In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically ...engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.
Trimeric Autotransporter Adhesins (TAA) found in Gram‐negative bacteria play a key role in virulence. This is the case of Burkholderia cepacia complex (Bcc), a group of related bacteria able to cause ...infections in patients with cystic fibrosis. These bacteria use TAAs, among other virulence factors, to bind to host protein receptors and their carbohydrate ligands. Blocking such contacts is an attractive approach to inhibit Bcc infections. In this study, using an antibody produced against the TAA BCAM2418 from the epidemic strain Burkholderia cenocepacia K56‐2, we were able to uncover its roles as an adhesin and the type of host glycan structures that serve as recognition targets. The neutralisation of BCAM2418 was found to cause a reduction in the adhesion of the bacteria to bronchial cells and mucins. Moreover, in vivo studies have shown that the anti‐BCAM2418 antibody exerted an inhibitory effect during infection in Galleria mellonella. Finally, inferred by glycan arrays, we were able to predict for the first time, host glycan epitopes for a TAA. We show that BCAM2418 favoured binding to 3′sialyl‐3‐fucosyllactose, histo‐blood group A, α‐(1,2)‐linked Fuc‐containing structures, Lewis structures and GM1 gangliosides. In addition, the glycan microarrays demonstrated similar specificities of Burkholderia species for their most intensely binding carbohydrates.
Burkholderia cenocepacia is known for its capacity of adherence and interaction with the host, causing severe opportunistic lung infections in cystic fibrosis patients. In this work we produced Giant ...Plasma Membrane Vesicles (GPMVs) from a bronchial epithelial cell line and validated their use as a cell-like alternative to investigate the steps involved in the adhesion process of B. cenocepacia. RNA-sequencing was performed and the analysis of the B. cenocepacia K56-2 transcriptome after the first contacts with the surface of host cells allowed the recognition of genes implicated in bacterial adaptation and virulence-associated functions. The sensing of host membranes led to a transcriptional shift that caused a cascade of metabolic and physiological adaptations to the host specific environment. Many of the differentially expressed genes encode proteins related with central metabolic pathways, transport systems, cellular processes, and virulence traits. The understanding of the changes in gene expression that occur in the early steps of infection can uncover new proteins implicated in B. cenocepacia-host cell adhesion, against which new blocking agents could be designed to control the progression of the infectious process.
Abstract
Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the ...in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound. Its action can be either bacteriostatic or bactericidal, depending upon the concentration used. The effect of DHA was also evaluated on two others B. cenocepacia clinical isolates and compared with one representative member of all the 17 Bcc species. To test whether DHA could have a therapeutic potential, we assessed its efficacy using a Galleria mellonella caterpillar model of B. cenocepacia infection. We observed that the treatment of infected larvae with a single dose of DHA (50 mM) caused an increase in the survival rate as well as a reduced bacterial load. Moreover, DHA administration markedly increases the expression profile of the gene encoding the antimicrobial peptide gallerimycin. Our results demonstrate that DHA has in vitro and in vivo antibacterial activity against Bcc microorganisms. These findings provide evidence that DHA may be a useful nutraceutical for the treatment of CF patients with lung infections caused by antibiotic multiresistant Bcc microorganisms.
Cell‐to‐cell early contact between pathogens and their host cells is required for the establishment of many infections. Among various surface factors produced by bacteria that allow an organism to ...become established in a host, the class of adhesins is a primary determinant. Burkholderia cenocepacia adheres to the respiratory epithelium of cystic fibrosis patients and causes chronic inflammation and disease. Cell‐to‐cell contacts are promoted by various kinds of adhesins, including trimeric autotransporter adhesins (TAAs). We observed that among the 7 TAA genes found in the B. cenocepacia K56‐2 genome, two of them (BCAM2418 and BCAS0236) express higher levels of mRNA following physical contact with host cells. Further analysis revealed that the B. cenocepacia K56‐2 BCAM2418 gene shows an on–off switch after an initial colonization period, exhibits a strong expression dependent on the host cell type, and enhances its function on cell adhesion. Furthermore, our analysis revealed that adhesion to mucin‐coated surfaces dramatically increases the expression levels of BCAM2418. Abrogation of mucin O‐glycans turns BCAM2418 gene expression off and impairs bacterial adherence. Overall, our findings suggest that glycosylated extracellular components of host membrane might be a binding site for B. cenocepacia and a signal for the differential expression of the TAA gene BCAM2418.
Burkholderia cenocepacia adheres to the respiratory epithelium of cystic fibrosis patients and causes chronic inflammation and disease. Our findings uncover the transcriptional alteration of BCAM2418 gene induced by the physical contact of the bacterium with bronchial epithelial cells. We found that overexpression of BCAM2418 gene augmented the bacterial cell adhesion to host cells, and it is dependent on recognition of O‐linked glycans from the host cell membranes.
EFG1 is a central transcriptional regulator of filamentation that is an important virulence factor of Candida albicans. This study serves to assess in vivo the applicability of the anti-EFG1 ...2′-OMethylRNA oligomer for inhibiting C. albicans filamentation and to attenuate candidiasis, using the Galleria mellonella model. For that, larvae infected with a lethal concentration of C. albicans cells were treated with a single dose and with a double dose of the anti-EFG1 2′OMe oligomer (at 40 and 100 nM). The anti-EFG1 2′OMe oligomer toxicity and effect on larvae survival was evaluated. No evidence of anti-EFG1 2′OMe oligomer toxicity was observed and the treatment with double dose of 2′OMe oligomer empowered larvae survival over 24 h (by 90%–100%) and prolonged its efficacy until 72 h of infection (by 30%). Undoubtedly, this work validates the in vivo therapeutic potential of anti-EFG1 2′OMe oligomer for controlling C. albicans infections.
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Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even ...after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3
generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses.
Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin.
It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.
Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the ...secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the ‘Sponge Microbiome Project’ dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.
Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure ...together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.
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