Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.     

Contribution of SecDF to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>resistance and expression of virulence factors

Background  

SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined.     

Burkholderia cenocepacia K56‐2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF‐associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56‐2, is a tumor necrosis factor receptor 1‐interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL‐8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

Background  

The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.     

Exploring the HME and HAE1 efflux systems in the genus <Emphasis Type="Italic">Burkholderia</Emphasis>

Background  

The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia.     

Extracellular Glutathione Decreases the Ability of Burkholderia cenocepacia to Penetrate into Epithelial Cells and to Induce an Inflammatory Response

Background

The airway surface liquid (ASL) of Cystic Fibrosis (CF) patients contains a lower concentration of reduced glutathione (GSH) with respect to healthy people. It is not known whether this defect may favor lung colonization by opportunistic pathogens.

Principal Findings

We have analyzed the effects of extracellular GSH on the ability of Burkholderia cenocepacia to penetrate and multiply in epithelial respiratory cells. Extracellular GSH proved to be able to drastically reduce the pathogen ability to adhere and invade airway epithelial cells. This effect is correlated to a GSH-dependent increase in the number of free thiols on the surface of epithelial cells, suggestive of a change in the oxidoreductive status of membrane proteins involved in B. cenocepacia recognition. Moreover, treatments with GSH led to a consistent reduction of the expression of IL-8, TNF-α and IL-1β in response to B. cenocepacia infection.

Conclusions and Significance

Extracellular GSH modulates the interaction between B. cenocepacia and epithelial respiratory cells and inhibits the bacterial invasion into these cells. This suggests that therapies aimed at restoring normal levels of GSH in the ASL might be beneficial to control CF lung infections.     

Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia(†)

One common mechanism of resistance against antimicrobial peptides in Gram‐negative bacteria is the addition of 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l ‐Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l ‐Ara4N synthesis and transfer to the LPS. The absence of l ‐Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi‐protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l ‐Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.     

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7–49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29–98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4–10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

     

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.     

Burkholderia cenocepacia disrupts host cell actin cytoskeleton by inactivating Rac and Cdc42

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex, is an opportunistic pathogen that causes devastating infections in patients with cystic fibrosis. The ability of B. cenocepacia to survive within host cells could contribute significantly to its virulence in immunocompromised patients. In this study, we explored the mechanisms that enable B. cenocepacia to survive inside macrophages. We found that B. cenocepacia disrupts the actin cytoskeleton of infected macrophages, drastically altering their morphology. Submembranous actin undergoes depolymerization, leading to cell retraction. The bacteria perturb actin architecture by inactivating Rho family GTPases, particularly Rac1 and Cdc42. GTPase inactivation follows internalization of viable B. cenocepacia and compromises phagocyte function: macropinocytosis and phagocytosis are markedly inhibited, likely impairing the microbicidal and antigen‐presenting capability of infected macrophages. The type VI secretion system is essential for the bacteria to elicit these changes. This is the first report demonstrating inactivation of Rho family GTPases by a member of the B. cepacia complex.     

First insights into the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates from HIV-infected Mexican patients and mutations causing multidrug resistance

Background  

The prevalence of infections with Mycobacterium tuberculosis (MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the Mycobacterium tuberculosis complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in katG, rpoB and inhA genes, resulting in isoniazid (INH) and rifampin (RIF) resistance.     

The interrelations of radiologic findings and mechanical ventilation in community acquired pneumonia patients admitted to the intensive care unit: a multicentre retrospective study

Background

Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.

Findings

Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [³H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro.

Conclusions

These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.     

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.     

Growth inhibition and ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors in Candida spp. isolates, including non-albicans organisms

Background  

Although Candida species are commensal microorganisms, they can cause many invasive fungal infections. In addition, antifungal resistance can contribute to failure of treatment.     

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection

Background

Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays.

Methods

B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase.

Results

ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs.

Conclusion

B. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.     

Immunochemical characterization of polysaccharide antigens from six clinical strains of Enterococci

Background  

Enterococci have become major nosocomial pathogens due to their intrinsic and acquired resistance to a broad spectrum of antibiotics. Their increasing drug resistance prompts us to search for prominent antigens to develop vaccines against enterococci. Given the success of polysaccharide-based vaccines against various bacterial pathogens, we isolated and characterized the immunochemical properties of polysaccharide antigens from five strains of Enterococcus faecalis and one strain of vancomycin-resistant E. faecium.