Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.     

Antimicrobial biotypes and colicine types: potential epidemiological markers of Salmonella agona

With the purpose of characterizing epidemiologic markers, 240 strains of S. agona isolated from differents sources (man, food and environment) and obtained from five Brazilian States (Minas Gerais, S?o Paulo, Rio de Janeiro, Pernambuco and Rio Grande do Sul) were analysed. The susceptibility to 15 antimicrobial agents and numeric codification of the resistance profiles allowed us to recognize 56 antibiotic resistance biotypes, while 40 strains were able to produce colicine, belonging to the types: Ia (55%); B (32.5%); Ib (10%) and untypable (2.5%). The application of these elements into intra-serotype differentiation is discussed.     

Serological Strains of Tobacco Ringspot Virus Transmitted by Xiphinema americanum

Five serological strains of tobacco ringspot virus isolated from naturally infected tobacco in North Carolina, and a strain isolated from watermelon in the Rio Grande Valley of Texas were transmitted from cucumber to cucumber by mass-screened and handpicked Xiphinerna americanum from North Carolina. The Eucharis mottle strain from Peru was not transmitted, indicating that a specific strain-vector relationship may exist between the geographically isolated strains from North and South America.     

The distribution of the P–M hybrid dysgenesis system in Drosophila melanogaster strains from Brazil

Wild-caught flies of Drosophila melanogaster from seven natural populations of extreme regions of Brazil (São Luís, MA; Teresina, PI; Rio Cipó, MG; Maringá, PR; São José do Rio Preto, SP; Joinville, SC; and Porto Alegre, RS) were studied with the purpose of evaluating hybrid dysgenesis due to mobilization of P elements and the regulatory capacity of the strains' cytotypes. Diagnostic crosses were made and the strains classified according to their P–M phenotypes. Four strains were classified as moderate P (MA, MG, PI, and SP), two as Q (PR and RS) and one as M′ (SC). Females of southern strains (PR, SC, and RS) presented in A crosses lower degrees of gonadal dysgenesis scores than those from northern strains (MA and PI).     

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.     

Autochthonous horizontal transmission of a CRF02_AG strain revealed by a human immunodeficiency virus type 1 diversity survey in a small city in inner state of Rio de Janeiro, Southeast Brazil

As part of an ongoing study on the features of AIDS spread towards small cities and rural areas, we present a molecular survey of human immunodeficiency virus type 1 (HIV-1) polymerase sequences recovered between 2004 and 2006 from 71 patients receiving care in the city of Saquarema, inner state of Rio de Janeiro. Phylogenetic reconstructions found the two prevalent lineages in the state (subtypes B [59 strains, 83.1%], F1 [6 strains; 8.4%], and BF1 recombinants [four strains; 5.6%]), as well as two (2.8%) CRF02_AG strains, which seems to be an emerging lineage in the capital. These CRF02_AG sequences were recovered from a married heterosexual couple who never traveled abroad, thus providing the first molecular evidence of autochthonous horizontal transmission of this lineage of major global importance. Also, three phylogenetic clusters of strains recovered from a total of 18.3% of the cohort were uncovered. Their close genetic relatedness suggests they were recovered from patients who probably took part in the same chain of viral spread. In conjunction with our previous surveys from inner Rio de Janeiro, these results suggest that although small cities harbor unique molecular features of HIV-1 infection, they also clearly reflect and may rapidly absorb the diversity recorded in large urban centers.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Ecological approach of macrolide-lincosamides-streptogramin producing Actinomyces from Cuban soils

We report in this study the frequency of Streptomyces strains to produce macrolide-lincosamide-streptogramin (MLS) antibiotics isolated from Cuban soils. The screening assay is based on the induction of MLS-resistance phenotype in a clinical isolated strain of Staphylococcus aureus S-18. Our results suggest that of 800 Streptomyces strains isolated from different soil samples, 6% were positives in the screening test used. The ferralitic red soil from Pinar del Río (north) provided the major percentage (3.6%) of MLS producing strains. The other soil samples tested belonging to Guira de Melena and Bauta in Havana, Matanzas City, Topes De Collantes (Villa Clara), and Soroa Mountains (Pinar del Rio) hill reached very low percentages.     

Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.     

Very large trypomastigotes as a morphological pattern of strains of Trypanosoma cruzi in the southern region of Brazil

The morphological patterns of blood trypomastigotes from five sylvatic Trypanosoma cruzi strains from Santa Catarina, South Brazil, were studied during the course of infection in experimentally infected mice. A predominance of stout trypomastigotes (greater than 70%) was observed during all over the acute phase in four strains of medium virulence. With the remaining strain, of high virulence, the slender forms predominating at the early infection stage were soon also replaced by stout forms. Since almost all T. cruzi strains displaying predominance of this peculiar morphological pattern have been isolated in South Brazil (Rio Grande do Sul, Santa Catarina) and since there are evidences pointing out to the existence of biological differences among these distinct blood parasites, the authors suggest further investigations of possible correlations between the morphological markers and clinical-epidemiological aspects of Chagas' disease.     

Genomic analysis of a nontoxigenic,invasive Corynebacterium diphtheriae strain from Brazil

We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.     

Antifouling activity as a function of population variation in <Emphasis Type="Italic">Sargassum vulgare</Emphasis> from the littoral of Rio de Janeiro (Brazil)

The brown seaweed Sargassum vulgare is abundant along the coast of Rio de Janeiro state. An investigation of the spatial variation of antifouling activity was conducted, in which algae were collected at five locations along the coast of Rio de Janeiro during October 2008. Hexane, dichloromethane, methanol and acetone/water extracts were prepared and screened for their bioactivities against the growth of five strains of marine fouling bacteria, five biofilm-associated microphytobenthic strains and attachment of the mussel Perna perna. The most active were the hexane extracts from Bananal algae that inhibited the growth of all microalgae tested; the methanol and dichloromethane extracts from Mar do Norte, which inhibited Vibrio aestuarianus and Pseudoalteromonas elyakovii and the polyphenol extracts from Ilha de Itacuruçá and Bananal that inhibited mussel attachment, respectively, by 64% and 71% compared to controls.     

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.     

Infectivity of two strains of Plasmodium vivax to Anopheles albitarsis mosquitoes from Colombia

Anopheles albitarsis obtained from Villavicencio, Colombia, were colonized in the laboratory using force-mating techniques. Laboratory reared mosquitoes were allowed to feed on Aotus monkeys infected with the Salvador II or the Rio Meta strains of Plasmodium vivax from El Salvador and Colombia, respectively. In comparison with other species, the An. albitarsis were less susceptible than Anopheles freeborni, Anopheles culicifacies and strains of Anopheles albimanus from El Salvador, Panama and Colombia and more susceptible than a strain of An. albimanus from Haiti.     

Population Dynamics of Pseudomonas syringae pv. tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto under Field Conditions

We examined the effects of the Pto resistance locus on the population dynamics of Pseudomonas syringae pv. tomato (Pst) strains in field experiments with the nearly isogenic tomato lines Rio Grande (RG, susceptible to Pst races 0 and 1) and Rio Grande-Pto (RG-Pto, resistant to Pst race 0, susceptible to Pst race 1). Pst strain SM78-1Sm^r (race 0) grew well under field conditions and caused ample bacterial speck disease on susceptible RG plants. In contrast, strain DC3000 failed to establish large populations when inoculated onto field grown RG plants. Mean population sizes of SM78-1Sm^r were 4–5 orders of magnitude larger on RG than RG-Pto plants indicating that RG-Pto plants were highly effective in attenuating pathogen population development. Most of the sampled leaflets from RG-Pto field plots harboured small numbers of SM78-1Sm^r. However, population sizes SM78-1Sm^r as large as 10⁵–10⁶ CFU were found on a few leaflets. Bacteria isolated from these leaflets had phenotypes characteristic of Pst race 1 strains. In growth chamber plant assays, the bacterial strains grew well and caused typical speck lesions on RG-Pto plants. The strains appeared to be race-shift mutants of SM SM78-1Sm^r. Interestingly, results from DNA hybridization experiments demonstrated that the race-shift mutants were deleted for the avirulence gene, avrPto but not for avrPtoB.     

