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The type VI secretion system (T6SS) has been regarded as a late-model virulence factor widely distributed in Acinetobacter baumannii (A. baumannii). This study aimed to elucidate the clinical manifestations, the genetic background and microbiological characteristics of A. baumannii isolates causing bloodstream infection (BSI), and assessed the impact of T6SS carrying state on the clinical course. In this study, Clinical samples of A. baumannii causing BSI were collected from a teaching hospital in China from 2016 to 2020 and a retrospective cohort was conducted. Experimental strains were categorized into T6SS positive and negative groups through PCR targeting on hcp gene. The antimicrobials sensitivity test, virulence genes, biofilm formation ability, serum resistance of A. baumannii strains and Galleria mellonella infection model were investigated. Independent risk factors for T6SS+ A. baumannii BSI and Kaplan-Meier curve through follow-up survey were analyzed. A total of 182 A. baumannii strains were isolated from patients with BSI during 5 years and the medical records of all patients were retrospectively reviewed. The proportion of T6SS+ isolates was 62.64% (114/182), which exhibited significantly higher resistance rates of commonly used antibacterial drugs compared to T6SS- group. We found that T6SS+ A. baumannii strains had significantly weaker biofilm formation ability compared to T6SS- A. baumannii. Despite no difference in the positivity rate of tested virulence genes in two groups, T6SS+ strains exhibited higher resistance to the serum and increased virulence in vivo compared to T6SS- strains, indicating that T6SS is likely to enhance the survival and invasive capabilities of A. baumannii in vivo. Indwelling catheter, respiratory diseases, ICU history, white blood cell count and percentage of neutrophils increasing were independent risk factors for T6SS+ A. baumannii BSI. At last, the Kaplan-Meier curve confirmed a higher mortality rate associated with T6SS+ A. baumannii BSI, suggesting that the presence of T6SS may serve as a prognostic factor for mortality. In conclusion, our study revealed that T6SS+ A. baumannii exhibited distinct clinical features, characterized by high antimicrobial resistance and enhanced virulence, providing valuable insights for clinical treatment considerations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jianming Cao reports financial support was provided by The Zhejiang Province Natural Science Foundation of China. Jianming Cao reports financial support was provided by The Wenzhou Science and Technology Bureau., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)