show the applicability of the FAME analysis to detect strain alterations and that this method is therefore a suitable, fast and feasible tool for quality assurance.

Species reassignment of Geotrichum bryndzae, Geotrichum phurueaensis, Geotrichum silvicola and Geotrichum vulgare based on phylogenetic analyses and mating compatibility.

The present classification of Galactomyces and its anamorph, Geotrichum, is based on various studies that used morphology, ecology, biochemistry, DNA-DNA reassociation comparisons and gene sequencing. In this study, the identities of strains of the Centraalbureau voor Schimmelcultures yeast culture collection, as well as seven strains from South Africa, were examined by analyses of the nucleotide divergence in the internal transcribed spacer regions of the nuclear rRNA gene (nrRNA) operon, the D1/D2 domains of the 26S rRNA gene and partial actin gene sequences as well as compatibility studies. The South African strains were assigned to species in the genus Galactomyces. The phylogenetic analyses and mating studies revealed that Geotrichum silvicola and Geotrichum bryndzae are synonyms of Galactomyces candidus and that Geotrichum vulgare is a synonym of Galactomyces pseudocandidus.

Control of T-2 toxin in Fusarium langsethiae and Geotrichum candidum co-culture.

Due to contamination of barley grains by Fusarium langsethiae, T-2 toxin can be present in the brewing process. It has been observed that the presence of the yeast Geotrichum candidum during malting can reduce the final concentration of this mycotoxin in beer. In this work, a co-culture method was carried out for both microorganisms in order to evaluate the effect on T-2 mycotoxin concentration in comparison with the pure culture of F. langsethiae in the same conditions. The microbial growth of both microorganisms was assessed using three different methods: dry weight, DOPE-FISH, and DNA quantification. In coculture, both microorganisms globally developed less than in pure cultures but G. candidum showed a better growth than F. langsethiae. The concentration of T-2 was reduced by 93 % compared to the pure culture. Hence, the interaction between G. candidum and F. langsethiae led to a drastic mycotoxin reduction despite the only partial inhibition of fungal growth.

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum.

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.

Relationship between growth behaviour, micro and macroscopic morphologies and freezing sensitivity of the ripening starter Geotrichum candidum is strain specific and mostly related to the morphotypes: the arthrospores/hyphae parameter.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml-1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes "yeast", "mould" or "intermediate". The two "mould" strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the "yeast" strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the "mould" morphotype respond similarly to freezing stress.

Geotrichum siamensis sp. nov. and Geotrichum phurueaensis sp. nov., two asexual arthroconidial yeast species isolated in Thailand.

Two asexual arthroconidial yeast strains, TM3-44(T) and LYSM5(T), were isolated, respectively, from estuarine water in a mangrove forest and soil in a terrestrial forest in Thailand. Analysis of the D1/D2 domains of the large-subunit rRNA gene sequences revealed that strain TM3-44(T) differed from the closest species in terms of pairwise sequence similarity, Dipodascus albidus, by 11.7% nucleotide substitutions, while strain LYSM5(T) was closest to Galactomyces geotrichum with only 2.9% nucleotide substitutions. The phylogenetic tree further demonstrated that strain TM3-44(T) was at a distant position from the closest species, D. albidus, and other related species in the Dipodascus clade, while strain LYSM5(T) clustered with G. geotrichum, it closest relative in the Galactomyces clade. The phenotypic characteristics of the two strains were typical of the genus Geotrichum. On the basis of the above findings, strain TM3-44(T) was assigned as a novel species of Geotrichum, for which the name Geotrichum siamensis sp. nov. is proposed. The type strain is TM3-44(T) (BCC 29903(T)=NBRC 104880(T)=CBS 10929(T)). Strain LYSM5(T) represented another novel species of Geotrichum, which was named Geotrichum phurueaensis sp. nov. The type strain is LYSM5(T) (BCC 34756(T)=NBRC 105674(T)=CBS 11418(T)).

Disseminated Geotrichum candidum infection in a patient with relapsed acute myelogenous leukemia following allogeneic stem cell transplantation and review of the literature.

We describe a woman with relapsed acute myelogenous leukemia after allogeneic stem cell transplantation who developed disseminated Geotrichum candidum infection during chemotherapy-induced neutropenia. The isolate was susceptible to voriconazole, amphotericin B, and micafungin in vitro. We review the literature regarding invasive infections with G. candidum, which predominantly affect immunocompromised hosts, and discuss potential therapies for this rare pathogen.

Geotrichum bryndzae sp. nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces.

Ten strains of an asexual arthroconidial yeast species were isolated from Bryndza, a traditional Slovak artisanal sheep cheese, which was manufactured from raw milk during a 4-month summer production period at two Slovakian sites (the northern RuZomberok and the central-southern Tisovec areas). Sequence comparison of the D1/D2 domains of the large-subunit rRNA gene revealed that this yeast represents a novel species of the genus Geotrichum, which contains anamorphs of the ascogenous genus Galactomyces, for which the name Geotrichum bryndzae sp. nov. is proposed (type culture CCY 16-2-1T=NRRL Y-48450T=CBS 11176T). The novel species is most closely related to Geotrichum silvicola NRRL Y-27641T, although yeasts with identical or very similar sequences have been found throughout the world.

Safety assessment of dairy microorganisms: Geotrichum candidum.

