Cholesterol and ergosterol affect the activity of Staphylococcus aureus antibiotic efflux pumps.

The aim of this study is to evaluate the effect of ergosterol on steroids and cholesterol efflux pumps in multidrug resistant strains of S. aureus. Were used RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein; and IS-58, which possesses the TetK tetracycline efflux protein; 1199B resists hydrophilic fluoroquinolones via a NorA-mediated mechanism and wild strain 1199B. The Minimal Inhibitory Concentration (MIC) was determined and the evaluation of possible inhibition of efflux pumps by reduction of MIC. Some of the detrimental effects on bacterial cells can be attributed to the detergent properties of cholesterol and ergosterol on account of their amphipathic structure. Besides the cholesterol did not affect directly the pump structure, a synergism was observed, maybe due the interaction with the cell membrane and interference in the lipid bilayer.

Novel DNA extraction assay for molecular identification of Aedes spp eggs.

Aedes aegypti and A. albopictus represent the two most important species of mosquitoes in relation to dengue virus transmission both in the Americas and Asia. However, the study of theses species generally requires the establishment of a colony for the larvae to hatch, or waiting for the adult development to perform its taxonomic classification, which is time consuming. Thus, the establishment of new methods aimed at obtaining DNA directly from the mosquito eggs is relevant. Accordingly, we compared a new approach based on Chelex(®) 100 resin with the standard STE method to extract DNA from the eggs of Aedes spp to molecularly identify these vectors. The Chelex(®) 100 resin approach was very efficient, as satisfactory amounts of DNA were obtained, making it possible to amplify and sequence a mitochondrial DNA barcode region widely used to identify species. The STE protocol yielded substantial amounts of DNA, but the 260/280 optical density ratio indicated a low quality, precluding amplification. This new method proved quite effective in obtaining DNA from even a single mosquito egg, and it can thus be applied in population genetic studies of various vector insects to enhance monitoring programs.

Subtyping Brazilian Yersinia pestis strains by pulsed-field gel electrophoresis.

We subtyped Brazilian Yersinia pestis strains by pulsed-field gel electrophoresis (PFGE). This was done with 22 Brazilian Y. pestis strains: 17 from an outbreak and 5 from endemic routine surveillance. The strains were divided into 2 groups (I and II), 8 subgroups (A-H) and 19 PFGE profiles or pulsotypes. PFGE did not separate outbreak from non-outbreak strains, as identical pulsotype patterns were found among outbreak strains and strains obtained from surveillance. However, it was able to detect intraspecific genetic diversity among Brazilian strains. This PFGE technique was able to differentiate a homogeneous group of Brazilian Y. pestis strains.

Genetic diversity of Yersinia pestis in Brazil.

Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence.

Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil.

AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates. MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.