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1.
Proc Natl Acad Sci U S A ; 114(25): 6557-6562, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28584102

RESUMEN

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including ß-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.


Asunto(s)
Complejo Burkholderia cepacia/química , Proteínas de Transporte de Membrana/química , Cristalografía por Rayos X/métodos , Periplasma/química , Factores de Virulencia/química
2.
mBio ; 7(6)2016 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-27879334

RESUMEN

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread. IMPORTANCE: Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.


Asunto(s)
Antiinfecciosos Locales/farmacología , Compuestos de Benzalconio/farmacología , Complejo Burkholderia cepacia/efectos de los fármacos , Farmacorresistencia Bacteriana , Antiinfecciosos Locales/metabolismo , Proteínas Bacterianas/análisis , Compuestos de Benzalconio/metabolismo , Biotransformación , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/metabolismo , Complejo Burkholderia cepacia/fisiología , Perfilación de la Expresión Génica , Viabilidad Microbiana/efectos de los fármacos , Proteoma/análisis
3.
Int J Antimicrob Agents ; 48(5): 521-527, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27665523

RESUMEN

The Burkholderia cepacia complex (Bcc) is notorious for the life-threatening pulmonary infections it causes in patients with cystic fibrosis. The multidrug-resistant nature of Bcc and differing infective Bcc species make the design of appropriate treatment regimens challenging. Previous synergy studies have failed to take account of the species of Bcc isolates. Etest methodology was used to facilitate minimum inhibitory concentration (MIC) and antimicrobial combination testing on 258 isolates of Bcc, identified to species level by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The most active antimicrobials were trimethoprim/sulphamethoxazole, doxycycline and minocycline (52.5%, 46.4% and 45.9% of isolates susceptible, respectively). Synergy was observed in 9.2% of the 1799 combinations tested; the most common synergistic combinations were tobramycin + ceftazidime, meropenem + tobramycin and levofloxacin + piperacillin/tazobactam (35.4%, 32.3% and 22.2% synergy, respectively). Antimicrobial susceptibility analysis revealed differences between Burkholderia cenocepacia and Burkholderia multivorans. Disparity in clinical outcome during infection with these two micro-organisms necessitates further investigation into the clinical outcomes of treatment regimens in light of species identification and in vitro antimicrobial susceptibility studies.


Asunto(s)
Antibacterianos/farmacología , Complejo Burkholderia cepacia/efectos de los fármacos , Interacciones Farmacológicas , Pruebas de Sensibilidad Microbiana/métodos , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/aislamiento & purificación , Femenino , Humanos , Masculino , Estudios Retrospectivos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto Joven
4.
Mar Drugs ; 14(5)2016 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-27128927

RESUMEN

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 µg/mL) and unreported antimicrobial activity against Bcc strains.


Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Lípidos/química , Lípidos/farmacología , Regiones Antárticas , Arthrobacter/química , Arthrobacter/genética , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/genética , Genes Bacterianos/genética , Filogenia , Pseudomonas/química , Pseudomonas/genética , Psychrobacter/química , Psychrobacter/genética , ARN Ribosómico 16S/genética
5.
Environ Microbiol Rep ; 7(2): 180-7, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25294803

RESUMEN

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacilli that are ubiquitous in the environment and have emerged over the past 30 years as opportunistic pathogens in immunocompromised populations, specifically individuals with cystic fibrosis (CF) and chronic granulomatous disease. This complex of at least 18 distinct species is phenotypically and genetically diverse. One phenotype observed in a subset of Burkholderia cenocepacia (a prominent Bcc pathogen) isolates is the ability to produce a melanin-like pigment. Melanins have antioxidant properties and have been shown to act as virulence factors allowing pathogens to resist killing by the host immune system. The melanin-like pigment expressed by B. cenocepacia is produced through tyrosine catabolism, specifically through the autoxidation and polymerization of homogentisate. Burkholderia cenocepacia J2315 is a CF clinical isolate that displays a pigmented phenotype when grown under normal laboratory conditions. We examined the amino acid sequences of critical enzymes in the melanin synthesis pathway in pigmented and non-pigmented Bcc isolates, and found that an amino acid substitution of glycine for arginine at amino acid 378 in homogentisate 1,2-dioxygenase correlated with pigment production; we identify this as one mechanism for expression of pigment in Bcc isolates.


