Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.     

Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil

Aims:  To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil.
Methods and Results:  Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl , tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S–23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S–23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh ^– Kanagawa-negative isolates.
Conclusions:  V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless.
Significance and Impact of the Study:  Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.     

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level

AIMS: In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. METHODS AND RESULTS: Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. CONCLUSIONS: Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.     

Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries

Yeasts of the genus Dekkera and its anamorph Brettanomyces represent a significant spoilage issue for the global wine industry. Despite this, there is limited knowledge of genetic diversity and strain distribution within wine and winery-related environments. In this study, amplified fragment length polymorphism (AFLP) analysis was conducted on 244 Dekkera bruxellensis isolates from red wine made in 31 winemaking regions of Australia. The results indicated there were eight genotypes among the isolates, and three of these were commonly found across multiple winemaking regions. Analysis of 26S rRNA gene sequences provided further evidence of three common, conserved groups, whereas a phylogeny based upon the AFLP data demonstrated that the most common D. bruxellensis genotype (I) in Australian red wine was highly divergent from the D. bruxellensis type strain (CBS 74).     

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis

Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small‐scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l^?1 (0·22 mol l^?1)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0·42 ± 0·01 g ethanol (g glucose)^?1 were observed for both yeasts in 1 : 10 hydrolysate. In small‐scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.     

Assessment of the microbial quality of irrigation water in a prairie watershed

Aims:  To assess levels of faecal contamination in the Qu'Appelle River (Saskatchewan, Canada) and its suitability for irrigation, by using the Colilert-18/Quanti-Tray technology.
Methods and Results:  Various sites located along the Qu'Appelle River were sampled weekly from May to August 2005–2007. A total of 594 freshwater samples were collected and analysed for enumeration of Escherichia coli using the Colilert-18. The false-positive rate for E. coli detection using Colilert-18 was at most 1·5%. Throughout the irrigation period (June to August), up to 85% of the water samples collected from one of the irrigation water-pumping sites exceeded the recommended limit of 100 CFU per 100 ml. Spikes in E. coli counts were generally concomitant with the sudden rise in river flows. A sub-sample of confirmed E. coli isolates were typed by randomly amplified polymorphic DNA (RAPD). RAPD analysis revealed a high degree of genetic diversity among E. coli isolates. A significant association between RAPD patterns and the month of E. coli isolation was demonstrated.
Conclusions:  Colilert-18 provides an effective means for assessing microbial quality of irrigation water.
Significance and Impact of the Study:  Qu'Appelle River is subject to variability of faecal contamination during irrigation times and monitoring throughout irrigation season is important for ensuring safe production practices.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine

The wine spoilage yeast Dekkera bruxellensis was evaluated for the production of 4-ethylphenol under low concentrations (0.02-20 g L(-1)) of glucose and fructose in synthetic media. Measurable amounts of 4-ethylphenol were produced over 0.2 g L(-1) of each sugar. The yeast growth rate and amount of biomass formed increased from 0.2 to 20 g L(-1) of glucose or fructose, being accompanied by increasing production of 4-ethylphenol. In red wines, the production of 4-ethylphenol was only observed in the presence of growing populations of indigenous or inoculated strains of D. bruxellensis. The production rate of 4-ethylphenol varied between 22 and 93 mug day(-1) either with inoculated strains or wild populations in bottled wines. The production rate of 4-ethylphenol as a function of the increase in the number of cells varied from 349 to 1882 mug L(-1) per one log CFU mL(-1). The effect of temperature on cellular viability and 4-ethylphenol production was tested in red wines with indigenous or inoculated strains of D. bruxellensis. Incubation temperatures of 15, 20 and 25 degrees C allowed cellular growth and volatile phenol production. Increasing incubation temperatures to 36 degrees C induced full viability loss of 10 strains of D. bruxellensis within <12 h.     

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.     

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.
Methods and Results:  Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi , the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia.
Conclusions:  The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical.
Significance and Impact of the Study:  This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.     

Genome survey sequencing of the wine spoilage yeast Dekkera (Brettanomyces) bruxellensis