Identification of entomopathogenic Bacillus isolated from Simulium (Diptera, Simuliidae) larvae and adults.

Entomopathogenic bacteria isolated from Simulium larvae and adults from breeding sites in the states of S?o Paulo and Rio de Janeiro, Brazil, were identified as 18 strains of Bacillus thuringiensis and one of B. sphaericus. Most of these strains were serotyped according to their flagellar antigens. However, nine of the B. thuringiensis samples, could not be serotyped and were designated as "autoagglutinating"; they were also shown to be toxic in preliminary tests against Aedes aegypti larvae. Additionally, B. sphaericus was also shown to be toxic towards Culex quinquefasciatus larvae.     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds

AIMS: To investigate the coagulase gene polymorphism of Staphylococcus aureus isolated from mastitic goat's milk. METHODS AND RESULTS: A typing procedure based on coagulase gene polymorphism was used to discriminate S. aureus isolated from goat mastitis. Thirty-six strains collected from goats belonging to herds from northern Ceara State and Serrana region of Rio de Janeiro State were analysed. Based on restriction fragment length polymorphism of 3' end coagulase gene, the goat strains were grouped into 11 types. In northern Ceara herds, the predominant type was found in 60% of the strains, while in the Serrana region herds the two most common accounting for 62.5% of the strains. CONCLUSIONS: The analysis of the coa gene proved to be useful for typing S. aureus from goat mastitis. Although the results showed that goats from the studied regions were infected by S. aureus strains harbouring more than one coagulase genotype, only one or two types predominated. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the prevalent strains within a herd or region is a necessity. The important virulence factors could be identified in such strains and this information can then be used as a specific base to develop S. aureus mastitis control measures.     

Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles

Corynebacterium ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals. Since pets are able to act as symptomless carriers, it is also essential to identify virulence potential for humans of these isolates. In this work the ability of C. ulcerans to bind to fibrinogen (Fbg), fibronectin (Fn) and Type I collagen as well the genetic relationship among strains isolated from human and asymptomatic dogs in Rio de Janeiro (Brazil) were analyzed. Five pulsed-field gel electrophoresis (PFGE) profiles were demonstrated (I, II, III, IV and V). In addition, the IV and V profiles exhibiting ≥85 % similarity were expressed by the BR-AD41 and BR-AD61 strains from companion dogs living in the same neighborhood. Independent of the PFGE-types, human and dog isolates showed affinity to Fbg, Fn and collagen. Heterogeneity of PFGE profiles indicated endemicity of C. ulcerans in the Rio de Janeiro metropolitan area. Differences in the expression of adhesins to the human extracellular matrix may contribute to variations in the virulence and zoonotic potential of C. ulcerans strains.     

Selection of rhizosphere-competent Pseudomonas strains as biocontrol agents in tropical soils

Pseudomonas strains were isolated from the rhizosphere of maize grown in yellow-red latosol from Rio de Janeiro, Brazil, to serve as a delivery system for heterologous genes and for risk assessment studies in tropical soils. Selected strains were modified by insertion of the cryIVB gene from Bacillus thuringiensis and tested for pathogenicity gene expression against larvae of a susceptible model species, Anopheles aquasalis. Modified strains Br8 and Br12 showed similar survival performance to their parental strains, and presented a viable density of 10⁷ c.f.u./g dry soil 30 days after release. A strain of P. fluorescens (Br12) that presented positive results for gene expression and the best survival performance, was selected for risk assessment studies in soil microcosms.