RESUMO
Helicobacter pylori is a Gram negative bacterium most frequently associated with human gastrointestinal infections worldwide. The increasing occurrence of antibiotic-resistant isolates of H. pylori constitutes a challenge. The eradication of the microorganism is currently being considered a "high priority" by the World Health Organization (WHO). In this context, bioactive compounds found in natural products seem to be an effective therapeutic option to develop new antibiotics against the pathogen. In this study, we investigated the effect of asclepain cI, the main purified proteolytic enzyme of the latex of petioles and stems from Asclepia curassavica L. (Asclepiadaceae), a South American native plant, against H. pylori; in order to obtain a natural therapeutic adjuvant and a safe nutraceutical product. Asclepain cI showed antibacterial activity against reference strains and drug-resistant clinical isolates of H. pylori in vitro. A range of minimal inhibitory concentration (MIC) from 1 to 2 µg/ml and minimal bactericidal concentration (MBC) from 2 to 4 µg/ml was obtained, respectively. The action of asclepain cI on the transcription of omp18, ureA, flaA genes showed a significantly decreased expression of the selected pathogenic factors. Furthermore, asclepain cI did not induce toxic effects at the concentrations assayed. Asclepain cI could be considered a highly feasible option to be used as a natural therapeutic adjuvant and a safe nutraceutical product against H. pylori.
RESUMO
Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori, to obtain natural food additives for the production of anti-H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 µg/mL and MBC from 312.5 to 625 µg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 µg/mL and MBC from 625 to 1,250 µg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp18, ureA, and flaA, with respect to a housekeeping gene (16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp18, ureA, and flaA. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 µg), clarithromycin (CLA, 15 µg), levofloxacin (LEV, 5 µg), and metronidazole (MTZ, 5 µg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori.
RESUMO
Clarithromycin resistance is an important factor of eradication failure. A commercially available fluorescent in situ hybridization (FISH) kit (creaFAST) was used to detect H. pylori infection and the resistance to clarithromycin in frozen biopsies. A total of 33 biopsies, H. pylori culture-positive, obtained from pediatric patients were retrospectively studied. Clarithromycin resistance was compared with MICs detected by E-test from H. pylori clinical isolates. All culture-positive biopsies were positive by FISH. Detection of clarithromycin resistance showed sensitivity of 90%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 86.7% compared with results obtained by E-test. Discrepant results were 2 biopsies, clarithromycin-susceptible by FISH but intermediate by E-test. In conclusion, FISH technology is a rapid, easy-to-implement, and reliable cultivation-independent method for routine application; however, when frozen biopsies are studied, some modification of the recommended procedure should be used to obtain better results.
Assuntos
Antibacterianos/farmacologia , Biópsia , Claritromicina/farmacologia , Helicobacter pylori/efeitos dos fármacos , Hibridização in Situ Fluorescente/métodos , Estômago , Criança , Pré-Escolar , Farmacorresistência Bacteriana/genética , Congelamento , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Kit de Reagentes para DiagnósticoAssuntos
Proteínas da Membrana Bacteriana Externa/genética , Claritromicina/farmacologia , Farmacorresistência Bacteriana/genética , Helicobacter pylori/genética , Argentina , Proteínas de Bactérias/genética , Dispepsia/microbiologia , Gastrite/microbiologia , Genótipo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Úlcera Péptica/microbiologia , Mutação Puntual/genética , RNA Ribossômico 23S/genética , Neoplasias Gástricas/microbiologiaRESUMO
Solid and liquid media supplemented only with a cyanobacterial extract (CE) and free of fetal calf serum (FCS), blood, and its derivatives support the growth of Helicobacter pylori. A total of 11 strains of H. pylori isolated from gastric biopsy samples were successfully subcultured in Mueller-Hinton agar supplemented with 0.4% CE. When this medium was used for primary isolation of H. pylori, a low isolation rate (30%) was observed because of the abundant growth of contaminants. The growth kinetics of eight isolates and H. pylori reference strain NCTC 11638 in Mueller-Hinton broth (MHB) supplemented with 0.7% CE were estimated by use of growth parameters, and the results were compared with those obtained with MHB-5% FCS. For four strains the cellular concentrations obtained with CE were statistically higher (P < 0.05) than those obtained with FCS, and in some cases these values were similar to the highest values reported in the literature. Depending on the strain, the specific growth rates obtained with CE were similar to or increased compared with those obtained with FCS. The replacement of FCS by CE in H. pylori cultures would facilitate the retrieval of cultures with high cellular densities as a source of cellular and extracellular proteins free of serum. Also, CE has advantages over conventional supplements, such as easier conservation and compliance with the pressing tendency at present to avoid the use of products derived from animals.