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PubMed2004

Mycobacterium cosmeticum sp. nov., a novel rapidly growing species isolated from a cosmetic infection and from a nail salon.

Four isolates of a rapidly growing Mycobacterium species had a mycolic acid pattern similar to that of Mycobacterium smegmatis, as determined by HPLC analyses. Three of the isolates were from footbath drains and a sink at a nail salon located in Atlanta, GA, USA; the fourth was obtained from a granulomatous subdermal lesion of a female patient in Venezuela who was undergoing mesotherapy. By random amplified polymorphic DNA electrophoresis and PFGE of large restriction fragments, the three isolates from the nail salon were shown to be the same strain but different from the strain from the patient in Venezuela. Polymorphisms in regions of the rpoB, hsp65 and 16S rRNA genes that were shown to be useful for species identification matched for the two strains but were different from those of other Mycobacterium species. The 16S rRNA gene sequence placed the strains in a taxonomic group along with Mycobacterium frederiksbergense, Mycobacterium hodleri, Mycobacterium diernhoferi and Mycobacterium neoaurum. The strains produced a pale-yellow pigment when grown in the dark at the optimal temperature of 35 degrees C. Biochemical testing showed that the strains were positive for iron uptake, nitrate reduction and utilization of d-mannitol, d-xylose, iso-myo-inositol, l-arabinose, citrate and d-trehalose. The strains were negative for d-sorbitol utilization and production of niacin and 3-day arylsulfatase, although arylsulfatase activity was observed after 14 days. The isolates grew on MacConkey agar without crystal violet but not on media containing 5 % (w/v) NaCl or at 45 degrees C. They were susceptible to ciprofloxacin, amikacin, tobramycin, cefoxitin, clarithromycin, doxycycline, sulfamethoxazole and imipenem. The name Mycobacterium cosmeticum sp. nov. is proposed for this novel species; two strains, LTA-388(T) (=ATCC BAA-878(T)=CIP 108170(T)) (the type strain) and 2003-11-06 (=ATCC BAA-879=CIP 108169) have been designated, respectively, for the strains of the patient in Venezuela and from the nail salon in Atlanta, GA, USA.

Bacterial Proteins↗

PubMedDate not supplied

Discriminatory power of RAPD, PCR-RFLP and southern blot analyses of ureCD or ureA gene probes on Helicobacter pylori isolates.

The genetic diversity of 33 Nigerian Helicobacter pylori isolates were studied using RAPD, PCR-RFLP and Southern blot analysis of ureA or ureCD gene probes. RAPD was able to distinguish the following number of isolates using the primers 3880: 5'-AAGAGCCCGT-3' (28), 3881 :5'-AACGCGCAAC-3' (33) and OPH8 :5'-GAAACACCCC-3' (25). Southern blot analysis using the ureCD probe was also able to distinguish the 12 isolates tested into ten different patterns. The PCR-RFLP technique distinguished all 33 isolates into six types. In conclusion, considering typeability, discriminatory power, and convenience, RAPD with the 3881 primer was considered the most useful technique.

Bacterial Proteins↗

PubMed2004

Molecular characterization of Brevibacillus laterosporus and its potential use in biological control.

Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.

Animals↗

PubMed2004

Comparison of methods for differentiation of Salmonella enterica serovar Enteritidis phage type 4 isolates.

OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.

Animals↗

PubMed2000

[Studies of molecular marker-assisted-selection for resistance to Fusarium wilt in watermelon (Citrullus lanatus) breeding].

The RAPD marker OPP01/700 proved to be linked to the Fusarium wilt resistant gene in watermelon wild germplasm PI296341 was cloned and sequenced, and transferred into a SCAR marker. The linked marker was tested to be one copy by Southern blotting. The technique of detection of the SCAR-amplified products was simplified. These techniques were applied in selecting resistant plants in F3 of the introgression of the disease resistant gene. It is the first report in this field of transfer ring a RAPD marker linked to disease resistance gene into SCAR marker, and establishing the technique of molecular marker-assisted-selection for breeding Fusarium wilt resistance cultivars.

