RESUMEN
Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Bacteroides fragilis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Proteínas Portadoras/aislamiento & purificación , Membrana Celular/efectos de los fármacos , Estrés Fisiológico/genética , Adaptación Fisiológica , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/química , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/química , Medios de Cultivo/química , Expresión Génica , Ontología de Genes , Anotación de Secuencia Molecular , Proteómica/métodosRESUMEN
As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/crecimiento & desarrollo , Animales , Infecciones por Bacteroides/genética , Infecciones por Bacteroides/metabolismo , Bacteroides fragilis/patogenicidad , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/efectos de los fármacosRESUMEN
The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs ≥ 4 µg/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 µg/ml). The inhibition by EDTA was a good predictor of the presence of metallo-ß-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.
Asunto(s)
Antibacterianos/administración & dosificación , Infecciones por Bacteroides/tratamiento farmacológico , Bacteroides fragilis/efectos de los fármacos , Bacteroides/efectos de los fármacos , Carbapenémicos/administración & dosificación , Resistencia betalactámica/genética , Argentina/epidemiología , Proteínas Bacterianas/genética , Bacteroides/crecimiento & desarrollo , Bacteroides/aislamiento & purificación , Infecciones por Bacteroides/epidemiología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/crecimiento & desarrollo , Bacteroides fragilis/aislamiento & purificación , Líquidos Corporales/microbiología , Estudios Transversales , Ácido Edético/farmacología , Femenino , Humanos , Estudios Longitudinales , Masculino , Pruebas de Sensibilidad Microbiana , Vigilancia de la Población , Resistencia betalactámica/efectos de los fármacos , beta-Lactamasas/genéticaRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Asunto(s)
Adhesión Bacteriana , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Laminina/metabolismo , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestructura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Polisacáridos Bacterianos/metabolismo , Factores de TiempoRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Asunto(s)
Adhesión Bacteriana , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Laminina/metabolismo , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestructura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Polisacáridos Bacterianos/metabolismo , Factores de TiempoRESUMEN
The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.
Asunto(s)
Antibacterianos/farmacología , Bacteroides fragilis/efectos de los fármacos , Clindamicina/farmacología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/citología , Bacteroides fragilis/genética , Bacteroides fragilis/crecimiento & desarrollo , Niño , Fragmentación del ADN , ADN Bacteriano/genética , Diarrea/microbiología , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Bacteroides fragilis strains isolated from different sources, i.e. 1 strain (AA1) from an aquatic environment, 1 strain from normal flora (118310) and the type strain (ATCC 25285) originally isolated from clinical material, were analysed for both cell envelope proteins composition and surviving under oxidative stress starvation. All strains examined showed a similar survival response when cultured in drinking water with a ten-fold decrease in viable counts per day during the 7 days of analysis. The outer membrane protein (OMP) profiles of all strains were quite similar during the stress period as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the periplasmic proteins of the strain 118310 showed two protein bands at 48 and 58 kDa, respectively, that were absent in the strains AA1 and ATCC 25285 during the incubation period in potable water. Whole cells and periplasmic 35S-labelled proteins from bacteria cultured in drinking water showed a significant increase in proteins at 16, 18, 24, 26, 35, 48, and 58 kDa and 18, 22, 24, 48, 58, and 70 kDa, respectively, in all strains when compared to cells grown in BHI-PRAS media as detected by autoradiography following SDS-PAGE. These data suggest that B. fragilis may have a synthesis mechanism that allows them to adapt to adverse environments.
Asunto(s)
Bacteroides fragilis/química , Bacteroides fragilis/crecimiento & desarrollo , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas Bacterianas/análisis , HumanosRESUMEN
The ability of strains of the B. fragilis group, isolated from the oral cavity and intestine of marmosets, to produce bacteriorin-like substances in solid medium, in terms of auto-, iso- and heteroantagonism, was evaluated. Antagonistic activity was exhibited by 52% of the intestinal strains, 3 of which showed autoantagonistic activity. Three out of 9 oral strains isolated, tested against themselves, showed simultaneous isoantagonism to 4 indicator strains; but not autoantagonism. The same 9 oral strains, when tested against 16 reference strains, revealed interspecific activity only against 2 Gram-positive microorganisms. Higher activity, evaluated by the size of the inhibition halo, was observed in BHI-S agar, and greatest inhibition was obtained after 72 h of incubation.
Asunto(s)
Antibiosis/fisiología , Bacteriocinas/biosíntesis , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/metabolismo , Callithrix/microbiología , Animales , Bacteroides fragilis/crecimiento & desarrollo , Bacteroides fragilis/aislamiento & purificación , Medios de Cultivo , Técnicas In Vitro , Enfermedades Intestinales/microbiología , Enfermedades de los Monos/microbiología , Enfermedades de la Boca/microbiologíaRESUMEN
The effects of subinhibitory concentrations of metronidazole on the general morphology, ultrastructure and charge and hydrophobicity of Bacteroides fragilis were investigated using transmission and scanning electron microscopy, and measurements of zeta-potentials and contact angles. Metronidazole treatment induced morphological alterations in B. fragilis. Cells became filamentous and showed a significant loss of cytoplasm. The surface anionogenicity and hydrophobicity of B. fragilis were enhanced by the drug treatment. Metronidazole, by modifying B. fragilis surface properties, could alter, indirectly, interactions with host cells.
Asunto(s)
Bacteroides fragilis/efectos de los fármacos , Metronidazol/farmacología , Bacteroides fragilis/crecimiento & desarrollo , Bacteroides fragilis/ultraestructura , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Propiedades de Superficie/efectos de los fármacosRESUMEN
The effects of subinhibitory concentrations of metronidazole, chloramphenicol, clindamycin and penicillin G on cell growth and neuraminidase activity of Bacteroides fragilis were tested in six strains from clinical specimens and two type collection strains. All showed significant inhibition of cell growth at one half the MIC after 24 h incubation. Metronidazole at one quarter the MIC was significantly inhibitory after 24 and 48 h incubation. The neuraminidase activity remained unaltered, in comparison with controls, when metronidazole and clindamycin were tested. Chloramphenicol reduced the enzyme activity at both one half and one quarter the MIC. On the other hand, penicillin G enhanced the neuraminidase activity, although the difference was not statistically significant. These in-vitro effects may simulate the behaviour of the bacteria in abscesses in vivo.
Asunto(s)
Bacteroides fragilis/crecimiento & desarrollo , Cloranfenicol/farmacología , Clindamicina/farmacología , Metronidazol/farmacología , Neuraminidasa/metabolismo , Penicilina G/farmacología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/enzimología , Recuento de Colonia Microbiana , Pruebas de Sensibilidad Microbiana , Neuraminidasa/biosíntesisRESUMEN
Stool samples from 196 healthy infants less than ten days of age were cultured to delineate bacterial colonization patterns of the intestine. By one week of age, isolation rates of Bacteroides fragilis and other anaerobic bacteria in term infants following vaginal delivery and formula feeding approach that of adults. Differences in gestational age, type of delivery, and type of feeding are associated with significantly different colonization patterns of anaerobic and aerobic bacteria in the first week after birth.