Analysis of the clonal diversity of Staphylococcus aureus methicillin-resistant strains isolated at João Pessoa, state of Paraíba, Brazil

To investigate the clonal diversity of Staphylococcus aureus strains isolated at João Pessoa, State of Paraíba, Brazil, digested genomic DNA were studied by pulsed-field gel electrophoresis (PFGE) in nine methicillin-resistant strains (MRSA) and three methicillin-sensitive strains (MSSA), selected among 67 isolates based on their antimicrobial susceptibility and epidemiology. The isolates were obtained between April and November 1992 from the Hospital of the Federal University of Paraíba, located in João Pessoa. Two MRSA isolates from the Oswaldo Cruz Hospital, São Paulo, Brazil, including an epidemic strain previously detected from different hospitals at the country were used as control. Five different patterns, were demonstrated by MRSA isolated in João Pessoa and these patterns were described in several epidemiologically unrelated hospitals in São Paulo. Our results suggest the interstate dissemination of a MRSA clone in João Pessoa which is similar to that described in other cities of Brazil.

Biological comparison between three clones of Trypanosoma cruzi and the strain of origin (Bolivia) with reference to clonal evolution studies

After isolating three clones of Trypanasoma cruzi (Bolivia), we first characterized them according to parasitaemia, pleomorphism and virulence, and then histopathologically. The study's interest lies on the hypothesis that clonal evolution of T. cruzi has a major impact on biologically relevant properties of this parasite. Data obtained from the studies of parasitaemia, pleomorphism and virulence showed no differences between the groups studied. As a final point, the histopathological study shows us a muscular tissue tropism both in clones and in their mother strain (Bolivia). In this paper, we conclude that Bolivia strain and clones isolated from it, pertaining to the same major clone share similar biological properties.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.

Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.

Short-term storage in vitro and large-scale propagation of grapevine genotypes

The objective of this work was to evaluate the large-scale propagation of grapevine genotypes after short-term storage in vitro. Microshoots from ten grapevine genotypes were used. The following storage temperatures were evaluated: 10, 20, and 25°C. After short-term storage, the shoots were propagated in up to five successive subcultures, to assess the large-scale propagation of the germplasm maintained under conditions of minimal growth. The propagated shoots were rooted in different concentrations of indolbutiric acid (IBA) and acclimatized in greenhouse. The best temperature for short-term storage in vitro and survival of the genotypes was 20°C. In the propagation phase, the highest number of shoots per explant was found in the subcultures 4 and 5, with averages of 4.9 and 4.8 shoots per explant, respectively. In the rooting phase, the best results for number of roots were obtained using a culture medium supplemented with 0.4 µmol L-1 of IBA, with an average of three roots per shoot. During the acclimation phase, a survival rate higher than 95% was achieved after 30 days in the greenhouse. Grapevine genotypes maintained for six months in vitro, at 20ºC, can be micropropagated in large scale.

Enraizamento de microestacas de mirtileiro provenientes de microjardim clonal semi-hidropônico

O uso de microjardins clonais hidropônicos tem sido relatado com sucesso para espécies florestais e pode vir a se tornar uma excelente alternativa para espécies frutíferas de difícil propagação, como é o caso do mirtilo. O objetivo deste estudo foi avaliar o enraizamento de microestacas de mirtileiro provenientes de dois sistemas de cultivo (convencional e semi-hidropônico), submetidas a diferentes concentrações de AIB (ácido indolbutírico). As microestacas de mirtileiro das cultivares Bluebelle e Woodard foram submetidas a diferentes concentrações de AIB (0; 500; 1.000; 1.500 e 2.000 mg.L-1), acondicionadas em caixas plásticas contendo vermiculita e, aos 90 dias de cultivo, avaliou-se o seu rendimento. O delineamento experimental foi o inteiramente casualizado, com vinte tratamentos, contendo quatro repetições, compostas por dez microestacas cada. Foram avaliados as porcentagem de sobrevivência e de enraizamento, o comprimento da maior raiz, o número de brotações, o comprimento médio das brotações, o número de folhas e as massas fresca e seca radiculares. O sistema semi-hidropônico proporcionou um rendimento de microestacas significativamente superior ao convencional; entretanto, este material apresentou menores porcentagens de sobrevivência e enraizamento. Todos os tratamentos, inclusive aquele sem a presença de AIB, apresentaram porcentagens de enraizamento superiores a 50%.

