Supernatant of platelet-Klebsiella pneumoniae coculture induces apoptosis-like death in Klebsiella pneumoniae

ABSTRACT Multidrug-resistant Klebsiella pneumoniae strains, especially carbapenem-resistant K. pneumoniae, have become a rapidly emerging crisis worldwide, greatly limiting current therapeutic options and posing new challenges to infection management. Therefore, it is imperative to develop novel and effective biological agents for the treatment of multidrug-resistant K. pneumoniae infections. Platelets play an important role in the development of inflammation and immune responses. The main component responsible for platelet antibacterial activity lies in the supernatant stimulated by gram-positive bacteria. However, little research has been conducted on the interaction of gram-negative bacteria with platelets. Therefore, we aimed to explore the bacteriostatic effect of the supernatant derived from platelet-K. pneumoniae coculture and the mechanism underlying this effect to further assess the potential of platelet-bacterial coculture supernatant. We conducted this study on the gram-negative bacteria K. pneumoniae and CRKP and detected turbidity changes in K. pneumoniae and CRKP cultures when grown with platelet-K. pneumoniae coculture supernatant added to the culture medium. We found that platelet-K. pneumoniae coculture supernatant significantly inhibited the growth of K. pneumoniae and CRKP in vitro. Furthermore, transfusion of platelet-K. pneumoniae coculture supernatant alleviated the symptoms of K. pneumoniae and CRKP infection in a murine model. Additionally, we observed apoptosis-like changes, such as phosphatidylserine exposure, chromosome condensation, DNA fragmentation, and overproduction of reactive oxygen species in K. pneumoniae following treatment with the supernatant. Our study demonstrates that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death, which is important for the antibacterial strategies development in the future. IMPORTANCE With the widespread use of antibiotics, bacterial resistance is increasing, and a variety of multi-drug resistant Gram-negative bacteria have emerged, which brings great challenges to the treatment of infections caused by Gram-negative bacteria. Therefore, finding new strategies to inhibit Gram-negative bacteria and even multi-drug- resistant Gram-negative bacteria is crucial for treating infections caused by Gram-negative bacteria, improving the abuse of antibiotics, and maintaining the balance between bacteria and antibiotics. K. pneumoniae is a common clinical pathogen, and drug-resistant CRKP is increasingly difficult to cure, which brings great clinical challenges. In this study, we found that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death. This finding has inspired the development of future antimicrobial strategies, which are expected to improve the clinical treatment of Gram-negative bacteria and control the development of multidrug-resistant strains.

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Reviewer comments:
Reviewer #1 (Comments for the Author): 1.In Figure 1A, the turbidity of the "S PLT +KP" group was significantly higher than that of the "KP" group.However, this difference is not reflected in Figure 1B, and the result is even reversed.When combined with the analysis of bacterial count results, turbidity detection does not appear to be a reliable method for determining inhibited bacterial growth.
Author Response: Thank the reviewer for pointing out this issue!We have examined Figure 1 carefully and repeated the bacteriostatic experiment.According to the results, we have re-adjusted Figure 1 as follows (Modified Fig. 1).In our research, we set up four groups, including Sn+KP as the experimental group (Sn: the supernatant prepared after co-culture of bacteria and platelets), S PLT +KP as the comparison group (S PLT : the supernatant obtained by physical lysis of platelets), KP group without any treatment as a negative control, IPM group was treated with antibiotic as a positive control.Firstly, we detected the OD 600nm value of each group at 24 h and drew the bacterial growth curve (FIG.1A).We found that the turbidity of Sn+KP group was significantly lower than that of S PLT +KP group and KP group.Therefore, we detected and statistically analyzed the turbidity of each group of bacteria at 8 h (FIG.1C and   D).At the same time, we also found the same changes in bacterial counting after dilution of each group of bacteria at 2 h, 4 h, 6 h, 8 h, and 10 h (FIG.1B).In the previous data, the photos shown in FIG.1A were indeed not clear enough.We provided the original photo as follows and repeated the experiment again, as shown in FIG.1C below.
We believe that the comparison of bacterial turbidity can directly reflect the proliferation of bacteria.In our research, we found that the turbidity of Sn+KP group was significantly lower than that of S PLT +KP group and KP group, which was consistent with the bacterial counting.These results indicated that the antibacterial effect of the supernatant of activated platelet was significantly better than that of the supernatant of physical lysis of non-activated platelets.Detailed explanations are provided on Page 5 and 6, line 133 to 156. 3. The authors should assess the direct inhibitory effect of platelets on bacteria, rather than solely focusing on the platelet-K.pneumoniae coculture supernatant.
2. The description of the experimental method for bacterial counting is unclear.The numbers of viable cells could be determined through serial dilution and plating assay.Author Response: We supplemented the description of the experimental method for bacterial counting.The modifications are shown on Page 15, line 441 and 445.