Breeding↗

PubMed2002

Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods.

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.

Arcobacter↗

PubMed1997

Classification of plant trypanosomatids (Phytomonas spp.): parity between random-primer DNA typing and multilocus enzyme electrophoresis.

The genetic polymorphism of 30 isolates of plant trypanosomatids colloquially referred to as plant trypanosomes was assayed by means of RAPD. The principle objectives of this study were to assess the discriminative power of RAPD analysis for studying plant trypanosomes and to determine whether the results obtained were comparable with those from a previous isoenzyme (MLEE) study. The principle groups of plant trypanosomes identified previously by isoenzyme analysis--intraphloemic trypanosomes, intralaticiferous trypanosomes and trypanosomes isolated from fruits--were also clearly separated by the RAPD technique. Moreover, the results showed a fair parity between MLEE and RAPD data (coefficient of correlation = 0.84) and the two techniques have comparable discriminative ability. Most of the separation revealed by the two techniques between the clusters was associated with major biological properties. However, the RAPD technique gave a more coherent separation than MLEE because the intraphloemic isolates, which were biologically similar in terms of their specific localization in the sieve tubes of the plant, were found to be in closer groups by the RAPD. For both techniques, the existence of the main clusters was correlated with the existence of synapomorphic characters, which could be used as powerful tools in taxonomy and epidemiology.

Animals↗

PubMed2001

Genetic heterogeneity and phylogenetic status of Leishmania (Leishmania) infantum zymodeme MON-1: epidemiological implications.

Leishinania (Leishmania) infantum zymodeme MON-1 is responsible for the majority of visceral leishmaniasis cases around the Mediterranean basin, albeit that it causes also cutaneous forms. The MON classification is based on starch gel multilocus enzyme electrophoresis (MLEE) typing. The aim of this work was to explore further the genetic diversity and phytogenetic status of this zymodeme by alternative typing techniques. Fourteen L. (L.) infantum/L. (L.) chagasi stocks identified as MON-1 by MLEE in reference laboratories, 3 L. infantum stocks attributed to other zymodemes (MON-24, MON-29, MON-33) and reference standard stocks belonging to other species (L. (L.) major, L. (L.) tropica and L. (L.) donovani) were characterized by 2 different markers: MLEE on cellulose acetate plates and Random Amplified Polymorphic DNA (RAPD). We have obtained 10 different genotypes with RAPD and 6 different genotypes with MLEE on cellulose acetate plates for the 14 L. infantum/L. chagasi MON-1 stocks studied. MLEE and RAPD data gave quite congruent phylogenetic results: L. infantum zymodeme MON-1 was shown to be polyphyletic and genetically heterogeneous. This work confirms the necessity of using different markers to build up a robust phylogeny. Finally the epidemiological and clinical implications of these results are discussed.

Animals↗

PubMed1998

Comparison of three methods for the molecular typing of Singapore isolates of enterococci with high-level aminoglycoside resistances.

Enterococci are frequently isolated as nosocomial pathogens and have often acquired intrinsic drug resistances. Molecular typing techniques have been developed to assist in epidemological and infection control measures. This study investigates enterococci with high-level aminoglycoside resistance (HLAR) from the National University Hospital (NUH) of Singapore, and evaluates and compares three methods for typing: restriction enzyme analysis by conventional gel electrophoresis [restriction fragment length polymorphism (RFLP)], pulsed-field gel electrophoresis (PFGE), and polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD). Fifty-two isolates of Enterococcus faecalis and 13 isolates of Enterococcus faecium were used for the study. The numbers of patterns obtained for E. faecalis and E. faecium were 26 and 4, respectively by the RFLP method, and very similar discrimination was obtained by PFGE. RAPD PCR results were not reliably reproducible. A single pattern type by RFLP accounted for 16 of the E. faecalis isolates, suggesting hospital spread.

