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PubMed2001

Molecular characterization of new clinical isolates of Candida albicans and C. dubliniensis in Japan: analysis reveals a new genotype of C. albicans with group I intron.

The genetic diversity of recent clinical isolates of Candida albicans in Japan was studied on the basis of amplified DNA band lengths determined with a specific PCR primer reported to have been designed to span a transposable intron region in the 25S rRNA gene. Our analyses of 301 clinical isolates of C. albicans showed that they could be classified into five genotypes: genotype A (172 isolates), genotype B (66 isolates), genotype C (56 isolates), genotype D (C. dubliniensis; 5 isolates), and a new genotype (designated genotype E; 2 isolates). The new genotype E was characterized to have a group I intron-like sequence, which is longer than hitherto reported ones and which has a nucleotide sequence length of 962 bp. Our analysis of the 962-bp sequence indicated that it is composed of an intron similar to that of C. dubliniensis of 621 bp with a 341-bp insertion. Analysis of the sequence of the internal transcribed spacer (ITS) region of the genotype E strain showed that its sequence is identical to those of strains of other genotypes, with only a few base substitution differences. Throughout the study, the possible horizontal transfer of the group I intron between C. dubliniensis and C. albicans was suggested. A high degree of correlation between the presence of a group I intron in C. albicans genotype E and susceptibility to the antifungal agent flucytosine was observed. The five isolates of C. dubliniensis examined in the present study showed genetic diversity when they were compared by randomly amplified polymorphic DNA fingerprinting pattern analysis, and this diversity was also confirmed by the analysis of ITS region sequences.

Antifungal Agents↗
PubMed2004

Characterization of molds from dry-cured meat products and their metabolites by micellar electrokinetic capillary electrophoresis and random amplified polymorphic DNA PCR.

Molds are common contaminants of dry-cured meat products in which mycotoxins could be synthesized if stored under favorable conditions. Thus, efficient and accurate characterization of the toxigenic molds from dry-cured meat products is necessary. A micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by 20 mold strains commonly found in dry-cured meat products. In addition, their random amplified polymorphic DNA (RAPD) genotypes were determined by using a PCR method. Although peak profiles of the secondary metabolites differed among mold strains of different species, they were similar in the same species. MECC analysis showed that 10 of the 20 molds tested produced mycotoxins, including patulin, penicillic acid, cyclopiazonic acid, mycophenolic acid, aflatoxin B1, sterigmatocystin, and griseofulvin. The RAPD analysis yielded a different pattern for each of the mold species tested. However, strains of the same species showed similar RAPD profiles. A high correlation between RAPD analysis and MECC was observed, since strains of the same species that showed similar RAPD patterns had similar profiles of secondary metabolites. RAPD patterns with primer GO2 and MECC profiles, either singly or combined, could be of great interest to distinguish toxigenic from nontoxigenic molds in dry-cured meat products.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Aspergillosis↗
PubMed2006

Development and application of RAPD-SCAR marker for identification of Phyllanthus emblica LINN.

Correct genotype identification of medicinal plant material remains important for botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated need for newer methods in quality control of botanicals. The present study was carried out to develop DNA based marker for identification of Phyllanthus emblica LINN. A putative marker (1.1 kb) specific for P. emblica was identified by Random Amplified Polymorphic DNA (RAPD) technique. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. The SCAR marker was found useful for identification of P. emblica in its commercial samples and Triphalachurna, a multi-component Ayurvedic formulation.

Base Sequence↗
PubMed2005

Biotypes and randomly amplified polymorphic DNA (RAPD) profiles of subgingival Candida albicans isolates in HIV infection.

A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.

AIDS-Related Opportunistic Infections↗
PubMed1997

A comparison of methods of phenotypic and genotypic fingerprinting of Exophiala dermatitidis isolated from sputum samples of patients with cystic fibrosis.

Phenotypic and genotypic characteristics of 11 strains of Exophiala dermatitidis were investigated. Ten strains (including three reference strains) were isolated from sputum samples of six patients with cystic fibrosis (CF) in Germany, and one reference strain was isolated from a patient with phaeohyphomycosis in Japan. The strains showed differences in their ability to assimilate sorbitol, palatinose, rhamnose, gluconate and melezitose, leading to the differentiation of seven auxotypes. The IC30 of amphotericin B, and ketoconazole and itraconazole, respectively, indicated susceptibility, whereas the IC30 of fluconazole and 5-fluorocytosine indicated resistance in all strains. Protein patterns in SDS-PAGE revealed no major differences. The glycoconjugate patterns distinguished the Japanese strain from the other strains. Cluster analysis of whole-cell fatty acid methyl ester (FAME) profiles with the Microbial Identification System (MIS) revealed two major clusters separating a reference strain and the Japanese strain from the other strains. Analysis of patterns resulting from random amplification of polymorphic DNA (RAPD) with two arbitrary primers showed four genotypes. Comparison of the results revealed no agreement between the different fingerprinting methods, except the separation of the Japanese strain from the European CF strains. As the results of assimilation tests seem to vary between different laboratories, the analysis of FAME profiles and RAPD analysis are recommended for typing E. dermatitidis.

