Rapid PCR Method for the Selection of 1,3-Pentadiene Non-Producing Debaryomyces hansenii Yeast Strains.

To prevent microbial growth and its consequences, preservatives such as sorbic acid or its salts, commonly known as sorbates, are added to foods. However, some moulds and yeasts are capable of decarboxylating sorbates and producing 1,3-pentadiene. This is a volatile compound with an unpleasant "petroleum-like "odour, which causes consumer rejection of the contaminated products. In this work, we studied the production of 1,3-pentadiene in 91 strains of the yeast Debaryomyces hansenii, and we found that nearly 96% were able to produce this compound. The sequence of the FDC1Dh gene was analysed showing differences between 1,3-pentadiene producer (P) and non-producer (NP) strains. A specific PCR assay with degenerated primers based on the gene sequence was developed to discern NP and P strains. It was tested on D. hansenii strains and on some physiologically related species frequently isolated from foods, such as D. fabrii, D. subglobosus and Meyerozyma guillermondii. This method could be applied for the selection of NP D. hansenii strains, useful in biotechnological food production and as a biocontrol agent.

Zygosaccharomyces rouxii strains CECT 11923 and Z. rouxii CECT 10425: Two new putative hybrids?

Based on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxii strains (CECT 11923 and CECT 10425) that clustered with hybrid strains (NCYC 1682, NCYC 3060 and NCYC 3061). Given the recently recognized important industrial role of hybrids, their detection is very useful. Based on the IGS1 rDNA region alignment of hybrid strains and the Z. rouxii CECT 11923 and CECT 10425, in this work, we developed a pair of Zygosaccharomyces hybrid-specific primers, HibZF/HibZR. Positive amplicons were only obtained in the Zygosaccharomyces spp. hybrids included in this study and the CECT 11923 and CECT 10425 strains analyzed here. In the present study, we applied molecular tools to highlight the nature of these strains; they are quite different from each other as well as from Z. rouxii type strain. Based on the presence of two heterologous copies of nuclear-encoded genes (SOD2 and HIS3), the sequences of divergent 5.8S-ITS rDNA, D1/D2 26S rDNA copies and, the amplification with species-specific primer for Z. rouxii and Z. pseudorouxii, we hypothesize that the CECT 11923 strain might be a hybrid strain. Whereas, CECT 10425, the sequence analysis of 5.8S-ITS rDNA and D1/D2 26S rDNA copies presented 99-100% sequence identity with Zygosaccharomyces sp. NBRC 10669 (LN849119.1) and Z. sapae ABT 301T. Nevertheless, we discard that it could be a Z. sapae strain based on the results obtained in this study. Namely, the amplification with hybrid-specific primer designed in this study, the number of divergent copies of HIS3 (2), the fact that it only possesses one SOD2 gene and the amplification with species-specific primer for Z. pseudorouxii, therefore it could be a new species or a hybrid strain.

Development of an affordable typing method for Meyerozyma guilliermondii using microsatellite markers.

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only four were used in this work. All microsatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction, which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair. Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool.

Development of species-specific primers for rapid identification of Debaryomyces hansenii.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

Assessment of the Factors Contributing to the Growth or Spoilage of Meyerozyma guilliermondii in Organic Yogurt: Comparison of Methods for Strain Differentiation.

In this work we analyze the spoiling potential of Meyerozyma guilliermondii in yogurt. The analysis was based on contaminated samples sent to us by an industrial laboratory over two years. All the plain and fruit yogurt packages were heavily contaminated by yeasts, but only the last ones, containing fermentable sugars besides lactose, were spoiled by gas swelling. These strains were unable to grow and ferment lactose (as the type strain); they did grow on lactate plus galactose, fermented glucose and sucrose, and galactose (weakly), but did not compete with lactic acid bacteria for lactose. This enables them to grow in any yogurt, although only those with added jam were spoiled due to the fermentation of the fruit sugars. Fermentation, but not growth, was strongly inhibited at 8 °C. In consequence, in plain yogurt as well as in any yogurt maintained at low temperature, yeast contamination would not be detected by the consumer. The risk could be enhanced because the species has been proposed for biological control of fungal infections in organic agriculture. The combination of the IGS PCR-RFLP (amplification of the intergenic spacer region of rDNA followed by restriction fragment length polymorphism analysis) method and mitochondrial DNA-RFLP makes a good tool to trace and control the contamination by M. guilliermondii.