Geotrichum candidum is a ubiquitous filamentous yeast-like fungus commonly isolated from soil, air, water, milk, silage, plant tissues, digestive tract in humans and other mammals. This species is widely used as adjunct culture in the maturation of cheese. The genus Geotrichum is composed of 18 species. A recent taxonomic revision concluded that the old Galactomyces geotrichum/G. candidum complex contained four separate species of which Galactomyces candidus sp. nov./G. candidum. M13 primer can be used for identifying species of the Geotrichum genus. Used in combination, RAPD-PCR and RAM-PCR permit strains to be differentiated. The species can be unambiguous differentiated from the two species most frequently described in human pathology: Geotrichum clavatum (reclassified Saprochaete clavata) and Geotrichum capitatum (reclassified Magnusiomyces capitatus/Saprochaete capitata). Sources of exposure are food ingestion--cheese consumption playing a major role--inhalation and contact. A bibliographic survey was conducted to assess corresponding hazards and risks. G. candidum infections (mainly pulmonary or bronchopulmonary, but also cutaneous, oral, disseminates) are very rare: fewer than 100 cases reported between 1842 and 2006. Moreover, cases were not all confirmed by repeated isolations and demonstration of the fungus' presence in tissues, a prerequisite to establish a true diagnosis of geotrichosis. Immunocompromised population was recently shown as a target for opportunistic infection. The most effective treatments include either azole drogs as ketonazole, iconazole and clotrimazole, or polyene antibiotics as amphotericin B, nystatin and pimaricin, or voriconazole-amphotericin B association. Less than 1 case/year of disease was possibly caused by G. candidum and it never included dairy products or foodborne infection. The risk of developing an infection due to G. candidum in connection with its technological use and consumption of dairy products is virtually nil. For these reasons, G. candidum should be proposed for QPS status.

Differentiation of closely related fungi by electronic nose analysis.

In this work the potential of electronic nose analysis for differentiation of closely related fungi has been described. A total of 20 isolates of the cheese-associated species Geotrichum candidum, Penicillium camemberti, P. nordicum, and P. roqueforti and its closely related species P. paneum, P. carneum as well as the noncheese-associated P. expansum have been investigated by electronic nose, GC-MS, and LC-MS analysis. The isolates were inoculated on yeast extract sucrose agar in 20-mL headspace flasks and electronic nose analysis was performed daily for a 7-d period. To assess which volatile metabolites the electronic nose potentially responded to, volatile metabolites were collected by diffusive sampling overnight onto tubes containing Tenax TA, between the 7th and 8th day of incubation. Volatiles were analyzed by gas chromatography coupled to mass spectrometry and the results indicated that mainly alcohols (ethanol, 2-methyl-1-propanol, and 3-methyl-1-butanol) and ketones (acetone, 2-butanone, and 2-pentanone) were produced at this stage. The volatile metabolite profile proved to be species specific. Nonvolatile metabolites were collected on the 8th day of incubation and mycotoxin analysis was performed by high pressure liquid chromatography coupled to a diode array detector and a time of flight mass spectrometer. Several mycotoxins were detected in samples from the species P. nordicum, P. roqueforti, P. paneum, P. carneum, and P. expansum. Differentiation of closely related mycotoxin producing fungi incubated on yeast extract sucrose agar has been achieved, indicating that there is a potential for predicting production of mycotoxins on food and feedstuffs by electronic nose analysis.

[The phylogenetic analysis of 15 Geotrichum strains based on 26S rRNA gene D1/D2 region sequencing].

The 26S rRNA gene D1/D2 domain sequences of 15 strains originally identified as Galactomyces geotrichum from the Chinese Industry Culture Collection (CICC) were determined. The results indicated that these strains differed from the type strain of Galactomyces geotrichum and other species of the genus remarkably. Two groups were recognized from the 15 strains which possibly represent 2 novel species of Galactomyces. Further molecular study is needed to confirm their taxonomic status.

Levels of cystathionine gamma lyase production by Geotrichum candidum in synthetic media and correlation with the presence of sulphur flavours in cheese.

Geotrichum candidum is a cheese-ripening agent with the potential to produce sulphur flavour compounds in soft cheeses. We aimed to develop an alternative test for predicting the aromatic (sulphur flavours) potential of G. candidum strains in soft cheese. Twelve strains of G. candidum with different levels of demethiolase activity (determined by a chemical method) in YEL-met (yeast extract, lactate methionine) medium were studied. We investigated cgl (cystathionine gamma lyase) gene expression after culture in three media - YEL-met, casamino acid and curd media - and then carried out sensory analysis on a Camembert cheese matrix. We found no correlation between demethiolase activity in vitro and cgl gene expression. Sensory analysis (detection of sulphur flavours) identified different aromatic profiles linked to cgl expression, but not to demethiolase activity. The RT-PCR technique described here is potentially useful for predicting the tendency of a given strain of G. candidum to develop sulphur flavours in cheese matrix. This is the first demonstration that an in vitro molecular approach could be used as a predictive test for evaluating the potential of G. candidum strains to generate sulphur compounds in situ (Camembert cheese matrix).

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese.

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol.

In this study, the M13 primer was used to distinguish Geotrichum candidum from the anamorphic and teleomorphic forms of other arthrospore-forming species (discriminatory power = 0.99). For intraspecific characterization, the GATA4 primer showed the highest level of discrimination for G. candidum among the 20 microsatellite primers tested. A molecular typing protocol (DNA concentration, hybridization temperature and type of PCR machine) was optimized through a series of intra- and interlaboratory trials. This protocol was validated using 75 strains of G. candidum, one strain of G. capitatum and one strain of G. fragrans, and exhibited a discrimination score of 0.87. This method could therefore be used in the agro-food industries to identify and to evaluate biodiversity and trace strains of G. candidum. The results show that the GATA4 primer might be used to differentiate strains according to their ecological niche.