Occurrence and dominance of yeast species in sourdough

AIMS: The aim of this work is to identify the dominant yeast species in homemade sourdoughs. METHODS AND RESULTS: PCR/restriction fragment length polymorphism analysis of internal transcribed spacer regions was used for the identification of isolates and the data were confirmed with phenotypic tests. The strains belonging to Saccharomyces cerevisiae were identified to strain level by analysis of inter-delta regions. CONCLUSION: This work shows that the dominant species in homemade sourdoughs can differ from each other. Saccharomyces cerevisiae was found to be the dominant species, followed by the Candida milleri, C. humilis, S. exiguus and Issatchenkia orientalis. The inter-delta regions of S. cerevisiae strains showed high polymorphism. SIGNIFICANCE AND IMPACT OF THE STUDY: Occurrence of single, non-Saccharomyces species and S. cerevisiae polymorphism in the yeast populations of sourdough samples.     

Yeast species associated with orange juice: evaluation of different identification methods

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.     

Molecular identification of yeasts from soils of the alluvial forest national park along the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen")

We analysed the diversity of yeasts from different soils in a river-floodplain landscape at the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen"). 136 strains were isolated, identification of species was done with molecular methods. Partial sequencing of the 26S rRNA gene resulted in 36 different sequences, they could be assigned to 16 genera, apart from two sequence types (from three isolates), which were not clearly assigned to any genus. 18 species were identified and confirmed by means of PCR fingerprinting. The most frequently isolated genus was Cryptococcus (61 isolates and 12 sequence types). Basidiomycetes dominated with about 60% above the members of the Ascomycetes. About half the yeasts was isolated from the litter, the quantity decreased with soil depth.     

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.     

Molecular divergence of the soil yeasts Williopsis sensu stricto

Fifty-three strains of saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA which comprised the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, Komagataea pratensis, and the Williopsis sensu stricto complex. Minisatellite primer M13 was proposed for the differentiation between twin species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.     

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques

AIMS: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. METHODS AND RESULTS: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. CONCLUSIONS: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.     

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.     

Molecular characterization of clinical Saccharomyces cerevisiae isolates and their association with non-clinical strains

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two baker's yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both baker's yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.     

Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci

Thirteen Staphylococcus aureus and S. epidermidis strains obtained from nose and hand of two employees and one patient of a medical ward as well as two S. hemolyticus strains were analysed according to their restriction fragment length patterns (RFLP) by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI and SstII. Species identification of the isolates was performed by a system which includes 20 biochemical reactions. Furthermore, the antibiotic resistance patterns of the strains were determined. While several isolates exhibited identical antibiotic susceptibilities and biochemical profiles, differences in the RFLP were obtained. In three cases, S. epidermidis strains colonizing the skin showed an identical restriction profile as isolates from the mucous membranes of the same person. We concluded that the analysis of staphylococcal strains by PFGE is an important epidemiological tool with high discrimination power.     

Molecular characterization of lactic acid bacteria from sourdough breads produced in Sardinia (Italy) and multivariate statistical analyses of results

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in sourdough used for the production of traditional breads (Carasau, Moddizzosu, Spianata, Zichi) in Sardinia. 16S rDNA sequencing and Randomly Amplified Polymorphic DNA (RAPD-PCR) was applied for the identification and typing of the LAB isolated from 25 samples of sourdoughs. Multivariate statistical techniques were applied to RAPD-PCR pattern to study the biological diversity of sourdough samples. Twelve different species of LAB were identified, and most isolates were classified as facultative heterofermentative lactobacilli. Lactobacillus pentosus dominated the lactic microflora of many samples while Lactobacillus sanfranciscensis was isolated only from a limited number of samples. Although heterofermentative species represented between between 30% and 60% of the isolates in Carasau, Spianata and Zichi sourdoughs, only 2% of the isolates from Moddizzosu sourdoughs were identified as heterofermentative LAB. RAPD-PCR with a single primer followed by cluster analysis did not allow the identification of the isolates at the species level. However, a multidimensional scaling/bootstrapping approach on the RAPD-PCR patterns uncovered the diversity of the LAB communities of LAB showing differences both within and between bread types.     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.     

Identification of Saccharomyces sensu stricto and Torulaspora yeasts by PCR ribotyping

18S rDNA + ITS1 and 25S rDNA PCR products covering more than 95% of the nuclear ribosomal DNA repeat unit of 28 Saccharomyces sensu stricto and Torulaspora yeasts and their anamorph forms were digested with Hae III, Msp I, Hinf I and Cfo I. Using combinations of two restriction enzymes, specific ribotyping patterns of six species were found. PCR ribotyping offers a convenient tool for quick identification of yeast isolates, but specificity of ribotyping patterns should be checked with a larger number of strains to avoid misidentification because of lack of variation within different taxa or because of strain-specific ribotyping patterns of species type strains.     

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Keratitis by Acanthamoeba triangularis: report of cases and characterization of isolates

Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.     

Application of a novel polyphasic approach to study the lactobacilli composition of sourdoughs from the Abruzzo region (central Italy)

AIMS: To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach. METHODS AND RESULTS: A polyphasic approach, consisting of a two-step multiplex polymerase chain reaction (PCR) system, 16S rRNA gene sequence analysis and physiological features, was applied to identify 127 isolates, representing about 37% of the presumptive lactobacilli collected from sourdough samples. Multiplex PCR successfully identified 111 isolates, while 16S rRNA gene sequencing was applied for the other 16 isolates, two of which could not be associated with any previously described lactic acid bacteria (LAB) species. Strain diversity was evaluated by phenotypic and random amplified polymorphic DNA-PCR analysis. Molecular detection of Lactobacillus group species was also performed on total DNA extracted from the doughs. CONCLUSIONS: Abruzzo region sourdough lactobacilli biodiversity, reflected in both Lactobacillus species composition and strain polymorphism, is similar to that of other Italian regions and is a source of novel LAB species. SIGNIFICANCE AND IMPACT OF THE STUDY: Within culture-independent methods, multiplex PCR is a rapid tool to study the lactobacilli population of sourdoughs.     

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.     

Identity, beer spoiling and biofilm forming potential of yeasts from beer bottling plant associated biofilms

Wild yeasts were isolated from process surfaces of two breweries. In total, 41 strains were obtained and differentiated by cultivation on CuSO₄ or crystal violet containing selective media, by fatty acid profiling and by a restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene. The restriction analysis showed the highest differentiating capacity and resulted in eleven groups. These groups were identified by the API ID 32 C kit or by sequencing the D1/D2 region of the 26S rRNA gene. Most of the wild yeasts were identified as Saccharomyces cerevisiae (46% of all isolates) and Candida pelliculosa (anamorph: Pichia anomala) (24%). No obvious differences were detected between the two breweries. While all of the S. cerevisiae isolates were able to grow in beer, only six out of 10 C. pelliculosa strains were able to tolerate this substrate. However, most of the C. pelliculosa strains showed biofilm formation in a microplate assay, but none of the S. cerevisiae isolates. Therefore, it is assumed that the former species is involved in attachment and primary biofilm formation on beer bottling plants, while S. cerevisiae is a late colonizer of a preformed biofilm but increased the beer spoiling potential of the biofilm.     

Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.