Aminoglycosides↗

PubMed2004

Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica).

A set of 146 Antarctic marine isolates from the Ross Sea was characterized by a combination of molecular techniques in order to determine the degree of inter- and intraspecific variability. Isolates were analyzed by amplified rDNA restriction analysis (ARDRA) using the tetrameric enzyme AluI, resulting in 52 different groups, corresponding to at least 52 different bacterial species, indicating a high degree of interspecific variability. The phylogenetic position of bacteria belonging to some ARDRA groups was obtained by sequencing of 16S rDNA. Random amplified polymorphic DNA (RAPD) analysis, carried out on the largest ARDRA groups, revealed a high intraspecific genetic variability, too. The analysis of plasmid content revealed the existence of horizontal gene transfer between strains belonging to the same and to different species. A comparison of the whole body of morphological, physiological and biochemical data was finally carried out.

Antarctic Regions↗

PubMed2004

Induced of plastid mutations in soybean plant (Glycine max L. Merrill) with gamma radiation and determination with RAPD.

The aim of our study was to induce with radiation of atrazine resistant and tolerated mutants in Coles, Amsoy-71 and 1937 soybean varieties. Atrazine that is photosynthetic inhibitor is the most important herbicide of S-triazin group, and shows toxic effect on soybean plant. For the improvement of the atrazine resistant plants with mutation breeding, the seeds belonging to the three varieties were irradiated with 200 Gy of gamma radiation dose. The irradiated seeds were sown in the field and at the end of harvesting season, every pod at node situated on the main stem was picked up separately and M2 generations were obtained. At the plants, which were obtained from M2 generation, chlorophyll mutants were determined and atrazine selection was made. The percentage of chlorophyll mutants for Amsoy-71, Coles and 1937 soybean varieties were found as 1.07, 1.48 and 1.32, respectively. At the end of atrazine selection, the percentages of atrazine resistant plants for Amsoy-71, Coles and 1937 soybean varieties were 0.80, 0.60 and 0.53, respectively. The percentages of atrazine tolerated plants were 1.07, 1.18 and 1.05, respectively as well. In our research; the differences among the mutants replying to atrazine in various concentrations were examined by using RAPD procedure as the molecular marker techniques in comparison with polymorphism. In the study done by using 14 primers; according to the amplification results, the differences between atrazine resistant plants were shown.

Atrazine↗

PubMed2002

DNA polymorphism in various goose lines by RAPD-PCR.

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.

Animals↗

PubMed2001

Molecular identification of a clinical isolate of non-pigmented and asporogenic Aspergillus fumigatus strain which cannot be identified by traditional morphological methods.

A non-pigmented and asporogenic fungal strain without taxonomically useful characteristics was isolated from sputum of a patient with chronic bronchitis. Although the taxonomic position could not be determined based on traditional morphological criteria, the fungus was identified as an Aspergillus fumigatus mutant strain by molecular techniques including ITS sequence analysis and PCR identification system using a PCR primer pair specific for A. fumigatus. Usefulness of these molecular techniques for the identification of non-classifiable fungal strains in a clinical laboratory is discussed.

Aspergillus fumigatus↗

PubMed2003

Specific detection and differentiation of two subspecies of Fusobacterium necrophorum by PCR.

The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.

Base Sequence↗

PubMed1998

Evolutionary distances and identification of Candida species in clinical isolates by randomly amplified polymorphic DNA (RAPD).

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the different species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epidemiological studies also depend on effective identification. To address this problem we established a RAPD method that enabled direct identification of five very common species of Candida. Initially, reference band patterns were established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. One of the primers, M2, showed remarkably conserved intra-specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific. Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single colonies yielded sufficient DNA for identification. The reference band patterns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional diagnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be consistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18S rRNA trees showed significant differences in tree topologies which indicated that RAPD data could not accurately measure the relative distances between different species. Also, computer simulations of RAPD random patterns were used to test whether the observed degree of RAPD band pattern similarities could occur at random. These simulations suggested that the level of inter-specific band pattern similarities observed in our data could be obtained at random, while intraspecific pattern similarities could not. RAPD would be helpful to discriminate between isolates but not to quantitate the differences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem of our identification method. Because of the repetitive character of the target sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with speciation events.

