Sérgio Olavo Pinto da Costa

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

ABSTRACT Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

Induction of ϱ− mutants in Saccharomyces cerevisiae by guanidine hydrochloride: I. Genetic analysis

Guanidine hydrochloride (GuHCl) induced in Saccharomyces cerevisiae cytoplasmic petite mutants (rho-) of the suppressive type. However, it was unable to induce the neutral type, even after prolonged incubation or increased drug concentration. No correlation was found between the degree of suppressiveness and the time of incubation of yeast cells with guanidine hydrochloride. The suppressiveness of rho- induced was not altered by further treatment with GuHCl, whereas it was reduced upon treatment with ethidium bromide (EtBr). Some mitochondrial genetic information was lacking in the rho- mutants induced by GuHCl, as demonstrated by the loss of the gene for erythromycin resistance and by reduced buoyant density of mitochondrial DNA of some rho-. There was no correlation between the degree of suppressiveness of the rho- induced by GuHCl and the buoyant density of the mutant mitochondrial DNA.

Non-chromosomal respiratory deficient mutants induced by guanidine hydrochloride in Saccharomycescerevisiae

Abstract Non-chromosomal petites can be produced in Saccharomyces cerevisiae by treatment with guanidine hydrochloride, a protein denaturing agent. Its efficiency in inducing petite mutants is comparable to the action of ethidium bromide. The high frequency of petite mutants observed is due to an induction effect rather than to a selection of preexisting mutants. Induction of petites by guanidine hydrochloride occurs even in non growing conditions, indicating that even parental cells are transformed in petites. Transformation depends upon the physiological properties of the cells, since repressed cells, cultivated in the presence of glucose, are more easily transformed than cells cultivated in ethanol.

Total protein electrophoresis and RAPD fingerprinting analysis for the identification of Aeromonas at the species level

Fifteen well-defined strains of Aeromonas of thirteen species were analyzed by SDS protein electrophoretic analysis (SDS-PAGE) and random amplified polymorphic DNA analysis (RAPD). The comparison between the patterns obtained by both methods allowed differentiating all the strains. Clusters formed by the unweighted pair group method with arithmetic averages applied to protein data correlates with the genetic and biochemical information about the species. The results show that protein fingerprinting has the potential to differentiate Aeromonas species, but the low qualitative variation indicates that this technique is not efficient for the characterization of strains within a species. Conversely, RAPD fingerprinting allows the identification of strains but the high variability limits its potential as an aiding method for species identification.

Environmental and genetic factors affecting mutability to aminoglycoside antibiotics among Escherichia coli K12 strains

Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2) plates, when compared to results obtained in experiments carried out with Nutrient agar (NA) plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin). These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains.

Effect of osmolarity on aminoglycoside susceptibility in Gram‐negative bacteria

The susceptibility of strains of Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Proteus mirabilis and Escheriehia coli to six aminoglycosides was tested in media of different osmolarity and ionic content. We observed that increasing osmolarity decreased susceptibility of these Gram‐negative bacteria to all antibiotics used. On the other hand, raising of ionic strength increased the susceptibility to tobramycin, neomycin and gentarnicin in all bacteria tested.

The effect of organic osmoprotectors on Aeromonas trota and A. hydrophila grown under high sodium chloride concentrations

The effect of organic solutes on the growth of Aeromonas trota and A. hydrophila was evaluated. Proline and glutamic acid were not effective as osmoprotectors, but betaine exerts osmoprotection allowing the growth of both strains in inhibitory concentrations of NaCl. Growth kinetics suggests that the halotolerance difference between the strains is associated with the synthesis of osmolytes rather than betaine uptake.

OppA Escherichia coli mutants have osmodependent resistance to aminoglycoside antibiotics

The oligopeptide permease (OppA) protein was found to be missing in the periplasmic fractions of Escherichia coli kanamycin-resistant mutants selected under high osmotic conditions. The growth behavior of one mutant in media containing kanamycin or the toxic peptide triornithine suggests that OppA and another cell envelope component contribute to the osmolarity-dependent aminoglycoside resistance of E. Coli.

Spontaneous Kanamycin-resistant Escherichia coli mutant with altered periplasmic oligopeptide permease protein (OPPA) and impermeability to aminoglycosides

Um mutante espontâneo de Escherichia coli foi selecionado com canamicina e mostrou resistencia cruzada a cinco outros aminoglicosideos e ausencia da proteina OppA. A incorporacao de diidroestreptomicina tritiada mostrou-se reduzida nesse mutante, implicando que o sistema de transporte de oligopeptideos esta envolvido na acumulacao de aminoglicosideos, embora aparentemente nao esteja relacionado com a alteracao de permeabilidade aos aminoglicosideos decorrente da adaptacao bacteriana a mudancas osmoticas.

Cell envelope components of Yersinia pestis grown in intraperitoneal diffusion chambers

The electrophoretic profiles of penicillin binding proteins (PBPs) and outer membrane proteins (OMPs) of Yersinia pestis EV 76 were determined following in vivo growth in diffusion chambers implanted in the peritoneal cavity of mice. In contrast to Y. pestis grown under in vitro conditions which activate the low calcium response (LCR) regulon there was no significant qualitative or quantitative change of the PBP profile of Y. pestis cells during growth in diffusion chambers for up to 72 h following implantation in mice. Three OMPs, with molecular weight of 100, 60 and 58 kDa, were expressed in Y. pestis cells grown for 24 h, but not at 48 h or at 72 h, in diffusion chambers. These results indicate that growth of Y. pestis in intraperitoneal diffusion chambers activates genes which might be relevant to the growth in the mammal host.