Multiplex PCR-based RFLP assay for early identification of prevalent Mycobacterium leprae genotypes.

Mycobacterium leprae is classified into four SNP genotypes and 16 subtypes (from 1A to 4P) that exhibit phylogeographical association reported from around the world. Among them, genotypes 1D and 3I represent more than 60% of M. leprae strains. Here, we report a new method for M. leprae genotyping which identifies the genotypes 1D and 3I by combining multiplex PCR amplification and restriction fragment length polymorphism (RFLP) of a M. leprae DNA amplicons using AgeI restriction enzyme. Agarose gel electrophoresis showed a deletion of 11 bp only among 3I genotypes by electrophoresis. When this multiplex PCR reaction is subjected to AgeI digestion, successful restriction digestion shows three bands for all the genotypes except 1D where only two bands were observed due to loss of restriction site. This method gives us the advantage of 1-step identification of the two most prevalent strains of M. leprae without using specialized equipments such as the Sanger sequencing system or quantitative PCR.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

Identification of atypical dermal leishmaniasis resolved by restriction fragment length polymorphism.

This case report series alerts to the atypical manifestations of dermal leishmaniasis in an area endemic for post kala-azar dermal leishmaniasis, the sequel to visceral leishmaniasis. We have reported two cases with multiple skin lesions, wherein the rK39 strip test, polymerase chain reaction and parasite load confirmed the presence of Leishmania parasites. The causative parasite was identified as Leishmania major by restriction fragment length polymorphism of the ribosomal DNA Internal Transcribed Spacer-1, overruling the clinical suspicion of post kala-azar dermal leishmaniasis. The third case presented with fever and extensive hypopigmented patches in the upper extremities; parasites were identified in blood and skin by polymerase chain reaction and typed by restriction fragment length polymorphism as Leishmania donovani, establishing this as a case of visceral leishmaniasis concomitant with dermal leishmaniasis, secondary to dissemination of viscerotropic L. donovani. The present case series emphasizes the importance of molecular tools to identify the Leishmania species in order to ensure appropriate treatment.

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while ß-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

Characterisation of the yeast and mould biota in traditional white pickled cheeses by culture-dependent and independent molecular techniques.

Artisanal white pickled cheese of Western Serbia is a product of complex microbial community which detection by culture-dependent method only is hampered by its limitations. Thus, in the present study, we used a culture-independent, semi-quantitative technique based on construction of an internal transcribed spacer (ITS)-clone library from metagenomic DNA. This approach, based on direct DNA extraction followed by amplification of fungal internal transcribed regions (ITS) cloned into plasmid and restricted by endonucleases, revealed greater species richness in analysed cheeses and their by-products (17 species in total) compared to the more commonly used techniques of the culture-dependent method (8 species) and LSU-DGGE (10 species). The most frequently occurring yeast species which are commonly associated with cheeses production were Debaryomyces hansenii, Kluyveromyces lactis and Candida zeylanoides. On the other hand, Yarrowia lipolytica and Galactomyces geotrichum were detected only in one cheese sample. Moreover, some species, mainly moulds (Filobasidium globisporum, Cladosporium sp., Aspergillus sp. or Alternaria sp.) were identified only by culture-independent methods. The discrepancies between the techniques were confirmed by low correlation factor and by different indices of general biodiversity and dominance of species. The ITS-clone library approach provides the opportunity to analyse complex fungal communities associated with food products.

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy.

BACKGROUND: Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. OBJECTIVE: To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. METHODS: The study group includes 404 participants of which 222 were leprosy patients (paucibacillary=87, multibacillary=135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. RESULTS: The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. CONCLUSION: The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response.

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.

Saccharomyces eubayanus and Saccharomyces uvarum associated with the fermentation of Araucaria araucana seeds in Patagonia.

Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species in A. araucana seeds and bark samples, led us to postulate a potential role for them as the original yeasts responsible for the elaboration of Mudai before the introduction of commercial S. cerevisiae cultures. The molecular and genetic characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus and S. pastorianus), allowed their ecology and evolution us to be examined.

