Baicalein Resensitizes Multidrug-Resistant Gram-Negative Pathogens to Doxycycline

ABSTRACT As multidrug-resistant pathogens emerge and spread rapidly, novel antibiotics urgently need to be discovered. With a dwindling antibiotic pipeline, antibiotic adjuvants might be used to revitalize existing antibiotics. In recent decades, traditional Chinese medicine has occupied an essential position in adjuvants of antibiotics. This study found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens. Mechanism studies have shown that baicalein causes membrane disruption by attaching to phospholipids on the Gram-negative bacterial cytoplasmic membrane and lipopolysaccharides on the outer membrane. This process facilitates the entry of doxycycline into bacteria. Through collaborative strategies, baicalein can also increase the production of reactive oxygen species and inhibit the activities of multidrug efflux pumps and biofilm formation to potentiate antibiotic efficacy. Additionally, baicalein attenuates the lipopolysaccharide-induced inflammatory response in vitro. Finally, baicalein can significantly improve doxycycline efficacy in mouse lung infection models. The present study showed that baicalein might be considered a lead compound, and it should be further optimized and developed as an adjuvant that helps combat antibiotic resistance. IMPORTANCE Doxycycline is an important broad-spectrum tetracycline antibiotic used for treating multiple human infections, but its resistance rates are recently rising globally. Thus, new agents capable of boosting the effectiveness of doxycycline need to be discovered. In this study, it was found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens in vitro and in vivo. Due to its low cytotoxicity and resistance, the combination of baicalein and doxycycline provides a valuable clinical reference for selecting more effective therapeutic strategies for treating infections caused by multidrug-resistant Gram-negative clinical isolates.

negative bacteria and then focus in on doxycycline. Through a series of assays, the authors show that co-treatment with dox and baicalein kills bacteria but does not kill or lyse mammalian cells. Baicalein damages A. baumannii outer and inner membranes and appears to specifically compete with magnesium to bind lipid A. The compound also likely interacts with bacterial lipids, especially PG. Transcriptomics comparing dox to dox + baicalein revealed many gene changes. Co-treatment also disrupted biofilms, reduced host responses to LPS, reduced mortality in wax-worms and reduced tissue colonization and inflammatory cytokines in mice. These data indicate that baicalein works through multiple mechanisms both at the level of the bacterium and on the host.
Major points 1) For the assays with florescent or luminescence probes, there need to be controls in which compound is incubated with the probe in the absence of cells at the concentrations of probe and compound used in the corresponding experiment. This is necessary to establish whether a compound on its own alters signal from the probe. These controls, or descriptions of the controls, were not found in the methods for Figure 7, for instance.
2) The transcriptomics are difficult to interpret without a baicalein-only sample.
Minor points 1) Figure 8 A and B -It would be more transparent to display Figure 8 A and B as bar graphs instead of line graphs, as the data are derived from different samples. Also, no error bars are visible, and the legend does not mention that there are error bars that are too small to be seen. 3) Ideally, bar graphs show individual data points so that readers can see data spreads.