The hemiascomycete yeast Dekkera bruxellensis, also known as Brettanomyces bruxellensis, is a major cause of wine spoilage worldwide. Wines infected with D. bruxellensis develop distinctive, unpleasant aromas due to volatile phenols produced by this species, which is highly ethanol tolerant and facultatively anaerobic. Despite its importance, however, D. bruxellensis has been poorly genetically characterized until now. We performed genome survey sequencing of a wine strain of D. bruxellensis to obtain 0.4x coverage of the genome. We identified approximately 3,000 genes, whose products averaged 49% amino acid identity to their Saccharomyces cerevisiae orthologs, with similar intron contents. Maximum likelihood phylogenetic analyses suggest that the relationship between D. bruxellensis, S. cerevisiae, and Candida albicans is close to a trichotomy. The estimated rate of chromosomal rearrangement in D. bruxellensis is slower than that calculated for C. albicans, while its rate of amino acid evolution is somewhat higher. The proteome of D. bruxellensis is enriched for transporters and genes involved in nitrogen and lipid metabolism, among other functions, which may reflect adaptations to its low-nutrient, high-ethanol niche. We also identified an adenyl deaminase gene that has high similarity to a gene in bacteria of the Burkholderia cepacia species complex and appears to be the result of horizontal gene transfer. These data provide a resource for further analyses of the population genetics and evolution of D. bruxellensis and of the genetic bases of its physiological capabilities.     

The yeast Dekkera bruxellensis genome contains two orthologs of the ARO10 gene encoding for phenylpyruvate decarboxylase

The yeast Dekkera bruxellensis possesses important physiological traits that enable it to grow in industrial environments as either spoiling yeast of wine production or a fermenting strain used for lambic beer, or fermenting yeast in the bioethanol production process. In this work, in silico analysis of the Dekkera genome database allowed the identification of two paralogous genes encoding for phenylpyruvate decarboxylase (DbARO10) that represents a unique trait among the hemiascomycetes. The molecular analysis of the theoretical protein confirmed its protein identity. Upon cultivation of the cell in medium containing phenylpyruvate, both increases in gene expression and in phenylpyruvate decarboxylase activity were observed. Both genes were differentially expressed depending on the culture condition and the type of metabolism, which indicated the difference in the biological function of their corresponding proteins. The importance of the duplicated DbARO10 genes in the D. bruxellensis genome was discussed and represents the first effort to understand the production of flavor by this yeast.     

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic

Aims:  Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods.
Methods and Results:  Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia , whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia . Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582^T and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same Eco RI ribotype profile and proved to be identical strains.
Conclusions:  Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification.
Significance and Impact of the Study:  To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.     

Molecular detection and identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines

In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.     

Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates

Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.     

Genetic characterization of spoilage pseudomonads isolated from retail-displayed beef

Aim:  This study genetically characterized Pseudomonas isolated from beef using the random amplification of polymorphic DNA (RAPD) method and correlate predominant genotypes with spoilage changes.
Methods and Results:  Pseudomonads were recovered from beef loins and steaks on days 0, 2, 4, 6, 8 and 10. A total of 309 pseudomonads were grouped into 50 RAPD types (>85% similarity). One major RAPD type contained 45% of the isolates comprising 71%, 45%, 31%, 35%, 50% and 37% of isolates from days 0, 2, 4, 6, 8 and 10, respectively, from steaks and 48% of the isolates recovered from beef loins. Nineteen RAPD types consisted of isolates that were shared between more than two sampling times, whereas the remaining 31 types were unique to one particular time.
Conclusions:  A genetically diverse Pseudomonas population was present on the loins and steaks at each sampling time. Although pseudomonads associated with beef loins were transferred to the steaks prepared from it, a genetically diverse Pseudomonas population emerged during the retail display.
Significance and Impact of the Study:  Information about the heterogeneous nature of Pseudomonas recovered from meat would help understanding the spoilage owing to predominant strains. The meat industry can use the knowledge to develop control strategies for prevalent spoilage strains.     

Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.     

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.     

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer

Objectives:  This study aims to establish three-dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two-dimensional (2D) models of the same cell lines and the primary tumours.
Methods:  3D models of ovarian and endometrial cancer cell cultures were established using a Rotary Cell Culture System. Immunohistochemical profiling and differential proteomics were used to characterize biological characteristics of multicellular spheroids (MCS) formed from these cultures. These were compared to characteristics of the same cells established in 2D and of the primary tumours from which the cell lines were derived.
Results:  MCSs from 3D cell cultures appeared histologically similar to the primary tumours. Immunohistochemical profiling of multiple markers, including CA125, BCL2 and p53, showed that patterns of protein expression in MCSs resemble those of the primary tumours. Proteomic profiling identified several differentially expressed protein markers between 2D and 3D cultures. These included prohibitin, which was down-regulated in 3D cultures suggesting cells proliferate less compared to 2D cultures; and VDAC1 and annexin 4, which were up-regulated in 3D cultures suggesting greater levels of apoptosis in 3D compared to 2D models.
Conclusion:  Establishing 3D models of cancer cell lines is likely to be of value for studying the molecular and biological mechanisms of ovarian/endometrial tumour progression and for testing novel molecular targets for cancer therapy.