Purpose. The presence of alginate-overproducing (Alg+) strains of Pseudomonas aeruginosa in cystic fibrosis patients is indicative of chronic infection. The Alg+ phenotype is generally due to a mutation in the mucA gene, encoding an innermembrane protein that sequesters AlgT/U, the alginate-specific sigma factor. AlgT/U release from the anti-sigma factor MucA is orchestrated via a complex cascade called regulated intramembrane proteolysis. The goal of this study is to identify new players involved in the regulation of alginate production.
Methodology. Previously, a mutant with a second-site suppressor of alginate production (sap), sap27, was isolated from the constitutively Alg+ PDO300 that harbours the mucA22 allele. A cosmid from a P. aeruginosa minimum tiling path library was identified via en masse complementation of sap27. The cosmid was transposon mutagenized to map the contributing gene involved in the alginate production. The identified gene was sequenced in sap27 along with algT/U, mucA, algO and mucP. The role of the novel gene was explored using precise in-frame algO and algW deletion mutants of PAO1 and PDO300.
Results/Key findings. The gene responsible for restoring the mucoid phenotype was mapped to lptD encoding an outer-membrane protein. However, the sequencing of sap27 revealed a mutation in algO, but not in lptD. In addition, we demonstrate that lipopolysaccharide transport protein D (LptD)-dependent alginate production requires AlgW in PAO1 and AlgO in PDO300.
Conclusion. LptD plays a specific role in alginate production. Our findings suggest that there are two pathways for the production of alginate in P. aeruginosa, one involving AlgW in the wild-type, and one involving AlgO in the mucA22 mutant.
Purpose. Burkholderia cenocepacia is among the most common members of the Burkholderia cepacia complex (Bcc) isolated from patients with cystic fibrosis (CF). The factors triggering the high rates of morbidity and mortality in CF patients are not well elucidated. In this study, we aim to highlight the genome diversity of two clinical isolates of B. cenocepacia through comparative genome analysis.
Methodology. The repertoire of virulence factors and resistance genes compared to reference strains J2315 and K56-2 was elucidated. The isolates were screened for the presence of phages and insertion sequences. Two methods were combined to obtain an accurate prediction of genomic islands (GIs): the cumulative GC profile and the IslandViewer web tool. To study evolutionary relatedness, whole genome-based single-nucleotide polymorphism (wgSNP) analysis was also performed with 43 publically available strains of the Bcc of various sequence types.
Results/Key findings. Genome-based species identification of the two isolates BC-AUH and BC-BMEH confirmed the species as B. cenocepacia. Both belonged to ST-602, a double-locus variant of ST-32 (CC31), genomovar IIIA, and carried a large number of antibiotic resistance genes. Eighteen GIs were predicted in BC-AUH and BC-BMEH, occupying 9.3 and 6.1 % of the respective genomes. Comparison to J2315 revealed 89 and 85 genes unique to BC-BMEH and BC-AUH, respectively. Additionally, 1823 intergenic SNPs were detected between BC-BMEH and BC-AUH.
Conclusion. This study mapped existing genetic variations in B. cenocepacia associated with notorious outcomes in CF patients, and the data obtained provide comprehensive, genome-inferred insights and multifactorial examination of an important human pathogen.
Purpose. Pseudomonas aeruginosa expresses a type III secretion system (T3SS) that activates the host inflammasome-mediated immune response. We examined the role of inflammasome activation in severe infection outcomes.
Methods. We infected C57BL/6 (B6) mice lacking inflammasome components ASC or caspase-1/11 with a highly virulent strain of P. aeruginosa, PSE9, using a mouse model of pneumonia. We evaluated inflammasome activation in vitro by infecting bone marrow-derived macrophages (BMDMs) with PSE9 and measuring cell death and release of inflammasome-dependent cytokines IL-18 and IL-1β. A bioluminescent reporter assay was used to detect activity of caspase-1 and caspase-3/7 in BMDMs from B6 and ASC-deficient mice.