Asunto(s)
Sustitución de Aminoácidos , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/enzimología , Homogentisato 1,2-Dioxigenasa/genética , Homogentisato 1,2-Dioxigenasa/metabolismo , Pigmentos Biológicos/análisis , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/crecimiento & desarrollo , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo
6.
J Clin Microbiol ; 51(11): 3849-51, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23966502

RESUMEN

Biofilm cultures of Burkholderia cepacia complex (BCC) infection have been found to generate the nonvolatile cyanide ion. We investigated if gaseous hydrogen cyanide (HCN) was a marker of BCC infection. Selected ion flow tube mass spectrometry analysis showed HCN was not elevated in the headspace of planktonic or biofilm cultures or in the exhaled breath of adult cystic fibrosis patients with chronic BCC infection. HCN is therefore not an in vitro or in vivo marker of BCC.


Asunto(s)
Biomarcadores/análisis , Infecciones por Burkholderia/diagnóstico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/aislamiento & purificación , Cianuro de Hidrógeno/análisis , Adulto , Pruebas Respiratorias/métodos , Fibrosis Quística/complicaciones , Femenino , Humanos , Masculino , Espectrometría de Masas
7.
Diagn Microbiol Infect Dis ; 77(2): 126-8, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23891221

RESUMEN

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) for identification of Bcc species compared with that of recA sequencing. MALDI-ToF was able of identifying 100% of Bcc isolates at the genus level, but 23.1% of Bcc isolates tested were not correctly identified at the species level. The misidentification occurred most frequently with Burkholderia contaminans (100%) and B. cepacia (33.3%).


Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Complejo Burkholderia cepacia/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/aislamiento & purificación , ADN Bacteriano/genética , Humanos , Reacción en Cadena de la Polimerasa , Rec A Recombinasas/genética , Análisis de Secuencia de ADN
8.
J Microbiol Methods ; 92(2): 145-9, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23201483

RESUMEN

The aim of this study was to establish the identification ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for bacteria of Burkholderia cepacia complex (Bcc) and to compare these results with those obtained by a molecular method (PCR-RFLP). A total of 57 isolates was used in the study. Isolates were collected from 31 patients attending the Regional Cystic Fibrosis Unit from January 2001 to December 2005. For phenotypic identification, both automated and manual systems were used. Using mass spectrometry, we identified all 57 isolates, previously identified by molecular method. Of these, 28 isolates were identified as B. cenocepacia, although not differentiated further into lineages. Moreover, other isolates were identified as B. cepacia (12 isolates), B. stabilis (12 isolates), and B. vietnamiensis (5 isolates). Our data indicate a good correlation between the two approaches.


Asunto(s)
Técnicas Bacteriológicas/métodos , Infecciones por Burkholderia/diagnóstico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adulto , Automatización/métodos , Complejo Burkholderia cepacia/química , Femenino , Humanos , Masculino , Factores de Tiempo
9.
J Clin Microbiol ; 48(11): 4186-92, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20810766

RESUMEN

Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infections with a number of bacterial pathogens. Among them, the Burkholderia cepacia complex (Bcc) bacteria, consisting of nine related species, have emerged as problematic CF pathogens due to their antibiotic resistance, incidence of nosocomial infection, and person-to-person transmission. Bcc organisms present the clinical microbiologist with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that reliably distinguish among these organisms. The complex taxonomy of the Bcc species colonizing the CF respiratory tract makes accurate identification problematic. Despite the clinical implications of Bcc identification, a clinical laboratory differentiation of species within the Bcc is lacking. Additionally, no commercial assays are available to further identify the Bcc species. In the current study, secretory proteins present in the cultured supernatants of Burkholderia cenocepacia and Burkholderia multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis. In total, 341 protein spots were detected, of which 23 were unique to each species, demonstrating that potential diagnostic candidates between these two members of the Bcc exist.


Asunto(s)
Proteínas Bacterianas/análisis , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/clasificación , Proteínas Bacterianas/metabolismo , Técnicas de Tipificación Bacteriana/métodos , Complejo Burkholderia cepacia/metabolismo , Medios de Cultivo/química , Electroforesis en Gel Bidimensional , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
10.
Microbiology (Reading) ; 156(Pt 4): 1084-1096, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20019083

