RESUMEN
During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to 3 different clones retrieved from 3 chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 y or the more recent isolation date after 12.5 y of chronic infection. The infection models proved useful to assess virulence potential diversification, but for some isolates the relative values diverged in C. elegans and G. mellonella. Results also reinforce the concept of the occurrence of clonal diversification and co-existence of multiple phenotypes within the CF lungs, also with respect to pathogenicity. No clear trend of decrease (or increase) of the virulence potential throughout long-term infection was found but there is an apparent tendency for a clone/patient-dependent decrease of virulence when the G. mellonella model was used. The sole avirulent variant in both infection hosts was found to lack the small third replicon previously associated to virulence. Although possible, the in vivo loss of this nonessential megaplasmid was found to be a rare event (1 among a total of 64 isolates examined).
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/patogenicidad , Fibrosis Quística/microbiología , Pulmón/microbiología , Animales , Infecciones por Burkholderia/complicaciones , Burkholderia cenocepacia/genética , Caenorhabditis elegans/microbiología , Enfermedad Crónica , Fibrosis Quística/complicaciones , Modelos Animales de Enfermedad , Genotipo , Humanos , Mariposas Nocturnas/microbiología , Fenotipo , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/microbiología , Estudios Retrospectivos , VirulenciaRESUMEN
Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF) patients. Bacteria of the Burkholderia cepacia complex are the most threatening pathogens in CF, and a better understanding of how these bacteria adapt to the CF airway environment and resist the host defense mechanisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies adopted by Burkholderia cenocepacia during long-term colonization, we carried out a phenotypic assessment of 11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient during 3.5 years of colonization of the lungs, until the patient's death with cepacia syndrome. Phenotypic characterization included susceptibility assays against different classes of antimicrobials and characterization of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition, growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed. The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However, the values for antimicrobial susceptibility and swarming motility for the first B. cenocepacia isolate, thought to have initiated the infection, were consistently above those for the clonal variants obtained during the course of infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization, described here for the first time, involved the alteration of membrane fatty acid composition, in particular a reduction of the degree of fatty acid saturation, in the B. cenocepacia variants retrieved, along with the deterioration of pulmonary function and severe oxygen limitation.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia , Fibrosis Quística/microbiología , Pulmón/microbiología , Antibacterianos/farmacología , Secuencia de Bases , Infecciones por Burkholderia/fisiopatología , Burkholderia cenocepacia/efectos de los fármacos , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/aislamiento & purificación , Burkholderia cenocepacia/fisiología , Membrana Celular/química , Fibrosis Quística/fisiopatología , Ácidos Grasos/análisis , Humanos , Enfermedades Pulmonares/microbiología , Lípidos de la Membrana/análisis , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenotipo , Polimorfismo GenéticoRESUMEN
Chronic respiratory infections caused by Burkholderia cenocepacia in patients with cystic fibrosis (CF) are characterized by low responsiveness to antibiotic therapy and, in general, to a more rapid decline of lung function. To get clues into the molecular mechanisms underlying the adaptive strategies employed to deal with the stressing conditions of the CF lung including antibiotic therapy, quantitative proteomics (2-D DIGE) was used to compare the expression programs of two clonal isolates retrieved from a chronically infected CF patient. Isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after 3 years of persistent infection and intravenous therapy with ceftazidime/gentamicin. This isolate exhibits higher resistance levels towards different classes of antimicrobials. Proteins of the functional categories Energy metabolism, Translation, Nucleotide synthesis, Protein folding and stabilization are more abundant in IST4113, compared with IST439, suggesting an increased protein synthesis, DNA repair and stress resistance in IST4113. The level of proteins involved in peptidoglycan, membrane lipids and lipopolysaccharide synthesis is also altered and proteins involved in iron binding and transport are more abundant in IST4113. The quantitative comparison of the two proteomes suggests a genetic adaptation leading to increased antimicrobial resistance and bacterial persistence in the CF airways.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/efectos de los fármacos , Burkholderia cenocepacia/genética , Proteómica , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/fisiopatología , Burkholderia cenocepacia/aislamiento & purificación , Burkholderia cenocepacia/metabolismo , Ceftazidima/administración & dosificación , Ceftazidima/uso terapéutico , Técnicas de Cultivo de Célula , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Electroforesis en Gel Bidimensional , Expresión Génica/efectos de los fármacos , Gentamicinas/administración & dosificación , Gentamicinas/uso terapéutico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Pruebas de Sensibilidad Microbiana , Sistema Respiratorio/microbiología , Sistema Respiratorio/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Burkholderia cepacia, a species found infrequently in cystic fibrosis (CF), was isolated from 85% of patients infected with bacteria of the B. cepacia complex that visited the major Portuguese CF center, in Lisbon, during 2003 to 2005. A detailed molecular analysis revealed that this was mainly due to two B. cepacia clones. These clones were indistinguishable from two strains isolated from intrinsically contaminated nonsterile saline solutions for nasal application, detected during routine market surveillance by the Portuguese Medicines and Health Products Authority.
Asunto(s)
Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Fibrosis Quística/epidemiología , Infecciones por Burkholderia/complicaciones , Humanos , Vigilancia de la Población , Portugal/epidemiología , Factores de TiempoRESUMEN
The E6 protein of human papillomavirus type 16 is essential for the oncogenic transformation process induced by these viruses. Here we expressed the E6 protein in Saccharomyces cerevisiae (which lacks p53) in order to determine if E6 interacts with normal cell functioning, independently of the p53 tumour suppressor factor. We observed a higher resistance to caffeine, hydrogen peroxide and to pheromone, but not to high temperature, starvation and osmostress. Measurement of the relative expression levels of target genes of the signalling pathways, involved in the latter stressful stimuli, led us to conclude that such pathways are differently regulated in the presence of E6.