Diversity and activities of yeasts from different parts of a Stilton cheese.

Blue cheeses are very complex food matrices presenting significant spatial differentiation between sections and the Stilton variety also has a hard brown crust making its matrix even more complex. The mycobiota communities in the three sections (blue veins, white core and outer crust) of a Stilton blue cheese were studied by employing culture-independent (TRFLP, DGGE) and culture-dependent analyses. Yeasts isolated from the cheese were studied for aroma production in a dairy model system with and without the starter Lactococcus lactis and filamentous fungus Penicillium roqueforti using SPME GC-MS. Significant qualitative and quantitative differences were observed in the yeast communities between the cheese sections with all the techniques. Yarrowia lipolytica presented strong synergistic activity with P. roqueforti enhancing the production of ketone aroma compounds, characteristic of blue cheeses. Culture techniques allowed the observation of the presence and uneven distribution of two different morphological groups of Debaryomyces hansenii in the different sections and of Trichosporon ovoides but failed to isolate Candida catenulata which dominated some parts of the cheese in the culture-independent analysis. This suggests that this species may be an important early coloniser but fails to survive into the final cheese. The study indicated that the yeast flora in the cheese sections differ including isolates that could affect their aroma profiles.

Genetic association of G896A polymorphism of TLR4 gene in leprosy through family-based and case-control study designs.

BACKGROUND: Polymorphisms in TLR4 may change the function of the protein and alter the efficiency of immune response of host to infection. The high relevance of host gene polymorphisms with outcome of Mycobacterium leprae infection led us to study the genetic association of TLR4 G896A polymorphism in order to identify its risk among contacts of affected leprosy patients. METHODS: For case-control study design a total of 628 individuals were recruited; 17 multicase leprosy families which included 32 case-parent trios were considered for family-based study. Genotyping was done using PCR-RFLP method. RESULTS: In case-control study AA genotype was positively associated while GA genotype was negatively associated with leprosy. In family based transmission disequilibrium test (TDT) analysis allele G was found to be over transmitted to the affected individuals. CONCLUSION: Case-control study suggests that homozygous AA genotype may confer susceptibility and heterozygous GA genotype may confer resistance to leprosy, while allele A was observed to increase risk and that of allele G may confer resistance to leprosy. No strong transmission disequilibrium was detected in family-based TDT analysis, possibly due to lower number of trios. In contrast to case-control data allele G was over transmitted to the affected ones in TDT analysis. To conclude, the frequencies of genotypes in household contacts were almost the same as in leprosy patients, suggesting that contacts with AA genotype may be at higher risk of leprosy and may therefore require prophylactic inputs.

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium.

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529(T) and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Trcek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529(T) and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA-DNA hybridizations confirmed their novel species identity by 73% DNA-DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529(T) and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)(5)-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529(T) and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529(T) and SKU 1109 is C(18:1ω7c) (60.2-64.8%). The DNA G+C content of LMG 1529(T) and SKU 1109 is 62.5 and 63.3mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529(T) (=NBRC 14815(T)=NCIMB 8752(T)).

Lack of effects of the TNF-alpha and IL-10 gene polymorphisms in Mexican patients with lepromatous leprosy.

Several human genetic variants have been associated with susceptibility or resistance to leprosy. The aim of this study was to assess whether gene polymorphisms of -308 G/A TNF-alpha and -819 T/C IL-10 are associated with lepromatous leprosy in Mexican mestizos patients from northwest Mexico. We genotyped these polymorphisms by means of polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) in 68 patients with lepromatous leprosy and 144 healthy Mexican Mestizos controls. We found that the -308G TNF-alpha allele was predominant in both cases (94.3%) and controls (92.3%) without statistical significance and the frequencies of -819C IL-10 allele were also similar for the cases (56.0%) and controls (59.0%). These negative findings suggest that other genes or polymorphisms may be important in the susceptibility to leprosy infection in the Mexican mestizos.

Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries.

A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii.

NINJURIN1 single nucleotide polymorphism and nerve damage in leprosy.

UNLABELLED: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, can damage the peripheral nervous system and represents one of the leading causes of nontraumatic neuropathy in some developing countries. The NINJURIN1 is a cell adhesion molecule that provides suitable substrates for repair of Schwann cells after peripheral nerve injury. The single nucleotide polymorphism NINJ1, is the result of a transversion of an adenine to a nucleotide polymorphic cytokine (A→C), responsible for an amino acid exchange of asparagine to alanine at position 110 of the protein (asp110ala). OBJECTIVES: The aim of this study was to investigate the importance of the polymorphism in the NINJ1 gene for neural impairment during leprosy course. METHODS: A single nucleotide polymorphism (asp110ala) was searched in 218 leprosy patients and 244 non-leprosy subjects using a polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: No statistical differences were observed in the frequency of the asp110ala SNP between leprosy patients versus non-leprosy and multibacillary versus paucibacillary clinical forms. The C allele (ala110) is increased among patients exhibiting nerve impairment (p=0.0379). Also, leprosy patients with the CC genotype (ala/ala) had a higher risk (OR=4.21) of developing nerve disability when compared those carrying the AA genotype (asp/asp) (OR=0.69). CONCLUSION: Our results show an association between the studied C allele (ala110) and damage nerve in leprosy patients. SIGNIFICANCE: Ninjurin analysis showed that asp110ala could be a valuable prognostic marker, since C allele (ala110) have increased susceptibility to nerve damage.

Monitoring a mixed starter of Hanseniaspora vineae-Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production.

The effect of simultaneous or sequential inoculation of Hanseniaspora vineae CECT 1471 and Saccharomyces cerevisiae T73 in non-sterile must on 2-phenylethyl acetate production has been examined. In both treatments tested, no significant differences in Saccharomyces yeast growth were found, whereas non-Saccharomyces yeast growth was significantly different during all days of fermentation. Independently of the type of inoculation, S. cerevisiae was the predominant species from day 3 till the end of the fermentation. The dynamics of indigenous and inoculated yeast populations showed H. vineae to be the predominant non-Saccharomyces species at the beginning of fermentation in sequentially inoculated wines, whereas the simultaneous inoculation of S. cerevisiae did not permit any non-Saccharomyces species to become predominant. Differences found in non-Saccharomyces yeast growth in both fermentations influenced the analytical profiles of final wines and specifically 2-phenylethyl acetate concentration which was two-fold increased in sequentially inoculated wines in comparison to those co-inoculated. In conclusion we have shown that H. vineae inoculated as part of a sequential mixed starter is able to compete with native yeasts present in non-sterile must and modify the wine aroma profile.

Interleukin 4-590T/C polymorphism and susceptibility to leprosy.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Cell-mediated (Th1) immune response and humoral (Th2) immune response play different roles in leprosy infection. Interleukin 4 (IL-4) is a typical Th2 cytokine. It is a critical mediator of the Th1/Th2 balance. OBJECTIVE: The objective of this study is to investigate the association between IL-4 gene -590T/C polymorphism and the susceptibility to leprosy in a Chinese population. METHODS: The IL-4 variant -590T/C was detected by polymerase chain reaction-restriction fragment length polymorphism in 432 leprosy cases and 465 age-matched healthy controls. Data were analyzed using the chi-square test. RESULTS: Frequencies of the IL-4-590TC and CC genotypes and the -590C allele were significantly lower in patients with leprosy than in healthy controls (odds ratio [OR]=0.74, 95% confidence interval [CI] 0.55-0.99, p=0.044; OR=0.46, 95% CI 0.25-0.84, p=0.010; and OR=0.68, 95% CI 0.54-0.86, p=0.001, respectively). CONCLUSIONS: Our data suggest that the -590T/C polymorphism of the IL-4 gene is associated with decreased susceptibility of leprosy.

Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques.

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.