Fig. 6: Schematic of the RIBEA method and identification of the clinical risk of evolved bacteria.

a Scheme of the RIBEA method developed in this study. We integrated serial passaging experiments to monitor the rapid bacterial evolution by using hypermutable strains in selective environments, such as different sites of infection and in the presence of antimicrobial agents; whole genome sequencing (WGS) to identify accumulated gene mutations during bacterial evolution; transposon-directed insertion sequencing (TraDIS) analysis to identify potential bacterial factors that contribute to survival in the selective environments (identification of resistance genes); and an in vivo model to evaluate the pathogenesis of the evolved bacteria and determine the potential clinical risk. RIBEA can be completed within approximately one month. b The mechanism by which pathogenicity develops in evolved bacteria. In this study, we revealed the pathogenesis mechanism and the potential clinical risk of the evolved non-HMV-Kp, a principal bacterial pathogen. Non-HMV-Kp does not exhibit typical clinical symptoms upon infection and can be eradicated by innate immune defences in immunocompetent hosts. In immunosuppressed (and/or immunodeficient) hosts, non-HMV-Kp infection can cause lower respiratory tract infections. Non-HMV-Kp, which has the potential for bacterial evolution (hyper- and high gene mutation frequencies), increases its clinical impact by spreading from the primary site (lung) to the blood with or without the development of antimicrobial (abxA) resistance, which leads to a decrease in the treatment efficacy of antimicrobial agents. Therefore, via RIBEA, we revealed the potential and the current clinical risks presented by bacteria that have a high frequency of gene mutations during infection.