Reviewer #2 (Comments for the Author):
This manuscript seeks to characterize the antibacterial activity of a natural product, baicalein, with particular focus on its ability to potentiate doxycycline activity in tetracycline-resistant Gram-negative bacteria. Baicalein is one of the active components in certain extracts used in Chinese traditional medicine that are thought to enhance the effectiveness of antibiotics in patients. As an isolated compound, it has been studied previously by others in order to evaluate its utility in various therapeutic indications, not all infectious disease. Its synergy with tetracyclines was studied in S aureus and was initially thought to result from inhibition of the TetK pump; it was then realized that baicalein could potentiate tetracycline in MRSA strains lacking TetK and that it could potentiate a variety of antibiotic classes in various bacterial species.
Li and colleagues undertook the currrent study with the idea that baicalein might restore the utility of doxycycline for treatment of Gram-negative infections. They found using checkerboard MICs that baicalein is technically synergistic with multiple antibiotics. The greatest effect is seen for doxycycline, where the MIC is reduced by up to 32 fold in some MDR strains of A baumannii. However, the resulting MIC is generally well above the clinical breakpoint. Moreover, these effects require fairly high concentrations of baicalein, 31.25 or 62.5 ug/ml. Although baicalein appears to be fairly well tolerated in mice, it seems unlikely that it will be feasible to reach these concentrations in plasma of patients treated with the proposed combination.
It is thus extremely unlikely that baicalein could be used clinically as an antibiotic partner to treat infections. Despite the very modest antibacterial activity of balicalein (MIC 250 ug/ml), one might feel it is worth studying, perhaps leading to novel approaches for designing agents with greater activity as standalone antibiotics or potentiators.
Much of the manuscript describes the attempt to determine the mechanism of action of baicalein and/or the mechanism by which it potentiates other antibacterial agents. Unfortunately, most of the mechanistic studies are not interpretable. In some studies, bacteria are treated with drugs for an hour or longer, with 4 hr for the gene expression studies. This is inappropriate, as the direct effects of antibacterial agents on bacterial physiology are generally apparent within 20-30 minutes. With longer times, the observed effects are indirect and shed no light on the molecular mechanism of the drug. Several experiments compared a subinhibitory treatment (single drug) to an inhibitory treatment (two drugs). It is likely that the effects seen are simply the effect of stress, growth inhibition or cell death. Any other inhibitory treatment might produce the same results.
I recommend shortening the manuscript substantially, focusing on the stronger parts of the work: in-vitro and in-vivo microbiology. I recommend delaying publication of the mechanistic studies until they have been conducted more carefully. These experiments should have included controls -i.e., comparator antibiotics with known mechanisms, and compounds previously characterized as OM disrupting agents, ionophores, or pore-forming agents. Bacterial viability should be monitored. Experimental conditions such as incubation times or concentration of reagents should be justified unless well supported by literature that is cited.
2. Fig 1B. Please explain the spider-web plots. A table showing FICI for each antibiotic would be much easier to read and would allow the MIC of each antibiotic alone to be included. Why was this experiment done with strain AB43Δcrispr-cas? What are the results for the drug-sensitive parent AB43? 3. Fig 1C and Table S2 show the same data for the same ten isolates of A baumannii, but in a different order. Please present both in the same order, perhaps numerical by strain ID. 4. Fig 1D requires some explanation. What does "inhibition rate" mean? How was the experiment done? 5. Fig 2. Eliminate panel B. The untreated bacteria are growing and are clearly not in stationary phase 6. Fig 2 C-F, hemolysis and cytotoxicity. Low levels of both hemolysis and cytotoxicity are seen at very high concentrations of doxycycline (256 or 512 ug/ml), except in the presence of extremely high concentrations of baicalein (250 ug/ml). Lines 638-641 interpret these observations as showing baicalein protecting mammalian cells from detrimental effects of doxycycline. Is it generally thought that cytotoxicity of doxycycline is clinically relevant? If not, I suggest rewriting this section to focus on the lack of cytotoxicity and hemolysis for baicalein at concentrations that are likely to be achieved in vivo -both alone and in the presence of doxycycline. 7. Fig 2G and G, serial passage studies. At the start of the experiment, the MIC of doxycycline is 128 ug/ml for both AB145 and KP1. The plot shows the MICs increasing by 32 or 64 fold during passage in doxycyline unless baicalein was included. That would mean the final MICs are 4 or 8 milligrams/milliliter -is that correct? Suggest plotting actual MICs on the Y axis, rather than fold increase. Were these extremely high MICs still seen after the final culture had been passaged on drug-free agar? Did the resistant isolates have elevated MICs for other antibiotics, or was the effect specific to doxycycline? 8. Fig 3 -8. Eliminate, as noted above. 9. Fig 9, Consider eliminating panels A and B, keeping the lung study. A: I am unaware of any situation in which Galleria infection is more predictive of human efficacy than mouse infection. B: The drug was administered directly to the site of infection (peritoneum), so this study is of little value. 10. Fig 9, cytokine study (panels G-I). This requires more explanation. Were cytokines determined in plasma from the same mice as in the efficacy study? If not, please describe the experiment (route of infection, route of drug administration, timing of sample collection, etc). Note that reduced cytokine reduction is consistent with smaller numbers of bacteria; these observations do not necessarily suggest an anti-inflammatory effect. 12. Eliminate figs S1 -S5 13. Technical details: Baicalein was prepared as a DMSO solution (line 269). What was the concentration of DMSO in MIC determinations? Cytotoxicity determinations presumably included serum in cell culture medium. What is the effect of added serum on the antibacterial activity of baicalein, doxycycline, or the combination? 14. Terminology: a. The term synergy should be avoided except where the experimental design allows one to distinguish between synergy and additivity. b. The term "the cell membrane" is used several times (line 103, lines 106-107, lines 210-211). This term makes no sense and should be replaced by a phrase that is consistent with Gram-negative bacteria possessing two membranes. For example, Lines 106-107, "In bacteria, the cell membrane is the first line of defense against antibiotics (21)." Suggest "In bacteria, the cell envelope is the first..." Also, check the reference. Ref 20 (Hurdle) might be more appropriate. 15. The writing is mostly clear and grammatical but does include some errors. Suggest editing for English. 16. Minor point: Reviewing would be made easier by labeling the figures (Fig 1, fig 2, etc) and by adding the legends at the bottom of each figure (similar to the supplemental material).
17. Reference list: Inclusion of doi numbers is much appreciated! Staff Comments:

