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PubMed2004

Microevolution between paired antral and paired antrum and corpus Helicobacter pylori isolates recovered from individual patients.

Sequence variations located at the signal sequence and mid-region within the vacA gene, the 3'-end of the cagA gene, the indel motifs at the 3'-end of the cag pathogenicity island and the regions upstream of the vacA and ribA genes were determined by PCR in 19 paired antral or antrum and corpus Helicobacter pylori isolates obtained at the same endoscopic session, and three antral pairs taken sequentially. Random amplification of polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (FAFLP)-PCR fingerprinting were applied to these paired clinical isolates. The FAFLP-PCR profiles generated were phylogenetically analysed. For the 22 paired isolates there were no differences within pairs at five of the genetic loci studied. However, six pairs of isolates (27%), of which four were antrum and corpus pairs, showed differences in the numbers of repeats located at the 3'-end of the cagA gene. RAPD-PCR fingerprinting showed that 16 (73%) pairs, nine of which were antrum and corpus pairs, possessed identical profiles, while six (27%) displayed distinctly different profiles, indicating mixed infections. Three of the six pairs showing differences at the 3'-end of the cagA gene yielded identical RAPD-PCR fingerprints. FAFLP-PCR fingerprinting and phylogenetic analysis revealed that all 16 pairs that displayed identical RAPD-PCR profiles had highly similar, but not identical, fingerprints, demonstrating that these pairs were ancestrally related but had undergone minor genomic alterations. Two antrum and corpus pairs of isolates, within the latter group, were isolates obtained from two siblings from the same family. This analysis demonstrated that each sibling was colonized by ancestrally related strains that exhibited differences in vacA genotype characteristics.

Antigens, Bacterial↗
PubMed2003

Molecular and physiological characterization of Italian isolates of Pyrenochaeta lycopersici.

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 degrees C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

DNA, Fungal↗
PubMed2005

Molecular mapping of a fertility restorer gene for Owen cytoplasmic male sterility in sugar beet.

We report here the molecular mapping of a fertility restorer gene (named Rf1) for Owen cytoplasmic male sterility in sugar beet. Eight AFLP and two RAPD markers, tightly linked to the Rf1 locus, were identified using bulked segregant analysis. Three AFLP markers, mAFEM972, mAFEM976 and mAFEM985, were found to co-segregate with the Rf1 allele in our mapping populations. With the help of RFLP markers, previously mapped on the sugar beet genome, we showed that Rf1 is positioned in the terminal region of linkage group Kiel III/Koeln IV. This map location agrees well with that found for the restorer gene X, which suggests that the Rf1 locus corresponds to the X locus. The availability of the molecular markers will facilitate the selection of maintainer-pollinator lines in breeding program and provide the foundation for map-based cloning of the Rf1 gene.

Beta vulgaris↗
PubMed2005

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov.

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.

Aflatoxin B1↗
PubMedDate not supplied

Typing of mutans streptococci by arbitrarily primed polymerase chain reaction.

The discriminative power of the arbitrarily primed polymerase chain reaction (AP-PCR) in differentiating between Streptococcus mutans and Strep. sobrinus species, serotypes and clones was investigated. Mutans streptococcal isolates (12(7)) obtained from 65 individuals (1-10 isolates per individual) were AP-PCR typed separately with two random primers, OPA-05 and OPA-13. Bacterial cell lysates were used as a template in PCR reactions, which made AP-PCR easy and fast to perform. Eighty-one isolates from 19 individuals were also ribotyped to compare the discriminative ability of ribotyping and AP-PCR techniques. AP-PCR performed with the two primers differentiated between Strep. mutans and Strep. sobrinus isolates, but neither primer detected serotype-specific amplification products. OPA-05 distinguished two main AP-PCR patterns among Strep. mutans isolates and one main pattern among Strep. sobrinus isolates, whereas OPA-13 found one main AP-PCR pattern among Strep. mutans isolates and two main patterns among Strep. sobrinus isolates. Ribotyping and AP-PCR revealed 40 and 33 different types among 81 selected isolates, respectively. Both techniques detected intra-individual heterogeneity in 16 out of 19 participants. The results indicate that AP-PCR has good discriminative ability in differentiating between mutans streptococcal clones and that the technique is suitable for epidemiological studies on mutans streptococci.

Bacterial Typing Techniques↗
PubMed2001

A novel EF-hand calcium-binding protein in the flagellum of the protozoan Tritrichomonas suis.

The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41 kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

Amino Acid Sequence↗
PubMed2001

Genetic diversity of Saccharomyces cerevisiae strains during the 24 h fermentative cycle for the production of the artisanal Brazilian cachaça.

