Molecular Basis Of The Pathogenicity And Niche Adaptation Of Burkholderia Cepacia Complex

Burkholderia cepacia complex (Bcc) is composed of17closely related species and found ubiquitously, where they can have both beneficial and detrimental effects on plants. Some can degrade natural and man-made pollutants; some can promote plant growth or can control plant diseases. Some members are also recognized as a plant pathogen and some opportunistic human pathogens. However, over the past decade, there have been several reports of emergence of multidrug resistant Bcc infecting the vulnerable people. This has increased the need for the risk assessment, molecular basis and niche adaptation of Bcc isolates resourced from soil, water, clinical as well as plant pathogen.To test the pathogenicity, an alternative, potential, easy accessible and high throughput lettuce infection model was developed in this study. Alfalfa, Galleria mellonella (wax moth) and rat agar bead model was also used to evaluate the virulence intensity among Bcc isolates comparing with lettuce. Moreover, the outer membrane (OM) proteins of Bcc were investigated for molecular basis by growing Bcc bacteria in host mimic media.1D-SDS-PAGE and Lipid chromatography tandem mass spectrometry (LC-MS/MS) peptides profiles and MASCOT search database as well as in silico functional analysis were performed. Furthermore, to find possible antibacterial agent to keep environment particularly hospital and industries hygienic, copper surfaces as antibacterial agent, first time, was evaluated against Bcc. Mode of action of dry metallic copper surfaces against Bcc was also evaluated at copper exposed Bcc cells, while stainless steel was used as a controlThe main results of these studies were as follows:1. The lettuce model takes～24h to launch experiment including bacterial growth using store-bought lettuce and monitor for up to5d post-infection. Total,265Bcc isolates were used to investigate the virulence intensity and efficacy of lettuce. All isolates belonging to B. anthina, B. stabilis and B. arboris were determined avirulent in lettuce except B. pyrrocinia, where out of184,50isolates showed weak,5 moderate and2isolates showed severe disease symptoms and127found totally avirulent. Selected isolates in rat infection model showed same level of infection as in lettuce infection model. The transmissibility marker BCAM0218, BCAM0224, BCAM0219and BCESM were exclusively absent in265isolates except in B. arboris in which BCAM0219were determined.2. In the further study,70more Bcc isolates resourced from different places e.g clinical, water, soil and plant pathogens were tested in lettuce. Following lettuce pathogenicity was also tested in alfalfa, Galleria mellonella and rat agar bead alternative infection model. The biofilm formation ability was also tested. Severe to moderate pathogenicity was observed for isolates of clinical and water origin compared to soil and plant pathogen, with the exception of a few clinical isolates exhibiting reduced pathogenicity. Moreover, among water isolates, pathogenicity was also higher than soil and plant resource isolates but less than clinical. The lettuce midrib tissues show remarkable invasion, localization, and replication of bacteria when observed by transmission electron microscopic study. Similarly, biofilm formation ability was also higher among water and clinical virulent isolates compared to soil and plant pathogen isolates. B. cepacia LMG1222was used as a reference strain in these infection models.3. In the OM study, shotgun proteomic analyses of OM by LC-MS/MS, overall identified>400proteins under water, Cystic Fibrosis (CF) mimic, water mimic, plant mimic and soil mimic media. Among the catalog of entire identified proteins, shared proteins as well as several proteins remarkably differentially expressed under each growth medium were detected. Shared proteins like ABC transporter proteins, efflux proteins, OmpW etc could be fundamental for growth and survival. While differentially expressed proteins e.g type3, type4, type6secretion system (T3SS, T4SS, T6SS), ZmpA, flagellin, porin, LysR etc under different host mimic medium, perhaps reflecting the evolution due to different environmental survival toward host-adaptation and may represent prime for further studies.4. Copper surfaces were tested against Bcc and it showed the strong antibacterial activity. Higher influx of copper ions into the bacterial cells and cells disintegration among the cells exposed to copper surfaces were noted, while no DNA damage was detected at specified time. At the existing level of knowledge, it looks that contact killing proceeds by successive membrane damage, copper influx into the cells, cell death, and DNA degradation.5. The genomics and transcriptomics studies of four B. seminalis strains for the purpose to unlock the ecology of Bcc are under process.