Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats,captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Two Specific Strains of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> causing Mucocutaneous Manifestations of Histoplasmosis: Preliminary Analysis of a Frequent Manifestation of Histoplasmosis in Southern Brazil

Objectives  Skin lesions, uncommon in US cases (<10%), occur in 38–85% of cases reported from Latin America. Although these differences may reflect reporting bias, delayed diagnosis, or differences in host immune response among different ethnic groups, they also could result from genetic differences changing the pathobiology of the organism. It is possible that genetic differences among strains of H. capsulatum may influence the pathogenesis and clinical manifestations of histoplasmosis. Methods  We examined the clinical features of patients with mucocutaneous manifestations of histoplasmosis and performed genetic analysis based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes of H. capsulatum isolates of patients. Two pairs of PCR primers were designed to develop and amplify the ITS regions of H. capsulatum, 5′-TACCCGGCCACCCTTGTCTA-3′ and 5′-AGCGGGTGGCAAAGCCC-3′. These primers were based on the ITS sequence of Ajellomyces capsulatus, the ascomycetous teleomorph form of H. capsulatum, deposited in the GenBank (accession number U18363). Eight patients attending a tertiary-care hospital in southern Brazil were enrolled into the study. All case patients had skin cultures growing H. capsulatum at the mycology laboratory. Results  Six of eight (75%) patients were HIV-positive and presented involvement of multiples organs by H. capsulatum. Two HIV-negative patients did not present evidence of involvement of other organs besides mucosa and skin. ITS sequencing of a DNA H. capsulatum fragment of 485-bp from isolates of 8 patients revealed two distinct strains. The 2 distinct fragments (Hc1, Hc2) differed from each other at 7 positions in the ITS regions. They were identical to strains of H. capsulatum isolated in patients from Colombia and Argentina, but different from strains isolated in US. Hc1 and Hc2 were isolated in 5 patients and 3 patients, respectively, with mucocutaneous manifestations of histoplasmosis. Both Hc1 and Hc2 strains were isolated in HIV-infected and non-HIV-infected patients. Conclusions  Mucocutaneous manifestations of histoplasmosis, which are frequently seen in Brazilian patients were caused by 2 specific strains in our institution. Those strains have been isolated in patients with these particular clinical features of histoplasmosis in Latin America. Our study suggests that unique pathogenic characteristics among the Latin American species of H. capsulatum might explain its increased dermatotropism.     

Assessment of the Genetic Diversity among Strains of Xanthomonas cynarae by Randomly Amplified Polymorphic DNA Analysis and Development of Specific Characterized Amplified Regions for the Rapid Identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Intraspecific Diversity of Vibrio vulnificus in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.     

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

Detection of Genetic Variation in Alternaria brassicae by RAPD Fingerprints

Genetic variation in fourteen isolates of Alternaria brassicae collected from different geographical regions of the world was determined by RAPD (random amplified polymorphic DNA) analysis. Twenty random primers were tried to amplify genomic DNA of A. brassicae. Based on the PCR (polymerase chain reaction) amplification of genomic DNA of A. brassicae with four oligonucleotide random primers, fingerprints were generated for each isolate and the amplifed products were compared. Using this technique, intra- and intercontinental genetic variation among isolates of A. brassicae could be distinguished.     

Biofilm formation,antibiotic susceptibility and RAPD genotypes in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> clinical strains isolated from single centre intensive care unit patients

