Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.
BackgroundChronic bacterial infections occur as a result of the infecting pathogen’s ability to live within a biofilm, hence escaping the detrimental effects of antibiotics and the immune defense system. Burkholderia pseudomallei, a gram-negative facultative pathogen, is distinctive in its ability to survive within phagocytic and non-phagocytic cells, to persist in vivo for many years and subsequently leading to relapse as well as the development of chronic disease. The capacity to persist has been attributed to the pathogen’s ability to form biofilm. However, the underlying biology of B. pseudomallei biofilm development remains unresolved.ResultsWe utilised RNA-Sequencing to identify genes that contribute to B. pseudomallei biofilm phenotype. Transcriptome analysis of a high and low biofilm producer identified 563 differentially regulated genes, implying that expression of ~9.5 % of the total B. pseudomallei gene content was altered during biofilm formation. Genes involved in surface-associated motility, surface composition and cell wall biogenesis were over-expressed and probably play a role in the initial attachment of biofilms. Up-regulation of genes related to two component signal transduction systems and a denitrification enzyme pathway suggest that the B. pseudomallei high biofilm producer is able to sense the surrounding environmental conditions and regulate the production of extracellular polymeric substance matrix, a hallmark of microbial biofilm formation.ConclusionsThe transcriptome profile described here provides the first comprehensive view of genes that contribute to the biofilm phenotype in B. pseudomallei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1692-0) contains supplementary material, which is available to authorized users.
A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.
Aims: Betanin is a red plant pigment belonging to the group called betalain. This present study aimed at investigating the effect betanin from beetroot (Beta vulgaris subsp. vulgaris) as a potential anti-infective agent against methicillinresistant Staphylococcus aureus (MRSA) using a Caenorhabditis elegans infection model. Methodology and results:The minimum inhibitory concentration of betanin against MRSA strain ATCC 33591 was determined to establish the non-inhibitory concentration. The minimum inhibitory concentration of betanin against MRSA was > 20 mg/mL. C. elegans were then infected with MRSA and treated with betanin at different concentrations (100, 200, 300 and 400 µg/mL). Betanin at 200 µg/mL significantly improved worm survival following infection whereby the mean time to death was extended about 76 h upon treatment. Intestinal colonization by MRSA of worms exposed to betanin extract was similar to non-betanin-treated infected worms. Conclusion, significance and impact of study:The enhanced survival of MRSA-infected worms upon betanin treatment was not a result of the activation of the host antimicrobial mechanism. Betanin from beetroot can be potentially used as a natural anti-infective agent as a mean to reduce antimicrobial resistance of S. aureus or used in combination with established antimicrobials to increase their effectiveness.
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