Relationships between phagocytosis, mucoid phenotype, and genetic characteristics of Klebsiella pneumoniae clinical isolates

Abstract INTRODUCTION The relationships between phagocytosis, and mucoid phenotype, plasmid profile and virulence, and resistance genetic characteristics of Klebsiella pneumoniae clinical isolates were evaluated. METHODS Thirty isolates were used to determine the mucoid aspect. Four were selected for analysis of phagocytosis by alveolar macrophages. RESULTS Thirty percent of the samples presented the mucoid phenotype. The phagocytosis rate ranged from 21.5% to 43.43%. Phagocytosis was not correlated with the plasmid profile, but was apparently correlated with mucoid phenotype and antibiotic susceptibility. CONCLUSIONS: Several virulence factors act in parallel in K. pneumoniae to impair host defense.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6 percent of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Polymorphism of the rDNA and tDNA loci in clinical isolates of Pseudomonas aeruginosa: a perspective for molecular epidemiology surveillance

The Gram-negative bacterium Pseudomonas aeruginosa has a wide environmental and ecological distribution. It is an opportunistic pathogen that acquires resistance to multiple antimicrobial agents and can infect plants, animals and humans. We used rDNA and tDNA PCR markers to characterize the bacterial diversity of P. aeruginosa strains isolated at a Brazilian teaching hospital (Oswaldo Cruz University Hospital, Recife, Brazil) between March 2003 and February 2004. Clonal groups of P. aeruginosa clinical isolates were identified from different patients in different hospital units using either rDNA or tDNA markers, or a combination of both in a duplex PCR. These PCR-typing methods together with drug-resistance profiles were used to trace the distribution of antibiotic resistant P. aeruginosa clones and to identify cross-infection of the same patient with a different bacterial clone after being moved to a different hospital unit. The data presented here demonstrates a rapid, reliable and useful method for epidemiological surveillance that can contribute to the control of P aeruginosa infections in hospital environments.

Identification in silico of putative damage responsive elements (DRE) in promoter regions of the yeast genome

We report an in silico analysis to identify nucleotide sequence motifs in DNA repair genes that may define a binding site for regulatory proteins during the induction of those genes by mutagens. The damage responsive elements (DRE) weight matrix generated in this analysis was used to search for homologous sequences in the promoter region of all genes, including putative gene and hypothetical open reading frames (ORFs), in the Saccharomyces Genome Data Base (SGD). The results demonstrated that over one third of the yeast genes in the database presented at least one 15-bp sequence in their promoter region with 85% or more of similarity to the DRE consensus sequence. The presence of the DRE sequence in the promoter region of regulatory genes and its high similarity to other well reported DNA binding sites points to its involvement in the general regulation of not only DNA repair genes but yeast genes in general

Genetic variability within Fusarium solani specie as revealed by PCR-fingerprinting based on pcr markers

O fungo Fusarium solani (teleomorfo Haematonectria haematococca) apresenta uma expressiva importância na agricultura por ser considerado patógeno para várias culturas de interesse econômico causando doença conhecida por podridão das raízes, além de ser patógeno aos animais e ao homem, provocando nestes últimos, micoses superficiais e sistêmicas. A complexidade associada a sua identificação correta através de métodos tradicionais justifica os esforços de usar marcadores moleculares para caracterização dos isolados. Neste trabalho, três métodos baseados na tecnologia da PCR (um por ribotipagem por PCR e dois por impressão genética por PCR) foram utilizados para investigar a variabilidade molecular de dezoito isolados de F. solani de quatro Estados brasileiros, coletados de diferentes substratos. A análise genética revelou a variabilidade intraespecífica dos isolados de F. solani, sem qualquer correlação para a origem geográfica e substrato. Seu polimorfismo foi observado até mesmo na seqüência conservada do locus do rDNA, e o marcador SPAR (GTG)5 mostrou o mais alto polimorfismo. Em conjunto, estes resultados poderão auxiliar nos estudos da relação entre variabilidade do perfil genético de isolados e os fenótipos de resistência de determinados cultivares às doenças provocadas pelo fungo, orientando programas de melhoramento vegetal.

The NADP+-dependent glutamate dehydrogenase of the yeast Kluyveromyces marxianus responds to nitrogen repression similarly to Saccharomyces cerevisiae

Glutamato desidrogenase dependente de NADP+ (NADP+-Gdh) constitui o primeiro passo enzimático no mecanismo de assimilacão de nitrogênio em Saccharomyces cerevisiae e o conhecimento de sua regulacão é chave na iniciativa de vários propósitos biotecnológicos, tais como a producão de proteína microbiana. A regulacão da atividade NADP+-Gdh em células de Kluyveromyces marxianus foi avaliada a partir de diferentes condicões de suprimento de amonia em cultivo em batelada. Os resultados mostraram que a atividade NADP+-Gdh de K. marxianus foi induzida em uma estreita faixa de concentracão de amonia no meio, sendo reprimida tanto por altas concentracões deste composto quanto pelo produto glutamato. Esta atividade não está associada ao crescimento celular e deve funcionar principalmente no rastreamento de pequenas quantidades de amonia após a parada do crescimento celular. Isto demonstra que NADP+-Gdh não deve ser a principal enzima de assimilacão de amonia em K. marxianus, como tem sido postulado para K. lactis, contudo deve estar submetida ao mesmo mecanismo regulatório encontrado em S. cerevisiae.

Expression of the bacterial recA gene impairs genetic recombination and sporulation in a Saccharomyces cerevisiae diploid strain

The Escherichia coli RecA protein (RecAp) has been demonstrated to induce mutagenesis in yeast cells, although there is still little information on the role of the RecAp in yeast recombination events. We evaluated spontaneous and induced general recombination in vegetative and meiotic cells of the XS2316 strain of the yeast Saccharomyces cerevisiae bearing the recA gene. We found that RecAp decreased reciprocal recombination, gene conversion and intrachromosomal recombination and promoted an increase in error-prone processes in both vegetative and meiotic cells, while its negative effect on meiotic recombination blocked ascospore formation

Production of extracellular lipase by the phytopathogenic fungus "Fusarium solani" FS1

A Brazilian strain of "Fusarium solani" was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.L(E-1) of lipase after 72 hours of cultivation at 25ºC in shake-flask at 120rpm in a medium containing 3(per cent) (w/v) peptone plus 0.5(per cent)(v/v) olive oil. Glucose (1(per cent) w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3(per cent)(w/v) resulted in a reduced lipase production while increased olive oil concentration (above o.5(per cent)) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30ºC and a good enzyme stability (80(per cent) activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50ºC. Lipase activity was stimulated by addition of n-hexane to the culture medium supernatatnts, in contrast to incubation with water-soluble solvents