Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification.

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

The role of structural evolution in the complexation and flocculation of heavy metals by the microbial product poly-γ-glutamic acid.

The process and mechanism of heavy metal flocculation with extracellular polymeric substances (EPS) secreted by microorganisms, are crucial to their fate in natural environment, wastewater treatment and soil bioremediation applications. However, the structural features of EPS and the relationship between these features and the flocculation process and mechanism remain unclear. In the present study, structural features of the microbial product poly-γ-glutamic acid (γ-PGA) complexed with the heavy metal ions Pb2+ and Cu2+ were characterized and the evolution of these features was identified as having a key role in the flocculation process and mechanism. The secondary structure of the γ-PGA-Pb complex changed significantly, while that of the γ-PGA-Cu complex was only slightly altered. The significant structural change in γ-PGA-Pb was found to be responsible for the combination of residual COOH and Pb2+, promoting the bridging of inter-colloids and faster growth of hydrodynamic diameter. If the conformation changed sufficiently, such as with the γ-PGA-Pb complex in the pH range 4.6-6.2, pH had no impact on the conversion ratio. The unchanged structure of γ-PGA-Cu prevented the flocculation process, although the coordination mode of γ-PGA-Cu resulted in a higher biosorption capacity. This in-depth molecular-level study provides insight into the γ-PGA flocculation mechanism, promoting the use of γ-PGA and γ-PGA producing microorganisms for application in various remediation strategies.