Influência genotípica e clonal de genótipos de bananeiras micropropagados a partir de gemas florais masculinas

Inflorescências masculinas de bananeiras apresentam potencial para serem usadas como explantes para a micropropagação de bananeiras. Este trabalho teve por objetivo avaliar a influência genotípica e clonal de bananeiras micropropagadas a partir de gemas florais masculinas em sucessivos subcultivos. Foram utilizados explantes florais de 15 clones plantados em campo, da variedade Preciosa e do híbrido FHIA 02, em meio de cultura de MS contendo 4,0 mg.L-1 de BAP. Por sete subcultivos sucessivos de 30 dias, os materiais foram avaliados quanto à taxa de multiplicação e contaminação microbiana. Ao final do período de multiplicação, o acúmulo de mudas produzidas, em razão dos sucessivos subcultivos utilizando esse tipo de explante, foi também avaliado. Verificou-se que os maiores índices de contaminação foram observados no primeiro subcultivo, com média superior a 50% de contaminação dos explantes. As melhores taxas de multiplicação foram alcançadas no terceiro subcultivo, com média de até 6,1 brotos por explante. Os clones que apresentaram maior acúmulo de mudas produziram 105 e 163 mudas por explante, para a variedade Preciosa e o híbrido FHIA 2, respectivamente, após sete subcultivos.

Limpeza clonal de mudas de videira infectadas por Xanthomonas campestris pv. viticola

O cancro bacteriano da videira é causado por Xanthomonas campestris pv. viticola (Xcv). Visando à limpeza clonal de mudas de 'Red Globe', foram estudados: tamanho ideal de ápices e gemas axilares para cultivo em meio de Galzy modificado (MGM); efeito da termoterapia (38ºC/30 dias); e ação de antibióticos na eliminação de Xcv em videiras infectadas. Os percentuais de contaminação por Xcv e de regeneração foram analisados, e as plantas obtidas foram indexadas em meio ágar nutritivo-dextrose-extrato de levedura-ampicilina (NYDAM), seguindo-se teste de patogenicidade. O cultivo de explantes com 3 mm possibilitou a obtenção de plantas livres da bactéria, com regeneração 14,3 vezes maior que explantes com 1 mm. A termoterapia de mudas infectadas, associada ao cultivo in vitro, não eliminou o patógeno. O cultivo de explantes com 10 mm, durante 40 dias em MGM + cefotaxima (300 mg L-1), proporcionou limpeza clonal das mudas. A indexação de plantas de videira regeneradas in vitro, quanto à infecção por Xcv utilizando NYDAM, seguida de teste de patogenicidade, é uma alternativa econômica e eficiente para produção de mudas de alta qualidade fitossanitária.

THE LYCHEE TREE PROPAGATION BY LAYERING

The aim of the study was to assess the influence of season and different substrates on rooting of air layers of lychee (Litchi chinensisSonn.) for the production of seedlings to ensure the formation of uniform and productive orchards. Air layers were done in plants of the Bengal cultivar using leafy and healthy woody branches, with about 0.010 to 0.015 m in diameter, in which were performed complete girdling with 0.020 m wide at a distance of 0.30 to 0.40 m below the apex. Then the branches were wrapped in moistened substrate. The layering was made at six times of theyear (January, March, May, July, September and November) and two substrates were used (coconut fiber and sphagnum) in a 6 x 2 factorial design in a randomized block with ten replicates. After 90 days, layers were separated from the matrix plant and evaluated for rooting and callus formation, root number, considering only the primary roots, length, area and volume of the roots, beyond the dry weight of roots and calluses. The months of January, March, September and November showed the best results for all analyzed variables related to rooting. With respect to the substrates, the only difference was in January and March to the root number and dry weight of roots, where the sphagnum showed the best results. The month of July was more conducive to the formation of calluses. The period between September and March was more suitable to the propagation of lychee, when there were rooting percentages above 90%, in addition to the formation of large amount of roots.