Objective

With the detection rate increasing each year, highly resistant and virulent CRKP has been a serious challenge to clinical treatment because of the high morbidity and mortality. Considering the virulence of CRKP is closely related to over-expression of siderophore, the high detection rate of entB and ybtS genes in highly virulent CRKP may be an important reason for the high virulence phenotype of CRKP. Therefore, in this study, single/double knockout and complemented strains of siderophore virulence genes entB and ybtS were constructed to clarify the effect of siderophore virulence genes on the virulence of CRKP.

Methods

1.The wire drawing experiment, mucus phenotype screening experiment, and PCR amplification were used to screen the target strain WT. the entB gene deletion strain △entB and the complementation strain C-△entB, ybtS gene deletion strain ΔybtS and complementation strain C-ΔybtS, entB and ybtS double gene deletion strain ΔentB + ybtS and complementation strain C-ΔentB + ybtS, were constructed by CrispR-Cas9 gene editing technology. PCR method was used to test whether the knockout and complementation were successful. 2. The colony morphology and mucus phenotype of the experimental strains were observed and the siderophore ability of the experimental strains was tested. Then the growth curves, biofilm-forming ability, and anti-serum killing ability of the strains were determined. 3. In order to understand the virulence of the experimental strain, the mouse intraperitoneal infection model was established to draw the survival curves and determine LD50 of experiment strains. Then to clarify the colonization ability of the experimental strains in the lung and liver of mice, the pathological biopsies were used to observe histopathological changes and ELISA method was used to determine the inflammatory factors IL-1β, LI-3 and TNF-α.

Results

1 CRKP-27 was screened as the target strain WT, which is characterized by positive wire drawing test, strong mucus, strong virulence and carrying both entB and ybtS genes. The single/double knockout and complemented strains of siderophore virulence genes entB and ybtS were successfully constructed. 2 Siderophore virulence genes entB and ybtS had no significant effect on the colony morphology, mucus phenotype (drawing test) and biofilm formation ability of CRKP strains. The CRKP strains with entB and ybtS genes could significantly increase siderophore production. Although both the entB and ybtS genes could impair the growth rate of the CRKP strain, the role of ybtS gene was relatively slow. entB and ybtS genes enhanced the antiserum killing ability of CRKP strains. 3 The presence of entB and ybtS genes reduced the survival rate of mice infected with CRKP strains. Histopathological changes and inflammatory factor levels in the lungs and livers of infected mice were enhanced by the presence of entB and ybtS genes. Mice infected with the same strain had higher histopathological changes and levels of inflammatory factors in the lungs than in the livers.

Conclusions

1.The siderophore virulence genes entB and ybtS have no significant effect on the colony morphology, mucus phenotype and biofilm formation ability of CRKP strains.2.The siderophore virulence genes entB and ybtS can significantly enhance the virulence of the CRKP strain, but weaken its growth ability.