Results/Key Findings. ASC−/− mice exhibited significantly improved survival relative to caspase-1/11−/− mice and B6 mice, demonstrating that ASC and caspase-1/11 play differential roles in P. aeruginosa infection. We found that ASC−/− BMDMs exhibited significantly reduced cell death relative to B6 BMDMs, while caspase-1/11−/− BMDMs were resistant to cell death. IL-18 and IL-1β were both detected from supernatants of infected B6 BMDMs, but cytokine release was abrogated in both ASC−/− and caspase-1/11−/− BMDMs. We detected a 2.5-fold increase in the activation of caspase-3/7 in PSE9-infected B6 BMDMs, but no increase in infected ASC−/− BMDMs. Cell death, cytokine release and caspase-3/7 activity were dependent on a functional T3SS.
Conclusions. Collectively, these results are consistent with a model whereby the T3SS apparatus of P. aeruginosa activates the caspase-1-dependent inflammasome and caspase-3/7 through an ASC-dependent mechanism. This activation may have implications for the outcomes of P. aeruginosa infections.
Purpose. The microbiota composition of faeces and colonic contents were analysed to investigate the mechaninsm by which fermented soybean meal improves intestinal microbial communities, growth and immunity in weaning piglets.
Methodology. Microbiota were investigated using16S rRNA gene sequencing and systematical bio-information Operational Taxonomic Units; α-diversity analyses indicated that fermented soybean meal increased bacterial species diversity.
Results. The levels of Actinobacteria and Proteobacteia in faeces, and Firmicutes and Tenericutes in the colon, increased significantly in piglets fed fermented soybean meal (P<0.05). The relative abundance of Clostridium sensu stricto1, Lachnospira and Bacteoides had positive correlations with diarrhoea in the piglets. Lactobacillus, Blautia and Clostridium sensu stricto1 levels were correlated with increases in the average daily feed intake of piglets. Lactobacillus and Lachnospira also had positive relationships with IgM levels, and lymphocytes levels were increased relative to Clostridium sensu stricto1. Lymphocyte numbers also increased with higher levels of Blautia and decreased with Clostridium sensu stricto1. Increased levels of Blautia were also correlated with significant increases in white blood cells.
Conclusion. The significant differences in faecal and colonic bacteria were correlated with enhanced immunity and overall improved health in the weaning piglets.
A prevailing opinion is that the strains of Pseudomonas aeruginosa that infects both plants and humans are two separate species. This study strongly disputes that notion until the modern molecular technology proves otherwise. This paper examines a spectrum of strains occurring in nature, their habitats, dissemination, their relationship to clinical strains, and the environmental conditions that favor their colonization of plants. The isolates were obtained from clinical specimens, plants, soil, and water. The identity of these strains was confirmed using pyocin typing and biochemical assays. The data reveal that agricultural soils, potted ornamental plants, hoses, fountains, and faucets frequently harbored P. aeruginosa. However, it was not commonly found in semi-arid areas, suggesting that moisture and high humidity is necessary for colonization and survival. Though found in soil, P. aeruginosa was seldom isolated on edible plant parts. The pathogenicity of various strains on plants was tested by inoculating vegetables, lettuce slices (Lactuca sativa L. "Great Lakes"), celery stalks (Apium graveolens L. var. Dulce], potato tuber slices (Solanum tuberosum L. "Whiterose"), tomato (Lycopersicon esculentum L. Mill), cucumber (Cucumis sativus L.), rutabaga (Brassica campestris L.), and carrot (Daucus carota L. var sativa). There was considerable variation in the strains' ability to cause rot, but no difference was observed between clinical isolates and others from agricultural fields, water, and soil. Two of the clinical isolates from burn patients, P. aeruginosa PA13 and PA14, exhibited the greatest virulence in causing rot in all the plants that were tested, especially on cucumber, lettuce, potato, and tomato. The study discusses how closely the epidemiology of P. aeruginosa relates to many plant pathogens, and the ability of human isolates to colonize plants and food material under favorable conditions. The biochemical and phenotypic similarity among strains from the clinical and agricultural material is strongly indicative that they are the same species and that plants and soil are natural reservoirs for P. aeruginosa.