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD–PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.Histoplasmosis is a serious community-acquired infection in the United States (28) and in certain countries of Latin America, where it is an especially significant problem in patients with AIDS (14). This disease is one of the most common systemic mycoses in Brazil, where epidemiological surveys carried out using the histoplasmin skin test have indicated that it is endemic in all areas surveyed (15). Data suggest that the numbers of cases of histoplasmosis in Brazil may be underestimated and that the areas where it is endemic are more widespread than previously thought.Histoplasma capsulatum is a dimorphic fungus that grows as a mold and produces aerial hyphae at 25 to 30°C, but it undergoes morphogenesis to a yeast phase at 37°C. The filamentous phase of this organism is usually found in soil enriched with several compounds, such as nitrogen and phosphate compounds. When conidial or hyphal fragments are inhaled by humans or animals, H. capsulatum changes to the yeast form and continues to replicate as a yeast. Although H. capsulatum has been recognized as an important fungal pathogen in immunocompromised hosts, particularly AIDS patients (27), there are significant gaps in our knowledge of this species'' epidemiology and pathogenesis. For instance, systemic histoplasmosis has been found in patients with AIDS who do not reside in regions where it is endemic (29), leading to the suggestion that the disease can result from reactivation of a previously acquired H. capsulatum infection. The clinical manifestations of histoplasmosis range from asymptomatic infections, mild flu-like symptoms, or pneumonia to a systemic disease involving the skin, brain, intestine, adrenal glands, and/or bone marrow (6). Importantly, diverse strains of H. capsulatum have been identified worldwide, and the strains vary in virulence. In addition to classical biochemical assays, distinctions between strains may be based on colony morphology or polymorphism of the genome (19).Multiple typing methods have been developed to study the epidemiology of H. capsulatum. These methods are based on phenotypic characteristics, such as antigenic profiles (13) and multilocus enzyme electrophoresis results (2), or on DNA-based analysis. Most recently, typing has been accomplished by analysis of fatty acid profiles of H. capsulatum (34). Molecular typing methods are generally considered to have advantages over phenotypic methods in terms of the stability of genomic markers and greater levels of typeability. Several genotype-based methods, such as hybridization of target genes (probes), chromosomal DNA typing, restriction fragment length polymorphism (RFLP) analysis, random amplified polymorphic DNA (RAPD) analysis, and sequencing, have been described for H. capsulatum (4, 5, 7, 8, 11, 17, 19). Despite the abundance of previously developed molecular techniques, there is limited information concerning comparisons of the results obtained with different methods using the same set of isolates. In H. capsulatum, no single approach based on DNA assays has been the dominant method.The current study was done to explore the diversity of H. capsulatum in Brazil and to determine the correlation between the results of three different molecular typing techniques. For this analysis, we used M13 PCR fingerprinting, PCR-RFLP analysis of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the rDNA gene, and analysis of the nucleotide sequence polymorphism of four partial genes. M13 PCR fingerprinting (25) is based on generation of multiple PCR products with different electrophoretic mobilities. PCR fingerprinting primers are typically designed using repetitive DNA sequences (31), and the products facilitate detection of two types of genetic variations: (i) differences in the length of DNA and (ii) alterations in the sequence of the priming regions. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region of the rDNA gene (9) involves use of a gene-specific PCR in combination with restriction digestion in order to generate highly stable and reproducible markers. Analysis of the nucleotide sequence polymorphism is based on the sequences of four partial protein-encoding genes (Arf, the H-anti gene, Ole, Tub1) (4). Additionally, to assess the utility of an assay to study the global epidemiology of the fungus, we performed a DNA sequencing analysis of the ITS1-5.8S-ITS2 region to compare the Brazilian H. capsulatum ITS1-5.8S-ITS2 sequences with sequences obtained for H. capsulatum strains isolated in other countries.     

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm,Bombyx mori,using random amplification of polymorphic DNA: Morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8–16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.     

Population Genetics of Vibrio vulnificus: Identification of Two Divisions and a Distinct Eel-Pathogenic Clone

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions Ι and ΙΙ). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions Ι and ΙΙ) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

Genetic and morphological comparisons ofGlomerella (Colletotrichum) isolates from maize and from sorghum

Various morphological and genetic characteristics were compared among six isolates ofColletotrichum from maize and six from sorghum. For the first time, a teleomorph was induced in sorghum isolates by pairing them on autoclaved sorghum leaves in a humidity chamber. The sorghum teleomorph was morphologically similar toGlomerella graminicola andGlomerella tucumanensis, the teleomorphs ofColletotrichum isolates from maize and from sugarcane, respectively. Mating tests demonstrated thatGlomerella isolates from maize and sorghum were not interfertile. Several small but consistent differences in the morphologies of the isolates from maize and from sorghum were observed which agreed with earlier reports. DNA fingerprints detected as restriction fragment length polymorphisms of mitochondrial DNA and random polymorphic DNA (RAPD) produced from nuclear DNA by the polymerase chain reaction could be used to reliably and unambiguously distinguish members of the two groups of isolates. Results of a statistical analysis of similarity of the RAPD fingerprints suggested that maize and sorghum isolates ofColletotrichum are only about 45% similar (±10%) and represent two distinct and separate genetic lineages. We conclude that isolates ofColletotrichum from maize and sorghum are sibling species since they are morphologically very similar but reproductively completely isolated.