Helicobacter pylori antimicrobial resistance rates in the central region of Portugal.

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal.

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus.

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Toxic effects of tamoxifen on the growth and respiratory activity of Bacillus stearothermophilus.

The anticancer drug tamoxifen (TAM) is used as first line therapy in breast cancer. Although tamoxifen is usually considered an estrogen antagonist, several studies suggest alternative mechanisms of action. Bacillus stearothermophilus has been used as a model to clarify the antiproliferative action of tamoxifen putatively related with drug-membrane interaction. According to previous data, TAM induces perturbation of membrane structure along with impairment of bacterial growth. The aim of this work was to correlate the effects of TAM on growth of intact B. stearothermophilus with the respiratory activity of isolated protoplasts of this bacteria. TAM inhibits bacterial growth and oxygen consumption of protoplasts as a function of concentration. Effects on oxygen consumption depend on the substrate used: NADH, allowing to study the full respiratory chain and ascorbate-TMPD to probe the final oxidase segment. The interaction of TAM with the respiratory components occurs at a level preceding the cytochrome oxidase segment.

Toxicity assessment of tamoxifen by means of a bacterial model.

A strain of Bacillus stearothermophilus was used as a model to study physical perturbations induced in the membrane by the cytostatic tamoxifen (TAM). This study was carried out using two lines of criteria: (1) bacterial growth, and temperature growth range, with determination of growth parameters as a function of TAM concentration; and (2) biophysical studies by differential scanning calorimetry (DSC) and by means of two fluorescent probes to evaluate perturbations promoted by the drug on the structural order of bacterial lipid membranes. The inhibition of growth induced by TAM, the structural bilayer disordering, and the shift in the phase transition temperature to a lower range were also determined in the presence of Ca2+, i.e., a natural membrane stabilizer, to elucidate further perturbing effects of TAM on membranes with putative implications in cell toxicity. Growth inhibition promoted by TAM is potentiated by an increase in growth temperature above the optimal range, but attenuated or relieved by the addition of 2.5 mM Ca2+ to the culture medium. Consistently, fluorescence polarization and DSC studies showed that Ca2+ ions (2.5 mM) effectively compensated for the destabilizing effects promoted by TAM in bacterial lipid membranes.

Cytotoxicity and membrane interaction of tamoxifen as affected by ca(2+) and mg(2+): use of a bacterial model system.

A strain of Bacillus stearothermophilus was used as a model to study the interaction of tamoxifen (TAM) with the membrane and the cytostatic antiproliferative effects not related to estrogen binding. TAM inhibits the growth of B. stearothermophilus as a function of concentration. The supplementation of the growth medium with Ca(2+) or Mg(2+) partially relieves the growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its propionic acid derivative (DPH-PA) reveals opposite effects induced by TAM and Ca(2+). The addition of Ca(2+) to liposomes of bacterial lipids promoted physical ordering as opposed to disordering induced by TAM. Thus, it is predictable that growth impairment induced by TAM is mediated through perturbations at the membrane level.

Lipid composition changes induced by tamoxifen in a bacterial model system.

A putative relationship between growth impairment of Bacillus stearothermophilus by tamoxifen (TAM) and TAM-induced perturbation of the physical properties of bacterial membrane lipids has been observed. The supplementation of the growth medium with Ca2+ (a membrane stabilizer) partially relieves growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium. B. stearothermophilus modifies the membrane lipid composition in response to the addition of TAM to the growth medium and the response is sensitive to Ca2+. Changes in lipid composition are observed in the acyl chains and in the polar head groups of phospholipids. The physical effects of alteration in these lipids was studied by fluorescence polarization of DPH and DPH-PA. Polar lipid dispersions from TAM-adapted cells grown in a Ca2+ medium show a shift of Tm to higher temperatures and a significant increase of the structural order as compared to lipids from control cells, suggesting that TAM-induced lipid composition changes compensate for the destabilizing effects of the cytostatic on membrane organization. The polar lipids from cells grown in the basal medium containing tamoxifen are also altered, but these alterations do not promote order increase of the bilayer in spite of a deviation of Tm to higher temperatures as detected by DPH. Data indicate that B. stearothermophilus controls the membrane lipid composition in response to tamoxifen, to compensate for TAM-promoted disordering in membranes and to provide an appropriate packing of phospholipid molecules in a stable bilayer, putatively disturbed by TAM incorporation.

Use ofBacillus stearothermophilus as a model to study tamoxifen-membrane interactions.

A strain ofBacillus stearothermophilus was used as a model to study the interaction of tamoxifen (TAM) with the cell membrane and the cytostatic antiproliferative effects not related to oestrogen binding. The bacterial growth in the presence of TAM was evaluated turbidimetrically and by viable cell counting. In parallel, partition coefficients of TAM in bacterial polar lipid bilayers were determined. Additionally, studies with fluorescent probes were carried out to investigate TAM effects on the physical state of the membrane lipid bilayer. TAM inhibits growth ofB. stearothermophilus and induces loss of cell viability as a function of concentration and the growth temperature. High partitioning of this drug in the bacterial lipid membranes was observed, reaching maximal values in the temperature range of the phase transition. Fluorescence polarizations of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its propionic acid derivative (DPH-PA) report significant structural disorder of the lipid bilayer induced by the cytostatic, particularly in the phase transition range. A putative relationship between growth impairment by TAM and the TAM-induced perturbation of the physical behaviour of bacterial membrane lipids is suggested.