(W0930-04-23) In Vitro Synergism of Depolymerase-Antibiotic Combination against Multidrug-Resistant Acinetobacter baumannii

Purpose: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii, which most commonly manifests as pneumonia, has posed significant clinical challenges and called for novel treatment strategies. One possible strategy involves exploiting phage-encoded depolymerases that degrade bacterial surface carbohydrates as antibiofilm agents and antimicrobial adjuvants. Currently, depolymerases have been widely reported to treat bloodstream infections due to their dependency on serum-mediated bacterial killing. To extend their application to low-serum local infections, like lung infections, the use of depolymerases in combination with antibiotics can result in a synergistic effect, leading to enhanced antibacterial activity and reduced antibiotic resistance. However, promising combinations with synergism, especially for local infection treatments, still need to be identified. Therefore, this study aims to screen and evaluate the antibacterial activity of a potential depolymerase-antibiotic combination against pneumonia-associated MDR A. baumannii in vitro.

Methods: In this study, Dpo71 was utilized as a model depolymerase, with the susceptible A. baumannii, MDR-AB2, serving as a model strain. The combined effects of Dpo71 with seven interested antibiotics on MDR-AB2 were evaluated. To mimic the lung infection environments, a low volume ratio of serum was added. As previously found in our research, Dpo71 only killed bacteria in the presence of serum, and a serum ratio as low as 5% was sufficient for Dpo71 to exert its bactericidal effect [1]. The minimum inhibitory concentrations (MICs) of interested antibiotics and the MIC change after combining with Dpo71 in the absence or presence of 5% serum were examined using the broth microdilution method [2]. A promising combination was selected for assessing in vitro synergism using the checkboard assay and time-kill assay. Furthermore, the effects of the selected depolymerase-antibiotic combination on biofilm formation and eradication were compared with the single treatments.

Results: The MICs of the seven interested antibiotics were unchanged with serum and also unchanged after combining with 10 μg/mL Dpo71 without any serum addition. However, in the presence of 5% serum, Dpo71 reduced the MICs of all antibiotics except levofloxacin (Table 1). Comparing the MIC change and the antibiotic susceptibility, Dpo71 reversed the resistance of ciprofloxacin, ceftazidime, and amikacin, with their MICs decreasing by over 16-fold. As reported, wild-type A. baumannii was re-sensitized to ceftazidime (CFZ) after losing the capsule as a mutant strain [3]. Based on this evidence, the Dpo71-CFZ combination in the presence of 5% serum was selected for further study. The checkboard assay confirmed the Dpo71-CFZ synergism with a fractional inhibitory concentration (FIC) index < 0.5 (Figure 1A). Moreover, the time-kill assay indicated a synergistic bactericidal effect of Dpo71-CFZ, with a 2-log more bacterial reduction than the sum of reductions caused by individual agents (Figure 1B). In the antibiofilm assay, the combined Dpo71-CFZ treatment significantly controlled planktonic bacteria and prevented biofilm formation compared to monotherapies (Figure 2A&B). Besides, Dpo71-CFZ also had a notable effect in removing early biofilms (Figure 2C). However, Dpo71-CFZ at the same concentrations did not eradicate mature biofilms significantly compared to Dpo71 alone treatment. This might be due to the insufficient degradation of the mature biofilm matrix by Dpo71 at 10 μg/mL, leading to reduced antibiotic activity against biofilm resident bacteria. Further increasing the concentration of Dpo71 to 40 μg/mL for enhanced biofilm matrix degradation, the activity of CFZ was re-sensitized, and the Dpo71-CFZ combination showed a significant eradication effect compared to monotherapies (Figure 2D).

Conclusion: This study has identified a promising Dpo71-CFZ combination that demonstrated a synergistic antibacterial effect against the MDR-AB2 strain in vitro. These findings provide a basis for further investigation of this combination strategy as a potential treatment for local infections associated with MDR A. baumannii.

References: [1] X. Chen et al. Phage-Derived Depolymerase as an Antibiotic Adjuvant Against Multidrug-Resistant Acinetobacter baumannii, Front Microbiol (2022); 13:845500.
[2] Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, Clinical Microbiology Procedures Handbook2016, pp. 5.16.11-15.16.23.
[3] F. Gordillo Altamirano et al. Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials, Nat Microbiol (2021); 6:157-161.

Acknowledgements:This work was supported by the University Grants Committee of Hong Kong (ref. 14112921). The support of HKPFS from the University Grants Committee to Honglan Wang was greatly acknowledged.

Table 1. MICs of interested antibiotics alone or in combination with 10 μg/mL Dpo71 in the absence or presence of 5% serum. ND: nondetectable.

Figure 1. Checkerboard assay (A) and time-kill assay (B) of the combined effects of Dpo71 with CFZ in the presence of 5% serum.

Figure 2. Evaluation of the bacterial growth (A) and biofilm formation (B) of MDR-AB2 after various treatments. Evaluation of the eradication effects on the early (C) and mature (D) biofilms of MDR-AB2 after various treatments. Treatments were given at 0h (B), 2 h (C), or 24 h (C) post-bacterial inoculation. Data are presented as mean ± s.d. (n = 3).