Effects of the type VI secretion system key genes hcp and vgrG3 on antimicrobial susceptibility in Acinetobacter baumannii ATCC 17978

The antimicrobial resistance of Acinetobacter baumannii ( A. baumannii ) is becoming an increasingly serious concern. This study aimed to investigate the effects of the type VI secretion system (T6SS)-related genes hcp and vgrG3 on antimicrobial susceptibility in A. baumannii and explore the underlying mechanisms. Using ATCC 17,978 as the parental strain, Δ hcp , Δ vgrG3 , and Δ hcp Δ vgrG3 knockout mutants, as well as vgrG3 -complemented strains (Δ vgrG3 -C and Δ hcp Δ vgrG3 -C), were constructed. Transcriptomic and proteomic analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation, antimicrobial susceptibility testing (AST), and ethidium bromide (EtBr) efflux assays were performed to evaluate the roles of hcp and vgrG3 in antimicrobial susceptibility. Compared with the wild-type (WT) strain, the Δ hcp mutant showed no significant changes in minimum inhibitory concentrations (MICs) for all tested antibiotics. In contrast, the Δ vgrG3 mutant exhibited increased MICs for multiple antibiotics, accompanied by enhanced expression of the resistance-nodulation-division (RND) efflux pump AdeIJK and enrichment of pathways associated with resistance to β-lactam antibiotics and cationic antimicrobial peptides (CAMPs). The Δ hcp Δ vgrG3 double mutant displayed further increased MICs for some antibiotics, which was potentially associated with elevated AdeIJK expression, major facilitator superfamily (MFS) efflux pump-related regulation, and alterations in lipopolysaccharide (LPS) biosynthesis pathways. Treatment with the efflux pump inhibitor Phenylalanine-Arginine β-Naphthylamide (PAβN) reduced the MICs of the Δ vgrG3 and Δ hcp Δ vgrG3 strains for some antibiotics. In addition, EtBr efflux assays demonstrated enhanced whole-cell efflux activity in the Δ vgrG3 and Δ hcp Δ vgrG3 strains. Complementation with vgrG3 restored the MICs and efflux activity to levels comparable to those of the WT strain. In conclusion, hcp and vgrG3 exhibit distinct effects on antimicrobial susceptibility in A. baumannii ATCC 17978. While hcp deletion had no apparent effect, vgrG3 deletion was associated with reduced susceptibility to multiple antimicrobials and increased AdeIJK efflux pump expression. However, the regulatory link between vgrG3 and AdeIJK requires further investigation.