Antifungal Agents↗
PubMed2001

Phenotypic and genotypic characterization of reference strains of the genus Aspergillus.

Twenty-five culture collection strains from four Aspergillus species (A. fumigatus n = 8, A. flavus n = 8, A. niger n = 4, A. nidulans n = 5) were characterized by four methods: (i) determination of patterns in an assimilation assay; (ii) protein pattern of whole mycelial cell lysates in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE); (iii) reactivity of a pool serum obtained from cystic fibrosis patients with mycelial lysates in the immunoblot; and (iv) random amplification of polymorphic DNA (RAPD) with eight primers having arbitrary or repetitive sequences. In the assimilation assay the A. fumigatus strains showed identical patterns in contrast to the strains of the species A. flavus, A. niger, and A. nidulans, which each showed four patterns. In the SDS-PAGE no differences in the band patterns in the A. fumigatus strains were found, in contrast to the A. flavus (three patterns), A. nidulans (five patterns) and A. niger strains (two patterns). The immunoblot patterns were characteristic for each species with bands at 62 and 17/18 kDa in the A. fumigatus strains, at 51 and 18 kDa in the A. flavus strains, at 51 kDa in the A. niger strains, and at 51, 40 and 17/18 kDa in the A. nidulans strains allowing, however, no intraspecies typing. In the RAPD assay four out of eight primers gave interpretable patterns with 3-20 bands. None of the primers showed sufficient discriminatory power when used alone. However, when combining the results of two of the primers (5'-GTA TTG CCC T-3' and 5'-GAT AGA TAG ATA GAT A-3') all strains except two A. fumigatus strains could be clearly separated from each other. It is concluded that the the RAPD assay showed the most discriminatory power in all Aspergillus species investigated. In contrast to the phenotypically similar A. fumigatus strains, the strains of the species A. flavus, A. nidulans and A. niger differed in their phenotypic characteristics. The presented data of strains from international culture collections may serve as basis for interlaboratory standardization of typing methods.

Aspergillosis↗
PubMed1996

Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward.

During a 2-month period, five patients suffering from invasive infections caused by Aspergillus flavus or Aspergillus fumigatus were identified in the Hematology Department of the University Hospital Dijkzigt (Rotterdam, The Netherlands). To study the epidemiological aspects of invasive aspergillosis, strains from these patients and from the hospital environment, isolated during extensive microbiological screening, were subjected to genotyping. A novel DNA extraction technique, involving freezing, grinding, and direct lysis in guanidium isothiocyanate-containing buffers of mycelial material, was applied. DNA isolation was followed by typing by random amplification of polymorphic DNA (RAPD) analysis. This showed that strains isolated from all patients infected with the same fungal species were genotypically distinct, thus providing evidence against the possibility of an ongoing, single-source nosocomial outbreak. Strains could also be differentiated from strains of geographically diverse origins. However, an A. flavus strain from one of the patients was also frequently encountered in the hospital environment. As all environmental strains were collected after this patient had been diagnosed with invasive disease, the epidemiological value of this observation could not be ascertained. Intensive investigations showed no single source of A. flavus or other aspergilli. RAPD genotyping proved that the outbreak of invasive aspergillosis in the hematology ward consisted of a series of unrelated events and was not due to a common source within the hospital. RAPD fingerprinting of aspergilli may greatly facilitate future investigations of the epidemiology of invasive disease caused by these pathogens.

Adult↗
PubMed2005

Evaluation of intra-specific diversities in Oenococcus oeni through analysis of genomic and expressed DNA.

In winemaking Oenococcus (O.) oeni is the most frequent species of lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). Several studies have demonstrated that O. oeni is a quite homogeneous species and strains are difficult to differentiate especially when isolates from the same region are analyzed. In this study, the molecular biodiversity of O. oeni isolated from wines of the same region (Aglianico produced in Basilicata Region, Southern Italy) was evaluated with the aim of designing a molecular approach for discrimination and characterization of the isolates at the strain level. Three molecular techniques were applied: random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction endonucleases analysis-pulsed field gel electrophoresis (REA-PFGE) and differential display PCR (DD-PCR). The results obtained by RAPD-PCR confirmed the difficulty in differentiating isolates. By means of REA-PFGE a higher polymorphism, often related to the origin (winery) of strains, was revealed. However, on analyzing strains isolated from the same winery, only in some cases was more than one REA-PFGE pattern obtained. By analyzing dendrograms constructed on the basis of DD-PCR profiles differentiation of strains isolated from the same winery, in some cases, could be accomplished. The reliability of the DD-PCR in the differentiation of closely related strains suggests that this method could represent an alternative and/or additional tool to other molecular methods, such as REA-PFGE, for fine characterization of oenococcal strains.

