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.

Educating in antimicrobial resistance awareness: adaptation of the Small World Initiative program to service-learning.

The Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society.

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.

Four new Candida cretensis strains isolated from Spanish fermented sausages (chorizo): taxonomic and phylogenetic implications.

Four yeast strains were isolated from Spanish traditional fermented sausages (chorizo) spoiled by gas production. Using the classical identification procedures, they were identified as Debaryomyces hansenii. However, they fermented galactose and did not produce positive results in Debaryomyces differential medium (DDM), a growth medium highly specific for this species. Phylogenetic analysis showed identical sequences for the D1/D2 domain of the 26S rRNA gene and almost identical sequences for the 5.8S-ITS region with those of the recently described yeast species Candida cretensis. This result was confirmed by sequencing the gene encoding actin of the type and the new strains. Candida cretensis is a new species included in the so-called Candida kruisii clade that was described from a single strain, isolated from a decaying mushroom in Crete, Greece. The discovery of new strains of C. cretensis in fermented food expands its physiological and ecological diversity. With the description of these new strains isolated from food, three groups of strains can be distinguished within C. cretensis according to the restriction patterns of the intergenic spacer rRNA gene region and on the basis of some physiological properties that are of ecological relevance.

PCR-RFLP analysis of the IGS region of rDNA: a useful tool for the practical discrimination between species of the genus Debaryomyces.

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.

A beta-glucuronidase-based agar medium for the differential detection of the yeast Debaryomyces hansenii from foods.

A selective and differential solid medium, Debaryomyces differential medium (DDM), was used for the isolation of Debaryomyces hansenii. This medium is formulated to allow detection of the beta-glucuronidase enzyme using the chromogenic substrate magenta-glucuro.CHA (5Br-6Cl-3indolyl-beta-D-glucuronide, cyclohexylammonium salt). Of the more than 120 microorganisms tested, including yeasts, bacteria, and a filamentous fungus, only D. hansenii produced violet colonies, thus permitting its easy discrimination from other organisms. When quality assessment tests were performed, optimal productivity and selectivity were obtained for D. hansenii. The medium was also satisfactory when used to test naturally contaminated food products.

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.

Occurrence of yeasts in municipal wastes and their behaviour in presence of cadmium, copper and zinc.

Seven yeasts strains have been isolated from sewage sludge. Also six samples of compost with different sieving, composting times and origins, have been analysed. Apparently, composting processes negatively affect the viability of yeasts, as none could be isolated from the compost samples. The margins of tolerance of the yeasts to Cd, Cu and Zn have been determined. The physiological response to metals was similar in all the species studied, and in general, kinetic parameters (mu and lag) were affected. Metal uptake ability was also studied and inter- and intra-specific heterogeneity was detected, thus indicating that both the tolerance to metals and the capacity of the uptake were dependent on ionic metal and yeast species. The effect of the presence of multi-metal ions on the uptake capacity of each individual metal was assayed for two selected yeasts, Pichia guilliermondii and Torulaspora delbrueckii. The uptake of each individual metal varied with the combination assayed, and when both strains were compared different results were also found.

Environmental adaptation factors of two yeasts isolated from the leachate of a uranium mineral heap.

Two yeasts from the effluent of a uranium mineral heap were identified as Candida sorbophila and Rhodotorula mucilaginosa. They are well adapted to such an environment as shown by their tolerance to metals and acid pH. However, different mechanisms could be involved in the adaptation: C. sorbophila could be considered to be more acid-tolerant as a function of its specific growth rate and respiratory behavior at acidic pH in an organic medium. However, R. mucilaginosa was capable of growing by 1 log unit when inoculated in a sterilized leaching effluent or in a ferrous mineral medium, without added organic compounds. Indeed, the ability of R. mucilaginosa to grow in a low-nutrient environment could be as important a factor as its acid tolerance for coping in the isolation habitat. In mixed cultures with sulfide-oxidizing bacteria, no effect was observed on the ferrous oxidation normally carried out by these bacteria. However, a negative effect on both yeasts, especially in C. sorbophila, was observed, when the bacteria were present.

Feasibility of copper uptake by the yeast Pichia guilliermondii isolated from sewage sludge.

Copper is one of the most abundant toxic heavy metals in municipal wastewaters and, in consequence, in sewage sludge and compost. The ability of a strain of the yeast Pichia guilliermondii, which was isolated from sewage sludge, to eliminate copper has been evaluated, using both viable and nonviable biomass. It has been found that raising concentrations of copper affected both morphology and physiological parameters of the viable yeast, and it is thought that a process of bioaccumulation may be involved in its copper uptake. The growth rate of nonadapted cells decreased with increasing concentrations of copper, mainly due to a decrease in the biomass yield. The cells could be adapted by training with increasing copper concentrations up to 317.7 mg/l. This adaptation was an all-or-nothing process: once cells had adapted, the biomass yield, metabolic flux and consequent growth rate were constant and independent of the external copper concentration. Also, it was determined that up to 20 mg of copper per gram of viable adapted biomass could accumulate from the medium (i.e., double the amount when using nonadapted viable biomass). Finally, adsorption data on nonviable cells were found to be well modeled by the Langmuir isotherm (qmax = 9.09 mg/g of biomass).