RESUMEN

Members of the Burkholderia cepacia complex (Bcc) are respiratory pathogens in patients with cystic fibrosis (CF). Close repetitive DNA sequences often associate with surface antigens to promote genetic variability in pathogenic bacteria. The genome of Burkholderia cenocepacia J2315, a CF isolate belonging to the epidemic lineage Edinburgh-Toronto (ET-12), was analysed for the presence of close repetitive DNA sequences. Among the 422 DNA close repeats, 45 genes potentially involved in virulence were identified and grouped into 12 classes; of these, 13 genes were included in the antigens class. Two trimeric autotransporter adhesins (TAA) among the 13 putative antigens are absent from the other Burkholderia genomes and are clustered downstream of the cci island that is a marker for transmissible B. cenocepacia strains. This cluster contains four adhesins, one outer-membrane protein, one sensor histidine kinase and two transcriptional regulators. By using PCR, we analysed three genes among 47 Bcc isolates to determine whether the cluster was conserved. These three genes were present in the isolates of the ET-12 lineage but absent in all the other members. Furthermore, the BCAM0224 gene was exclusively detected in this epidemic lineage and may serve as a valuable new addition to the field of Bcc diagnostics. The BCAM0224 gene encodes a putative TAA that demonstrates adhesive properties to the extracellular matrix protein collagen type I. Quantitative real-time PCR analysis indicated that BCAM0224 gene expression occurred preferentially for cells grown under high osmolarity, oxygen-limited conditions and oxidative stress. Inactivation of BCAM0224 in B. cenocepacia attenuates the ability of the mutant to promote cell adherence in vitro and impairs the overall bacterial virulence against Galleria mellonella as a model of infection. Together, our data show that BCAM0224 from B. cenocepacia J2315 represents a new collagen-binding TAA with no bacterial orthologues which has an important role in cellular adhesion and virulence.


Asunto(s)
Adhesinas Bacterianas/genética , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/microbiología , Genoma Bacteriano , Secuencias Repetidas en Tándem , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/clasificación , Estudio de Asociación del Genoma Completo , Humanos , Estructura Terciaria de Proteína
11.
Anal Bioanal Chem ; 394(8): 2161-71, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19579020

RESUMEN

A methodology for the discrimination of Burkholderia cepacia complex (Bcc) clinical isolates at the species level and at the ribopattern level using Fourier transform infrared (FTIR) spectroscopy and chemometrics analysis was assessed in this study. Different Bcc sequential isolates collected at the Santa Maria Hospital (HSM), in Portugal, from clinically infected cystic fibrosis (CF) patients were previously classified by established molecular methods at the species level and differentiated at the strain level, based on their ribopatterns. A set of 185 of these isolates, representing four different Bcc species (Burkholderia cepacia, Burkholderia cenocepacia (recA lineages III-A and III-B), Burkholderia multivorans and Burkholderia stabilis), was analyzed by FTIR and results were processed with chemometric methods. Ten reference strains of these species were used to test the FTIR method. The discrimination at the species level led to misclassification error rates of 10% and 32% for the HSM isolates and reference strains, respectively, clearly indicating that the FTIR classification method was unable to generalize results for the reference strains. Infrared spectra of HSM isolates were further analyzed in terms of the discrimination according to the ribopattern. Results showed misclassification error rates of 4%, 2%, and 8% for B. cepacia, B. cenocepacia III-A, and B. cenocepacia III-B ribopatterns, respectively. These results demonstrated good FTIR spectroscopy discrimination capacity at the ribopattern level, for the HSM isolates but showed difficulty at the species level, especially when the reference strains were included. Remarkably, this methodology was found to discriminate isolates belonging to the same species and ribopattern that were collected from the same patient during prolonged colonization, opening the door to the identification of chemical modifications resulting from adaptation strategies to the CF lung stressing environment, in particular to aggressive and prolonged antibiotic therapy.


Asunto(s)
Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/clasificación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/microbiología , Humanos , Análisis de Componente Principal , Ribotipificación
12.
Mol Microbiol ; 72(5): 1137-46, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19400793

RESUMEN

The interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an alpha-helical conformation interact with exopolysaccharides through electrostatic and other non-covalent interactions.


Asunto(s)
Catelicidinas/antagonistas & inhibidores , Polisacáridos Bacterianos/farmacología , Animales , Complejo Burkholderia cepacia/química , Dicroismo Circular , Fibrosis Quística/microbiología , Escherichia coli/efectos de los fármacos , Haplorrinos , Humanos , Pruebas de Sensibilidad Microbiana , Microscopía de Fuerza Atómica , Conformación Molecular , Pongo pygmaeus , Pseudomonas aeruginosa/química , Rhodospirillaceae/química , Espectrometría de Fluorescencia
13.
Microb Pathog ; 45(5-6): 331-6, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18771721

RESUMEN

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.


Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Complejo Burkholderia cepacia/fisiología , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/genética , Caenorhabditis elegans , Evaluación Preclínica de Medicamentos , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Alineación de Secuencia
14.
Innate Immun ; 14(3): 127-44, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18562572

RESUMEN

The Burkholderia cepacia complex comprises 10 closely related Gram-negative organisms all of which appear capable of causing disease in humans. These organisms appear of particular relevance to patients with cystic fibrosis. Lipopolysaccharide (LPS) is an important virulence determinant in Gram-negative pathogens. In this review, we highlight important data within the field commenting on LPS/lipid A structure-to-function relationships and cytokine induction capacity of Burkholderia strains studied so far.


Asunto(s)
Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Animales , Infecciones por Burkholderia/inmunología , Infecciones por Burkholderia/metabolismo , Complejo Burkholderia cepacia/efectos de los fármacos , Complejo Burkholderia cepacia/patogenicidad , Defensinas/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Humanos , Transducción de Señal
15.
Carbohydr Res ; 343(1): 81-9, 2008 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-18028888

RESUMEN

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.


Asunto(s)
Complejo Burkholderia cepacia/química , Polisacáridos Bacterianos/química , Biopelículas , Conformación de Carbohidratos , Dimetilsulfóxido , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Viscosidad , Agua
16.
Infect Immun ; 75(6): 2740-52, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17296759

RESUMEN

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Amantadina/análogos & derivados , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Infecciones por Burkholderia/prevención & control , Complejo Burkholderia cepacia/química , Dipéptidos/administración & dosificación , Enfermedades Pulmonares/prevención & control , Administración Intranasal , Amantadina/administración & dosificación , Amantadina/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Infecciones por Burkholderia/inmunología , Dipéptidos/inmunología , Modelos Animales de Enfermedad , Inmunización , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/microbiología , Ratones , Ratones Endogámicos BALB C
17.
Cell Microbiol ; 9(2): 532-43, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17002785

RESUMEN

In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with this decline but little is known about the molecular pathogenesis of Bcc LPS. In this study we have investigated the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed. The inflammatory response (TNFalpha, IL-6) was measured in human MonoMac 6 monocytes and inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized. LPS from all clinical Bcc isolates induced significant pro-inflammatory cytokines and utilized TLR4 and CD14 to mediate activation of mitogen-activated protein kinase pathways, IkappaB-alpha degradation and NFkappaB activation. However, LPS from different clinical isolates of the same clonal strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. LPS from clinical isolates of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. This study suggests that LPS alone from clinical isolates of Bcc is an important virulence factor in CF and utilizes TLR4-mediated signalling pathways to induce a significant inflammatory response.


Asunto(s)
Complejo Burkholderia cepacia/química , Citocinas/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 1/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Infecciones por Burkholderia/inmunología , Células Cultivadas , Humanos , Lipopolisacáridos/química , Transducción de Señal/fisiología , Receptor Toll-Like 4/inmunología
18.
Carbohydr Res ; 341(18): 2954-8, 2006 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-17078936

RESUMEN

The O-chain polysaccharide of the lipopolysaccharide from the endophytic bacterium Burkholderia cepacia strain was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be the following: -->2)-beta-D-Ribf-(1-->6)-alpha-D-Glcp-(1-->.


Asunto(s)
Complejo Burkholderia cepacia/química , Lipopolisacáridos/química , Secuencia de Carbohidratos , Cromatografía de Gases , Resonancia Magnética Nuclear Biomolecular
19.
J Clin Microbiol ; 44(3): 1159-64, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16517920

RESUMEN

Using the established commercial system Sherlock (MIDI, Inc.), cellular fatty acid methyl ester analysis for differentiation among Burkholderia cepacia complex species was proven. The identification key based on the diagnostic fatty acids is able to discern phenotypically related Ralstonia pickettii and Pandoraea spp. and further distinguish Burkholderia pyrrocinia, Burkholderia ambifaria, and Burkholderia vietnamiensis.


Asunto(s)
Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/clasificación , Ácidos Grasos/análisis , Infecciones por Burkholderia/microbiología , Humanos , Fenotipo , Especificidad de la Especie
20.
J Bacteriol ; 187(24): 8247-55, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16321929

RESUMEN

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.


Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Complejo Burkholderia cepacia/enzimología , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/fisiología , Animales , Proteínas Bacterianas/metabolismo , Western Blotting , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/genética , Caseínas/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Ligasas/genética , Ligasas/metabolismo , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , ARN Bacteriano/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transcripción Genética
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