Project description:Pseudomonas aeruginosa is a major cause of infection in hospitalised patients, with a large genome which makes it highly versatile and resistant to most antimicrobial agents. Ceftazidime-avibactam (CZA) offers an alternative treatment, but resistance is quickly evolving. There is limited knowledge on the exact resistance mechanisms to this drug, or on cross-resistance to meropenem (MEM). This laboratory experiment aimed to decipher these mechanisms in order to provide guidance for the best treatment choice in meropenem pre-treated P. aeruginosa infections. Six clinical isolates of P. aeruginosa were subjected to multistep resistance selection in sub inhibitory concentrations of CZA and MEM. MICs were also determined in the presence of the efflux pump inhibitor phenyl-Arginine-β-Naphthylamide (PAβN). Molecular analyses were performed by whole genome sequencing, whole -gene- and -protein expression profiles. CRISPR/Cas9 genome editing was performed using a two-plasmid method for selected mutations
Project description:ErfA is a transcription factor of Pseudomonas aeruginosa. We here define the genome-wide binding sites of ErfA by DAP-seq in Pseudomonas aeruginosa PAO1 and IHMA87, Pseudomonas chlororaphis PA23, Pseudomonas protegens CHA0 and Pseudomonas putida KT2440.
Project description:Whole-genome sequencing is an important way to understand the genetic information, gene function, biological characteristics, and living mechanisms of organisms. There is no difficulty to have mega-level genomes sequenced at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. The shotgun sequencing method failed to dissect this genome. After insisting for 10 years and going over 3 generations of sequencing techniques, we successfully dissected the PaP1 genome with 91,715 bp in length. Single-molecule sequencing revealed that this genome contains lots of modified bases, including 51 N6-methyladenines (m6A) and 152 N4-methylcytosines (m4C). At the same time, further investigations revealed a novel immune mechanism of bacteria, by which the host bacteria can recognize and repel the modified bases containing inserts in large scale, and this led to the failure of the shotgun method in PaP1 genome sequencing. Strategy of resolving this problem is use of non-library dependent sequencing techniques or use of the nfi- mutant of E. coli DH5M-NM-1 as the host bacteria to construct the shotgun library. In conclusion, we unlock the mystery of phage PaP1 genome hard to be sequenced, and discover a new mechanism of bacterial immunity in present study. Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:In this research, Pseudomonas aeruginosa underwent evolution in the supernatant of either S. aureus (SA) or K. pneumoniae (KP). Through this evolution process, we enriched a sufficient number of bacterial cells along with their corresponding proteins. Following 15 cultivation cycles, notable changes were observed in the phenotypes of P. aeruginosa, with a series of mutations identified through whole-genome sequencing (WGS). Proteomics analysis unveiled that the supernatant-evolved mutants displayed distinct regulation patterns in crucial pathways (including the type VI secretion system, biofilm formation, phenazine biosynthesis, translation, beta-lactam resistance, and O-antigen biosynthesis) in comparison to both the ancestral strain and the unmodified medium-evolved (UmMd-evolved) strain.