DNA, Bacterial↗
PubMed2006

Fine-scale genetic structure and gene dispersal inferences in 10 neotropical tree species.

The extent of gene dispersal is a fundamental factor of the population and evolutionary dynamics of tropical tree species, but directly monitoring seed and pollen movement is a difficult task. However, indirect estimates of historical gene dispersal can be obtained from the fine-scale spatial genetic structure of populations at drift-dispersal equilibrium. Using an approach that is based on the slope of the regression of pairwise kinship coefficients on spatial distance and estimates of the effective population density, we compare indirect gene dispersal estimates of sympatric populations of 10 tropical tree species. We re-analysed 26 data sets consisting of mapped allozyme, SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) or AFLP (amplified fragment length polymorphism) genotypes from two rainforest sites in French Guiana. Gene dispersal estimates were obtained for at least one marker in each species, although the estimation procedure failed under insufficient marker polymorphism, limited sample size, or inappropriate sampling area. Estimates generally suffered low precision and were affected by assumptions regarding the effective population density. Averaging estimates over data sets, the extent of gene dispersal ranged from 150 m to 1200 m according to species. Smaller gene dispersal estimates were obtained in species with heavy diaspores, which are presumably not well dispersed, and in populations with high local adult density. We suggest that limited seed dispersal could indirectly limit effective pollen dispersal by creating higher local tree densities, thereby increasing the positive correlation between pollen and seed dispersal distances. We discuss the potential and limitations of our indirect estimation procedure and suggest guidelines for future studies.

French Guiana↗
PubMed2005

Phenotypic and molecular characterization of Candida dubliniensis isolates from clinical specimens in Kuwait.

OBJECTIVE: This study was carried out to characterize Candida dubliniensis using phenotypic and molecular methods and to determine the occurrence of C. dubliniensis in clinical specimens in Kuwait. MATERIALS AND METHODS: A total of 880 clinical specimens for isolation of fungi were processed according to standard procedures. Of these, 390 germ-tube-positive clinical isolates of Candida species were examined for rough colonies with hyphal fringes and chlamydospore production on simplified sunflower seed agar for their presumptive phenotypicidentification as C. dubliniensis. The identification of C. dubliniensis isolates was further confirmed by the Vitek 2 yeast identification system, semi-nested (sn) PCR amplification of high-copy rDNA and direct DNA sequencing of the internally transcribed spacer 2 (ITS2) region. RESULTS: Of the 390 isolates of Candida species investigated, 12 were identified as C. dubliniensis, giving an overall occurrence of 3%. All the C. dubliniensis isolates formed rough colonies with hyphal fringes and abundant chlamydospores on sunflower seed agar, did not assimilate trehalose, lactate and alpha-methyl-D-glucoside, and were isolated from human immunodeficiency virus (HIV)-negative patients. Four C. dubliniensis isolates utilized D-xylose. The species-specific primer derived from the ITS2 sequence of C. dubliniensis and used together with the panfungal reverse primer in the reamplification step of the snPCR specifically amplified rDNA from reference and clinical C. dubliniensis isolates and not from C. albicans or other Candida species. The identity of two representative isolates was confirmed by DNA sequencing of the ITS2 region. CONCLUSIONS: The identity of 12 C. dubliniensis isolates was first established by phenotypic characteristics and then by snPCR using species-specific primers derived from ITS2 sequences. The recovery of C. dubliniensis from HIV-negative patients from Kuwait reinforces the existing view that this novel yeast species has a worldwide distribution and its occurrence is not restricted to any particular immunocompromised population.

Antifungal Agents↗
PubMed2005

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces.

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.

Basidiomycota↗
PubMed2003

Adhesion and cell-surface-hydrophobicity of sequentially isolated genetic isotypes of Candida albicans in an HIV-infected Southern Chinese cohort.

