Zhilang Qiu,1,2,* Jun Ran,1,2,* Yifan Yang,1,2,* Yue Wang,1,2 Yang Zeng,1,2 Yinhui Jiang,1 Zuquan Hu,1 Zhu Zeng,1 Jian Peng1,2 

1Key Laboratory of Infectious Immune and Antibody Engineering of Guizhou Province, Cellular Immunotherapy Engineering Research Center of Guizhou Province, School of Biology and Engineering/School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, 550025, People’s Republic of China; 2The Engineering Research Center of Health Medicine Biotechnology of Institution of Higher Education of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550025, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jian Peng; Zhu Zeng, Email jianpeng@gmc.edu.cn; zengzhu@gmc.edu.cn

Purpose: The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) poses great difficulties in clinical treatment, and has been listed by the World Health Organization as a class of pathogens in urgent need of new antibiotic development. In our previous report, the novel antimicrobial peptide Cec4 showed great potential in decreasing the clinical CRAB biofilm, but its mechanism of action is still illusive. Therefore, in order to evaluate the clinical therapeutic potential of Cec4, it is necessary to explore the mechanism of how Cec4 decreases mature biofilms.
Methods: Key genes involved in the removal of CRAB biofilms by Cec4 were analyzed using transcriptomics. Based on the results of the bioinformatics analysis, the CRISPR-Cas9 method was used to construct the deletion strain of the key gene. The pYMAb2 plasmid was used for the complementation strain construction. Finally, the roles of key genes in biofilm removal by Cec4 were determined by crystal violet staining, podocyte staining, laser confocal imaging, and MBC and MBEC50.
Results: Combined with transcriptome analysis, we hypothesized that OmpH is a key gene involved in the removal of CRAB biofilms by Cec4. Deletion of the OmpH gene did not affect A. baumannii growth, but decreased A. baumannii capsule thickness, increasing biofilm production, and made biofilm-state A. baumannii more sensitive to Cec4.
Conclusion: Cec4 decreases biofilms formed by CRAB targeting OmpH. Deletion of the OmpH gene results in an increase in biofilms and greater sensitivity to Cec4, which enhances the removal of A. baumannii biofilms by Cec4.

Keywords: Acinetobacter baumannii, antibiofilm mechanism, antimicrobial peptide, OmpH