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Nosocomial Legionella pneumophila serogroup 5 outbreak associated with persistent colonization of a hospital water system.

An outbreak of infections caused by Legionella pneumophila serogroup 5 was detected in a university hospital, and nosocomial reservoirs of the legionella epidemic were examined. Clinical isolates from two patients who had been affected by the L. pneumophila serogroup 5 outbreak, and from another patient with a legionella infection caused by the same serogroup 3 years later, were compared to L. pneumophila serogroup 5 isolates from the hospital water supply by two molecular methods, amplified fragment length polymorphism (AFLP) analysis and random amplified polymorphic DNA analysis (RAPD). Genotyping confirmed the epidemiological linkage of the first two patients, and linked their infections with the hospital water supply. The third clinical strain, which was also linked to the hospital water, was very similar to the epidemic strain. Even though the water distribution system was sanitized (superheat and flush sanitation), the epidemic strain was shown to be persisting in the hospital water outlets several years after its initial discovery.

Adult↗

Nucleic acid evolution and minimization by nonhomologous random recombination.

We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure-activity relationships among evolved sequences.

Base Sequence↗

[Cluster analysis of Dendrobium by RAPD and design of specific primer for Dendrobium candidum].

OBJECTIVE: To analyze the genetic relationship of Dendrobium and design a specific primer to differentiate Dendrobium candidum effectively. METHOD: Random amplified polymorphic DNA (RAPD) technique was used to analyze the genetic relationships of 26 species of Dendrobium and one species of Ephemerantha. Dendrogram was constructed by UPGMA. According to the sequence of DNA fragment selected, Sangon 18 primer had been extended from 3' extreme to 20 bp in order to form a specific primer. RESULT AND CONCLUSION: This primer can be used to distinguish Dendrobium candidum from other Dendrobium effectively. Such a technique provides a new way for the identification of Chinese traditional medicines.

Cluster Analysis↗

Detection of RAPD markers correlated with chloroquine resistance in Plasmodium falciparum.

We described the use of the random amplified polymorphic DNA (RAPD) technique on Plasmodium falciparum DNA to detect genetic markers for chloroquine-resistant strains. Fourteen RAPD primers were tested, three of which generated banding patterns correlated with chloroquine resistance. To measure this correlation, the RAPD profiles were analyzed using the Nei and Li similarity coefficient. Detection of distinctive RAPD bands allowed us to synthesize specific PCR primers to be used on whole-blood samples. Two primer sets were synthesized and tested on sensitive and resistant strains for their ability to amplify the DNA fragment corresponding to the RAPD marker. These results suggest that RAPD and PCR techniques can be used as powerful tools for the detection of genetic markers associated with drug resistance.

Animals↗

Study of Corynebacterium diphtheriae strains isolated in Romania, northwestern Russia and the Republic of Moldova.

A selection of 167 Corynebacterium diphtheriae strains isolated in Romania, the Russian Federation and the Republic of Moldova were analysed by biotyping, phage typing, the toxin production test and by molecular techniques such as ribotyping, pulsed field gel electrophoresis and random amplified polymorphic DNA, in order to establish the epidemiological relatedness, genetic divergence and strain circulation within and between the bordering countries. Using a set of five digoxigenin-labeled oligonucleotides and BstEII digestion, 34 ribotypes were identified. The strains isolated in the epidemic areas (Russia and Moldova) were very closely related but different from those isolated in Romania. C1 and C5 were the main ribotypes identified in these areas. Neither ribotype was found in Romania, where the main circulating types were C3 and C7. Field inversion gel electrophoresis was more discriminative than ribotyping and revealed 54 macrorestriction profiles after SfiI restriction. Both methods showed a significant homogeneity of the strains from epidemic areas and a large diversity among the Romanian strains. Random amplification was useful as an identification method for the epidemic strains, but not for the Romanian ones which displayed a large number of amplification profiles. The phenotypic methods associated with molecular typing techniques enabled distinguishing between strains, detecting the epidemic clone, and sustaining the absence of transmission across borders.

Bacterial Typing Techniques↗

Prospective epidemiologic analysis of triazole-resistant nosocomial Candida glabrata isolated from patients at a comprehensive cancer center.

OBJECTIVE: The emergence of Candida glabrata infections among patients with compromised immunity has become a serious concern, especially at centers caring for individuals with cancer. METHODS: During a prospective evaluation of Candida species associated with either clinically significant colonization or infection, 26.9% of C. glabrata isolates showed in vitro resistance to fluconazole (MIC of > or = 64 microg/ml). RESULTS: Antifungal susceptibility profiles and genetic fingerprinting analysis performed by randomly amplified polymorphic DNA (RAPD) techniques confirmed low-probability of phenotypic and genotypic relatedness among nosocomial C. glabrata isolates. CONCLUSIONS: Presence of polyclonal strains of C. glabrata in patients at our hospital was probably related to selection of resistant yeasts from environmental pool rather than monoclonal expansion or clustering of multi-drug resistant C. glabrata in high-risk patients.

Adult↗

The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology.

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious or uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred 'amplification method' for most diagnostic applications.

Bacteria↗

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera.

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.

Animals↗

Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti strains by Medicago sativa.