AIMS: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cachaça production. METHODS AND RESULTS: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle. CONCLUSION: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cachaça fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Spontaneous fermentations to produce cachaça, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.

Alcoholic Beverages↗
PubMed2004

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis.

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

Actinomycetales↗
PubMed2001

Randomly amplified polymorphic DNA (RAPD) profiles for the distinction of Lactobacillus species.

Forty-one type and reference strains of Lactobacillus were evaluated using their randomly amplified polymorphic DNA band profiles. Developed bands for each strain were distinct and enabled discrimination. The best correlations were obtained applying the Pearson product moment correlation coefficient (r) together with the unweighted pair group method using arithmetic averages algorithm. All of the strains were clearly differentiated at and below the 72% similarity value. Species discrimination might be possible making use of the distinctly polymorphic bands amplified specific to a strain.

Bacterial Typing Techniques↗
PubMed2002

Approaches to investigating the ecology of plasmids in marine bacterial communities.

To better understand prokaryotic gene flux in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of plasmid populations in marine bacterial communities, information on the distribution, diversity, and ecological traits of marine plasmids is necessary. This mini-review highlights recent insights gained into the molecular diversity and ecology of plasmids occurring in marine microbial communities.

DNA↗
PubMed2000

Genetic and antigenic characteristics of Neisseria meningitidis strains isolated in the Czech Republic in 1997-1998.

Strains of Neisseria meningitidis isolated from patients and asymptomatic individuals who had been in contact with patients were investigated using four typing methods with the aim of identifying any heterogeneity and/or homogeneity among the strains. In 1993, a dramatic change in the incidence and severity of invasive meningococcal disease in the Czech Republic occurred as a consequence of the appearance of a Neisseria meningitidis strain of phenotype C:2a:P1.5,2 and electrophoretic type ET-15, belonging to the ET-37 complex, which had not previously been identified in this country. Presented here are the results of a study of the relationships between 58 Neisseria meningitidis isolates collected between January 1997 and June 1998. Forty-nine isolates originating from patients with invasive meningococcal disease and nine from healthy contacts were analyzed using the following four methods: whole-cell enzyme immunoassay, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. A high prevalence of phenotype C:2a:P1.5,2 electrophoretic type ET-15 was confirmed among the patients' strains. Nevertheless, during the study period, they became heterogeneous. Strains isolated from healthy contacts showed greater heterogeneity in serological phenotypes and electrophoretic types from the beginning of the study, and electrophoretic type ET-15 strains were less frequent. Within the electrophoretic type ET-15 clone, strains showing the identical serological phenotype (with the exception of one isolate) were indistinguishable using randomly amplified polymorphic DNA analysis, while pulsed-field gel electrophoresis revealed heterogeneity with 12 pulsed-field gel electrophoretic types identified. The strains from the same cluster displaying the same serological phenotype were indistinguishable with any of the methods used.

Cluster Analysis↗
PubMed2000

Isolation of a single strain of Helicobacter pylori from the antrum and body of individual patients.

OBJECTIVES: This study aimed to determine intra-patient colonization patterns of Helicobacter pylori strains based on DNA fingerprinting and antibiotic susceptibility. METHODS: Two biopsies, one from the antrum and one from the body of the stomach, were taken from 97 patients. Prior informed consent was obtained. The status of cagA gene of H. pylori strains was analysed by using the polymerase chain reaction (PCR) technique, while DNA fingerprints were generated by PCR-based, random amplified polymorphic DNA (RAPD) fingerprinting. The antibiotic susceptibility of the H. pylori isolates was examined by the disk diffusion method. RESULTS: A total of 51 pairs of H. pylori strains were isolated from both antrum and body specimens of 51 patients. This included two patients who were endoscoped twice because of treatment failure. All strains were positive for cagA gene by PCR. These 51 patients were found to harbour a single strain of H. pylori with identical or highly similar DNA profiles by PCR-based RAPD fingerprinting. In four of the 51 pairs, the DNA patterns of H. pylori from antrum and body showed minor differences, while three pairs of strains with different metronidazole sensitivities showed identical DNA fingerprints. Interestingly, the two treatment failure patients remained colonized with the strains that had the same RAPD fingerprinting patterns before and after treatment. CONCLUSION: The present study demonstrates that a single H. pylori strain colonizes a single stomach. However, this single genotypic strain may exhibit different metronidazole susceptibility in different parts of stomach.

Adult↗
PubMed1999

Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD).