Objectives of the study were to investigate the variability in yeast adhesion and cell-surface-hydrophobicity (CSH) during human immunodeficiency virus (HIV) disease progression, using a total of 60 sequential Candida albicans isolated from oral rinse samples of seven HIV-infected individuals with (4) and without (3) clinical symptoms of oropharyngeal candidosis. Significant differences in the adhesion to buccal epithelial cells (BECs) during sequential visits were observed for all genetic isotypes in five of the seven individuals and three isotypes belonging to the sixth individual. A single isotype of patient HK1 and another of HK4 (genotype I) demonstrated significant variations in their CSH during sequential visits whereas no such differences were noted for the remaining genotypes. On Spearman correlation analysis an isotype from HK1 demonstrated a significant increased adherence to BECs and CSH during HIV disease progression whereas no such correlation was noted for the remaining isotypes studied. No significant differences in adherence to BECs or CSH values were observed between the symptomatic oral candidosis and the asymptomatic carrier group. Further, on regression analysis only the single isotype of HK1 demonstrated a significant positive correlation between adherence to BECs and CSH whereas no such correlation was observed when all tested Candida isolates were pooled and evaluated as a single, large group.

AIDS-Related Opportunistic Infections↗
PubMed1999

Molecular nature of RAPD markers from Haemophilus influenzae Rd genome.

Despite the widespread application of the random amplified polymorphic DNA (RAPD) technique, there is no experimental evidence of the molecular mechanism of random amplification starting from a complex template. To investigate this mechanism, we cloned and sequenced 23 selected RAPD bands amplified from Haemophilus influenzae Rd genomic DNA using eight decamer primers different in GC content and/or nucleotide sequence. As the whole genome sequence of H. influenzae Rd has been reported, the exact nucleotide sequence of each primer-template annealing site was identified. Results showed that, on an average, a homology of eight base pairs was involved in priming events and that the number of nonhomologous base pairings declined exponentially from the 5' end of the primer to its 3' end. The interaction between the primer and the template DNA was stabilized by the formation of secondary structures, and a perfect match of the 3' terminal region of the primer was not necessary for successful amplification. The complexity of the annealing process suggested that, in the studied reaction conditions, many primer-template annealing sites were extended in the first cycles and that differences in the efficiency of priming and replication processes led to amplification of RAPD fragments. Moreover, the distribution of the amplified regions on the H. influenzae chromosome was analyzed.

Base Sequence↗
PubMed2005

Polyphasic study of wine Lactobacillus strains: taxonomic implications.

One hundred and seventy-eight lactobacilli isolated from wine were characterized by a polyphasic approach. Strains were phenotypically identified at genus and species level by classical tests including the analysis of cell morphology, homo/heterofermentative character, sugar fermentation patterns, growth at different temperatures and the optical nature of the isomer of lactic acid produced from glucose. Molecular techniques such as random amplification of polymorphic DNA (RAPD), amplified 16S rDNA restriction analysis (16S-ARDRA), PFGE-RFLP and ribotyping were used to characterize strains, and their potential for identification and/or typing was evaluated. The information obtained with these techniques was processed with the BioNumerics software in order to analyse relationships existing between isolated strains and various reference species of the genus. Then, taxonomic dendrograms were obtained, and this information allowed the proposal of molecular procedures suitable for the identification and typing of these wine micro-organisms. The techniques useful for both identification and typing were RAPD and ribotyping, while 16S-ARDRA was only useful for identification and PFGE-RFLP only for typing purposes. The wine strains were identified as Lactobacillus brevis (19 strains), Lactobacillus collinoides (2 strains), Lactobacillus hilgardii (71 strains), Lactobacillus paracasei (13 strains), Lactobacillus pentosus (2 strains), Lactobacillus plantarum (34 strains) and Lactobacillus mali (10 strains).

Bacterial Typing Techniques↗
PubMed2002

Diversity and genetic distance in populations of Steindachnerina in the upper Paraná river floodplain of Brazil.

Whereas four species of the genus Steindachnerina occur in the Paraná river basin, S. insculpta was the only endemic species of the region under analysis, which is the third lower section of the upper Paraná river. Among other factors, this species has been characterised by the absence of spots in the basal region of the dorsal fin. However, various specimens with this characteristic appeared in the region after the construction of the Itaipu Hydroelectric Plant in 1982. An analysis of the genetic variability of Steindachnerina populations with or without spots is provided. Specimens were collected in different sites of the floodplain of the upper Paraná river and samples were compared by random amplified polymorphic DNA (RAPD) technique and morphological analyses. Ninety-eight amplified loci with nine random primers were analysed in 19 specimens of each phenotype. Data for genetic distance showed great divergences between the two phenotypes and indicate two different species. Spotted specimens may be identified as S. brevipinna, found in the region downstream Sete Quedas Falls. The species must have overcome the geographical barrier during the building of the Itaipu hydroelectric dam that submerged the waterfalls and which became an obstacle between the upper and middle Paraná river some 150 km downstream. Since phenotypes do not share dominant alleles, absence of gene flow has been suggested.

Animals↗
PubMed2001

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks.

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.

Ascomycota↗