Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Antibiotic susceptibility and genotype patterns of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from urinary tract infected patients

Thirty nine isolates of Escherichia coli, twenty two isolates of Klebsiella pneumoniae and sixteen isolates of Pseudomonas aeruginosa isolated from urinary tract infected patients were analyzed by antimicrobial susceptibility typing and random amplified polymorphic DNA (RAPD)-PCR. Antibiotic susceptibility testing was carried out by microdilution and E Test methods. From the antibiotic susceptibility, ten patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). Furthermore, genotyping showed seventeen RAPD patterns (seven for E. coli, five for K. pneumoniae and five for P. aeruginosa respectively). In this study, differentiation of strains of E. coli, K. pneumoniae and P. aeruginosa from nosocomial infection was possible with the use of RAPD.     

Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.     

Virulence Analysis and Oligonucleotide Fingerprinting to Detect Diversity Among Indian Isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis> Causing Chickpea Wilt

Virulence analysis of 64 isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from major chickpea growing states of India on 14 varieties, including 10 international differentials revealed that the isolates from each state were highly variable. Based on the reactions on international differentials, more than one race was found to be prevalent in every state. Majority of the isolates were not matched with the race specific reactions. Therefore, some of the cultivars, namely, GPF 2, DCP 92-3, and KWR 108 should be included as new differentials to obtain clear-cut differential responses. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and simple sequence repeat (SSR) markers were used to assess the genetic diversity of these isolates. Unweighted paired group method with arithmetic average (UPGMA) cluster analysis was used to divide the isolates into distinct clusters. The clusters generated by RAPD grouped all isolates into three categories at 25% genetic similarity and into two major categories at 30% genetic similarity. ISSR and SSR analyses also grouped all the isolates into two major categories. Majority of the isolates from Punjab and a few from Rajasthan were grouped in one category while the isolates from all other states were grouped in another suggesting the existence of diverse genetic populations of the pathogen at the same location. Some of the RAPD (OPM 6, OPI 9, P 17, OPN 4, OPF 1, P 17, P 21, and SC 1), ISSR (ISSR 7, ISSR 11, and ISSR 12) and SSR (MB 17) markers clearly distinguished area specific isolates.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

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.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Prevalence of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae in Trakya University Hospital,Turkey

The prevalence of extended-spectrum beta-lactamase (ESBL) production by 194 nosocomial isolates of Enterobacteriacea recovered from 1995 to 1999 was investigated. The ESBL production was determined by the double-disk synergy test and was confirmed by the E-test ESBL strip. Twenty-three isolates (21 Klebsiella pneumoniae, one Escherichia coli, one Providencia rettgeri) were found as ESBL-producers (11.8%). These isolates were also usually resistant to non-betalactam antibiotics. Most of them contained a beta-lactamase with a pI of 7.6. All the strains conjugally transferred their ESBLs to recipient E. coli. Contrary to others, ESBL-producing K. pneumoniae strains isolated in 1999 were resistant to ciprofloxacin, and had the identical plasmid profiles suggestive of an outbreak. Ciprofloxacin resistance in these strains could not be transferred. In conclusion, K. pneumoniae was the main ESBL-producing species among nosocomial isolates of Enterobacteriacae in our hospital.     

Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections.     

Prevalence of the <Emphasis Type="Italic">bla</Emphasis><Subscript>SHV</Subscript> Gene in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Isolates Obtained from Hospital and Community Infections and from the Microbiota of Healthy Individuals in Recife,Brazil

The aim of this study was to determine the prevalence of the bla _SHV gene in Klebsiella pneumoniae isolates from hospital and community infections and from the normal microbiota of healthy individuals in Recife, PE, Brazil. Fifty-two K. pneumoniae isolates were analyzed regarding the presence of the bla _SHV gene, using PCR, and eight isolates were analyzed by DNA sequencing. This gene was detected in 16 isolates from hospital infections, four from community infections, and nine from the normal microbiota. This was the first study to find the bla _SHV gene in K. pneumoniae isolates from the normal microbiota. Through DNA sequencing of eight K. pneumoniae isolates from hospital and community infections, with a resistance phenotype indicative of extended-spectrum β-lactamase production, a new SHV variant named SHV-122 was found. We also detected the presence of bla _SHV-1, bla _SHV-11, bla _SHV-28, and bla _SHV-108. The results show that in Recife, Brazil, K. pneumoniae isolates that presented resistance to oxyimino-β-lactams had high prevalence and diversity of the bla _SHV gene. We also conclude that there was a high presence of the bla _SHV gene among isolates from the normal microbiota of healthy individuals.     

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.     

Species attribution and distinguishing strains of Oenococcus oeni isolated from Chinese wines

Summary Twenty-four strains of Oenococcus oeni were isolated from different Chinese wines. Differentiation of isolates was carried out by analysis of total soluble cell protein patterns and random amplified polymorphic DNA (RAPD) patterns. The results indicated that the total soluble cell protein patterns could be used to distinguish different genera but fail to distinguish different strains. It was also found that strain RAPD pattern can successfully distinguish isolates by UPGMA analysis. The RAPD profiles (107 different prints) were strain specific and two main groups of strains were screened.     

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.     

Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America

Crinipellis perniciosa has been classified into at least four known biotypes associated with members of unrelated plant families. In this study, genetic variability is shown for 27 C (Cacao), 4 S (Solanum), and 7 L biotype (Liana) isolates of C. perniciosa collected from different regions of Brazil and South America. The objective was to investigate the genetic variability of the pathogen in the cacao-producing region of Bahia, Brazil, and elsewhere, through microsatellite analysis, and attempt to identify possible correlations between host specificity and electrophoretic karyotypes. The PCR-banding patterns were found to vary both within and between the different biotypes, and a correlation was established between the PCR-banding patterns and the chromosomal-banding patterns of each isolate. Microsatellite and chromosomal patterns among all of the L and S biotype isolates were distinctly different from the C biotypes analysed. A higher degree of genetic and chromosomal variability was found among C biotype isolates from the Amazon in comparison with C biotype isolates from Bahia, which seems to be comprised of only two main genotypes. This finding has important implications to the current cacao-breeding programme in Brazil.     

Genetic diversity of Leishmania infantum field populations from Brazil

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.     

Typing of clinical and environmental strains of Aeromonas spp. using two PCR based methods and whole cell protein analysis

Two PCR based typing methods i.e. random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR were evaluated for typing of 42 Aeromonas isolates from clinical and environmental sources and whole cell protein (WCP) profiles were analyzed. Both RAPD and ERIC-PCR showed a high level of genetic diversity. Numerical index of the discriminatory (D) values were 0.94 and 0.96 (>0.90) for RAPD and ERIC-PCR, respectively. No correlation in banding pattern and evidence of genetic similarity was found between Aeromonas isolates from environmental and clinical sources. Therefore these techniques are highly reproducible and sensitive methods for typing the Aeromonas isolate from different sources. WCP profile showed two major variable regions i.e. 20 kDa to 45 kDa region and 70 kDa to 85 kDa region. Though WCP profiling had less discriminatory power, use of this method in combination with other established typing methods such as RAPD and ERIC-PCR may be helpful for reliable typing of Aeromonas isolates or to identify new proteins with pathogenic potential.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.