Similarity among a number of aquatic microbial communities was examined using randomly amplified polymorphic DNA (RAPD), a common polymerase chain reaction (PCR)-based DNA fingerprinting technique. After amplification of whole-community DNA extracts, the PCR products were resolved by agarose gel electrophoresis and the band patterns compared to determine percent similarity. Twelve different primers were used to amplify approximately 100 fragments (total) from each DNA sample; the bands were scored as present or absent and the similarity between each sample was determined using Jaccard's coefficient. From this information. dendrograms were constructed and a bootstrapping procedure was used to assess how well supported the tree topologies were. Principal component analyses were also conducted as a means of visualizing the relationships among samples. Results obtained for two different experimental systems (a pair of tidal creeks and several wells in a shallow groundwater aquifer) correlated well with the temporal and spatial variations in environmental regime at the sites confirming that arbitrarily primed PCR-based DNA fingerprinting techniques such as RAPD are useful means of discriminating among microbial communities and estimating community relatedness. Moreover, this approach has several advantages over other DNA-based procedures for whole-community analysis; it is less laborious and uses smaller quantities of DNA, making it amenable to sample-intensive monitoring, and it does not depend on culturing or the use of selective PCR primers.

Bacteria↗
PubMed1995

Random amplified polymorphic DNA fingerprinting of Campylobacter jejuni and C. coli isolated from human faeces, seawater and poultry products.

The polymerase chain reaction (PCR) technique was used to obtain randomly amplified polymorphic DNA (RAPD) profiles from 64 type and serotype reference strains and 114 isolates of Campylobacter jejuni and C. coli from food, seawater and human faeces. Genetic diversity was detected among the strains as a total of 118 different RAPD profiles were obtained, each one containing from 4 to 11 bands between 0.30 and 1.50 kb. The discriminatory power of a random 10-mer primer (sequence 5'-CAATCGCCGT-3') was assessed. In general, no profiles were common to strains of the same Penner serogroup, but occasional strains from different Penner serotypes shared identical band profiles. RAPD analysis also differentiated between the species, and after numerical analysis, five main clusters were defined at the 40% similarity level, corresponding to C. jejuni, C. coli and C. lari with some exceptions. RAPD profiling of Campylobacter is highly discriminatory and is a valuable new alternative to traditional typing in epidemiological studies.

Campylobacter↗
PubMed2003

Madurella mycetomatis strains from mycetoma lesions in Sudanese patients are clonal.

Molecular diversity among clinical isolates of Madurella mycetomatis, the prime fungal agent of human mycetoma in Sudan, could possibly explain the diverse clinical presentations of this severely debilitating infectious disease. In addition, culture-independent DNA-mediated typing tests need to be developed for this organism, since M. mycetomatis DNA, but not the organism itself, can be identified in soil, the material from which infections are thought to originate. A collection of 38 different clinical M. mycetomatis isolates was characterized by large-scale random amplification of polymorphic DNA using 20 different primer species. These analyses, involving at least 2,600 annealing sites, showed a complete lack of DNA fingerprint variation among the various isolates. From the resulting homogeneous DNA fingerprints, seven fragments were cloned and sequenced, and novel, species-specific PCR restriction fragment length polymorphism (RFLP) tests were designed. The seven PCR RFLP tests were successfully performed on the 38 different M. mycetomatis strains. However, again all M. mycetomatis DNA patterns obtained appeared to be identical, whereas patterns produced using DNAs from other fungal species were clearly discriminatory. These results suggest that there is little genetic variation among clinically relevant M. mycetomatis strains from Sudan. The data tentatively imply that different manifestations of mycetoma are due to differences in host susceptibility rather than differential virulence of the causative agent.

DNA Primers↗
PubMed2000

The use of anonymous DNA markers in assessing worldwide relatedness in the yeast species Pichia kluyveri Bedford and Kudrjavzev.

Pichia kluyveri, a sexual ascomycetous yeast from cactus necroses and acidic fruit, is divided into three varieties. We used physiological, RAPD, and AFLP data to compare 46 P. kluyveri strains collected worldwide to investigate relationships among varieties. Physiology did not place all strains into described varieties. Although the combined AFLP and RAPD data produced a single most parsimonious tree, separate analysis of AFLP and RAPD data resulted in significantly different trees (by the partition homogeneity test). We then compared the distribution of strains per band to an expected distribution. This suggested we could separate both the AFLP and RAPD datasets into bands from rapidly and slowly changing DNA regions. When only bands from slowly changing regions (from each dataset) were included in the analysis, both the RAPD and AFLP datasets supported a single tree. This second tree did not differ significantly from the cladogram based on all of the DNA data, which we accepted as the best estimate of the phylogeny of these yeast strains. Based on this phylogeny, we were able to demonstrate the strong influence of geography on the population structure of this yeast, confirm the monophyly of one variety, question the utility of maintaining another variety, and demonstrate that the physiological differences used to separate the varieties did not do so in all cases.

Americas↗