We analysed the genetic diversity of 270 Sinorhizobium meliloti strains isolated from nodules of three different Medicago sativa varieties, planted in three different Italian soils, combining the Analysis of Molecular Variance (AMOVA) with the Random Amplified Polymorphic DNA (RAPD) technique to estimate variance among RAPD patterns with the aim to draw an objective description of the population genetic structure. Results indicated that a general intraspecific genetic diversity was globally distributed among all the population, however a very high level of diversity was found among strains nodulating different Medicago sativa varieties. Moreover the distribution of the RAPD haplotypes among the plant varieties also showed to be non-random. The overall data indicated that the plant genotype is a major factor in shaping the genetic structure of this natural Rhizobium population.

Analysis of Variance↗

Molecular typing of Aspergillus species.

In the last four years, several molecular typing methods have been applied to various Aspergillus species, primarily A. fumigatus and A. niger. Many 'clones' or DNA types exist in the environment and in patients. The most discriminatory methods appear to be restriction fragment length polymorphism analysis with or without specific probes and random amplification of polymorphic DNA analysis. Insufficient work on interlaboratory reproducibility has been done. Most patients are infected by single DNA types, although occasionally multiple isolates are detected. Little work has been done with regard to detailed hospital epidemiology using molecular typing methods.

Aspergillus↗

Detection of two fungal biocontrol agents against root-knot nematodes by RAPD markers.

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 mug, respectively.

DNA, Fungal↗

Molecular characterization of a salt-tolerant bacterial community in the rice rhizosphere.

The diversity of salt-tolerant bacteria present in the rhizosphere of Oryza sativa was investigated. Fourteen bacterial strains, isolated after enrichment in nitrogen-free, semi-solid medium and showing tolerance to 3% NaCl, were analyzed by restriction patterns produced by amplified DNA coding for 16S rDNA (ARDRA) with enzymes Sau3AI, AluI and RsaI which showed that they were represented by 4 ARDRA types. Biodiversity among the 14 strains was also analyzed by the random amplified polymorphic DNA (RAPD) technique with a 10-mer primer. Partial nucleotide sequence of 16S rDNA assigned these clusters to Serratia marcescens, Pseudomonas aeruginosa, Alcaligenes xylosoxidans and Ochrobactrum anthropi. Notably, all four bacterial species are potential human pathogens that infect immunocompromised patients.

Alcaligenes↗

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

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

DNA, Fungal↗

Molecular fingerprinting methods for the discrimination between C. albicans and C. dubliniensis.

Opportunistic fungal pathogens are becoming increasingly important causes of both community-acquired and nosocomial infections. The most important fungal pathogens are yeast species belonging to the genus Candida. These species show differences in levels of resistance to antifungal agents and mortality. Consequently, it is important to correctly identify the causative organism to the species level. Identification of Candida dubliniensis in particular remains problematic because of the high degree of phenotypic similarity between this species and Candida albicans. However, as the differences between both are most pronounced at the genetic level, several studies have been conducted in order to provide a specific and rapid identification fingerprinting molecular test. In most candidal infectious, no single DNA fingerprinting technique has evolved as a dominant method, and each method has its advantages, disadvantages and limitations. Moreover, the current challenge of these techniques is to compile standardized patterns in a database for interlaboratory use and future reference. This review provides an overview of most common molecular fingerprinting techniques currently available for discrimination of C. albicans and C. dubliniensis.

Candida↗

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis.

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.

Cluster Analysis↗

Characterization of trichomonad species and strains by PCR fingerprinting.

The random amplified polymorphic DNA (RAPD) technique was used for phylogenetic analysis of trichomonads, for intraspecies genealogical study of Trichomonas vaginalis strains, and for assessment of intrastrain polymorphism in Trichomonas vaginalis. The phylogenetic tree for 12 trichomonad species showed certain discrepancies with current models of trichomonad evolution. However, it shows that RAPD traits retain phylogenetically relevant information. The results of intraspecies analyses of 18 Trichomonas vaginalis strains suggested some concordance between the genetic relationship of strains and their geographic origin. They also suggested a concordance between the strain genetic relationships and the resistance to metronidazole. A concordance was also found with respect to the severity of disease observed in donor patients but not with the results of laboratory virulence assays. No concordance was found between genetic relationship of strains and strain infection with a dsRNA Trichomonas vaginalis virus (TVV). The latter suggests that TVV might be transmitted horizontally among Trichomonas vaginalis populations. The identity of RAPD patterns of clones isolated from in vitro cultures and those of the cultures reisolated independently from the same patient within a period of six weeks suggests that individual Trichomonas vaginalis strains are not polymorphic and that the RAPD patterns are stable. Therefore, the RAPD technique seems useful for addressing various clinically relevant issues.

Animals↗

AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum).

The red flour beetle (Tribolium castaneum) is a major pest of stored grain and grain products and a popular model species for a variety of ecological, evolutionary, and developmental biology studies. Development of a linkage map based on reproducible and highly polymorphic molecular markers would greatly facilitate research in these disciplines. We have developed a genetic linkage map using 269 amplified fragment length polymorphism (AFLP) markers. Ten previously known random amplified polymorphic DNA (RAPD) markers were used as anchor markers for linkage group assignment. The linkage map was constructed through genotyping two independent F(2) segregating populations with 48 AFLP primer combinations. Each primer combination generated an average of 4.6 AFLP markers eligible for linkage mapping. The length of the integrated map is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. A cluster of loci on linkage group 3 exhibited significant segregation distortion. We have also identified six X-linked and two Y-linked markers. Five mapped AFLP fragments were sequenced and converted to sequence-tagged site (STS) markers.

Animals↗

DNA typing methods for differentiation of yeasts related to dry-cured meat products.

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.

DNA Primers↗