Quantitative analysis of morphological changes in yeast colonies growing on solid medium: the eccentricity and Fourier indices.

The colony shape of four yeast species growing on agar medium was measured for 116 days by image analysis. Initially, all the colonies are circular, with regular edges. The loss of circularity can be quantitatively estimated by the eccentricity index, Ei , calculated as the ratio between their orthogonal vertical and horizontal diameters. Ei can increase from 1 (complete circularity) to a maximum of 1.17-1.30, depending on the species. One colony inhibits its neighbour only when it has reached a threshold area. Then, Ei of the inhibited colony increases proportionally to the area of the inhibitory colony. The initial distance between colonies affects those threshold values but not the proportionality, Ei /area; this inhibition affects the shape but not the total surface of the colony. The appearance of irregularities in the edges is associated, in all the species, not with age but with nutrient exhaustion. The edge irregularity can be quantified by the Fourier index, Fi , calculated by the minimum number of Fourier coefficients that are needed to describe the colony contour with 99% fitness. An ad hoc function has been developed in Matlab v. 7.0 to automate the computation of the Fourier coefficients. In young colonies, Fi has a value between 2 (circumference) and 3 (ellipse). These values are maintained in mature colonies of Debaryomyces, but can reach values up to 14 in Saccharomyces. All the species studied showed the inhibition of growth in facing colony edges, but only three species showed edge irregularities associated with substrate exhaustion.

Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS).

Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z. bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster.

Molecular characterization of a novel splice site mutation within the CYBB gene leading to X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency that affects the oxidative mechanism of microbial killing of phagocytic cells. The defect is characterized by a lack or severely reduced superoxide anion (O2-) production by phagocytes. Seventy percent of CGD cases are X-linked (X-CGD) and they are caused by mutations in the gene encoding for gp91(phox), one of the two subunits of the flavocytochrome b558 of the NADPH oxidase. We identified an abnormal transcript arising from a novel splice site mutation within the gene encoding gp91(phox), which suggested that the mutation affected normal mRNA splicing. Thus, the effect of this mutation leads to the complete absence of the flavocytochrome b558 in neutrophil membranes, which caused the biochemical phenotype X91 degrees-CGD in this family. These molecular findings help to explain the early onset and severe phenotype in this X-CGD kindred.

X-Linked Chronic Granulomatous Disease: First Report of Mutations in Patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91, the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91°-CGD phenotype. The lack of gp91 protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

X-linked chronic granulomatous disease: first report of mutations in patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91(phox), the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91(o)-CGD phenotype. The lack of gp91(phox) protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

Recurrent eosinophilic cystitis in a child with chronic granulomatous disease.

Eosinophilic cystitis is an uncommon disease in children, and its association with chronic granulomatous disease (CGD) has been previously reported in only five patients. In all those patients the disease showed either a self-limited benign course or a rapid response to corticosteroid treatment. The authors describe a child with X-linked CGD who developed eosinophilic cystitis with a recurrent course and difficult therapeutic management. The authors also discuss the pathogenesis of granuloma formation in CGD and review the literature for current therapies for these complications.

Bruton tyrosine kinase gene mutations in Argentina.

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.

Wiskott-Aldrich syndrome in Argentina: 17 unique, including nine novel, mutations.

Wiskott-Aldrich syndrome (WAS), is an X-linked immunodeficiency disease caused by mutations of the WAS protein (WASP) gene, characterized by thrombocytopenia, eczema and recurrent infections. X-linked thrombocytopenia (XLT) is a milder form with only platelet abnormalities. Cumulative mutation data have revealed that WASP genotypes are highly variable among WAS patients. By SSCP analysis, we determined the location of the mutation in 23 WAS patients from 17 unrelated families with variable clinical phenotypes. Direct sequence analysis of genomic DNA showed 9 novel mutations (Q52H, G70W, 393del7, Ex 7 Ex11del, IVS 8+1G-->C, 925delG, 959ins38, 1380del8, and IVS 2+2T-->C) and 8 known mutations distributed throughout the WAS gene. This is the first report of WAS gene mutations from a Latin American country.