Candida↗

PubMed2001

Concordance between genetic relatedness and phenotypic similarities of Trichomonas vaginalis strains.

BACKGROUND: Despite the medical importance of trichomoniasis, little is known about the genetic relatedness of Trichomonas vaginalis strains with similar biological characteristics. Furthermore, the distribution of endobionts such as mycoplasmas or Trichomonas vaginalis virus (TVV) in the T. vaginalis metapopulation is poorly characterised. RESULTS: We assayed the relationship between 20 strains of T. vaginalis from 8 countries using the Random Amplified Polymorphic DNA (RAPD) analysis with 27 random primers. The genealogical tree was constructed and its bootstrap values were computed using the program FreeTree. Using the permutation tail probability tests we found that the topology of the tree reflected both the pattern of resistance to metronidazole (the major anti-trichomonal drug) (p < 0.01) and the pattern of infection of strains by mycoplasmas (p < 0.05). However, the tree did not reflect pattern of virulence, geographic origin or infection by TVV. Despite low bootstrap support for many branches, the significant clustering of strains with similar drug susceptibility suggests that the tree approaches the true genealogy of strains. The clustering of mycoplasma positive strains may be an experimental artifact, caused by shared RAPD characters which are dependent on the presence of mycoplasma DNA. CONCLUSIONS: Our results confirmed both the suitability of the RAPD technique for genealogical studies in T. vaginalis and previous conclusions on the relatedness of metronidazol resistant strains. However, our studies indicate that testing analysed strains for the presence of endobionts and assessment of the robustness of tree topologies by bootstrap analysis seem to be obligatory steps in such analyses.

Animals↗

PubMed1996

A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene.

A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.

Amino Acid Sequence↗

PubMed2004

Sphingomonas phyllosphaerae sp. nov., from the phyllosphere of Acacia caven in Argentina.

Two bacterial strains (FA1 and FA2(T)) were isolated from the phyllosphere of a leguminous tree, Acacia caven, in central Argentina. The strains were Gram-negative, strictly aerobic, rod-shaped, motile and formed yellow-pigmented colonies on nutrient agar. The two-primer RAPD patterns of the two strains were identical, suggesting that they belong to the same species. The complete 16S rRNA gene sequences of the two strains were obtained and comparisons demonstrated that they cluster phylogenetically with the species of the genus Sphingomonas sensu stricto. Strain FA2(T) was most closely related (97.6 %) to Sphingomonas adhaesiva. 16S rRNA gene sequence similarities to all other established Sphingomonas species ranged from 94.4 % (to Sphingomonas echinoides) to 97.6 % (to S. adhaesiva). Strains FA1 and FA2(T) were catalase-positive and oxidase-negative. Aesculin was hydrolysed, gelatin and urea were not. beta-Galactosidase was produced. From 51 compounds tested 21 were used as single sources of carbon. The major respiratory lipoquinone was ubiquinone-10. The predominant cellular fatty acids were 16 : 0, 18 : 1omega7c and 16 : 1omega7c (from summed feature 3). Hydroxy fatty acids 14 : 0 2-OH and 15 : 0 iso 2-OH were present as well (from summed feature 4). The polar lipids detected in strain FA2(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and two unidentified phospholipids. The DNA G+C content of strain FA2(T) was 61 mol%. DNA-DNA hybridization experiments showed 27.6 % relatedness between strain FA2(T) and S. adhaesiva DSM 7418(T). Based upon phenotypic and molecular evidence, a novel species of the genus Sphingomonas is proposed, Sphingomonas phyllosphaerae sp. nov., with strain FA2(T) (=LMG 21958(T)=CECT 5832(T)) as the type strain.

Acacia↗