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If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. Overall response to Reviewer 1: Thank you for reviewing our manuscript and providing some kind comments. In the revised version, the new text is highlighted in yellow, and the underline indicates the revised text to identify better the changes made to the previous version. Our point-to-point response is provided below in red text.

Comments of Reviewer 1
In what follows, we would like to answer the questions you mentioned and give a detailed account of the changes made to the original manuscript. Response: Since we did not express it clearly, we are sorry for your misunderstanding.
Actually, in fluorescence or luminescence assay, the corresponding concentration of drugs (without cells) were incubated with probes as blank control, and the relative fluorescence intensity was calculated using the analyte value minus the blank controls.
To be more explicit and to follow the reviewer's concerns, we have added a brief description as follows: "In fluorescence or luminescence assay, the corresponding concentration of drugs (without cells) were incubated with probes as blank control, and the relative fluorescence intensity was calculated using the analyte value minus Because our focus herein is on the mechanism of action of the baicalein combined with doxycycline against doxycycline-resistant gram-negative bacteria, in the following research, we conducted an analysis focused on gene transcription levels of the combination compared to the doxycycline alone.   isolates were high-MIC of doxycycline-resistant strains (≥128 µg/mL) and they are all pandrug-resistant strains, the synergistic MIC of which is above the clinical breakpoint. This could be due to the pandrug-resistant and high-MIC of doxycycline strains with a robust antibiotic-resistance gene system that limits baicalein and doxycycline's combined effects.
An excessive concentration of baicalein is used in the combination, which causes it challenging to reach effective drug concentrations in the plasma of patients.
Nevertheless, we recognized this limitation should be mentioned in the paper, so we added the following sentence "This research reveals that baicalein could be considered a lead compound that merits further optimization and development as a candidate adjuvant that helps combat antibiotic resistance." (Page 2, line 35-37).
" Due to its low cytotoxicity and resistance, the baicalein/doxycycline combination provides a valuable clinical reference for selecting more effective therapeutic strategies for treating multidrug-resistant gram-negative clinical isolate infections." "Scanning electron microscopy (SEM). Doxycycline (64 µg/mL), baicalein (125 µg/mL), or their combination was used to treat bacterium solution (OD600 = 0.5) for 1 h, followed by fixed overnight using 2.5% glutaraldehyde at 4°C. After thoroughly rinsing thrice using PBS, bacteria were subjected to 10 min gradual dehydration using ethanol (30%, 50%, 70%, 80%, 90%, 95%) and 10 min dehydration using 100% ethanol thrice. The samples were centrifuged at 4,000 g for 10 min to remove fixatives, followed by resuspension of bacterial pellets in PBS (1 mL). After processing, each sample was dried using a critical point dryer, coated with goldpalladium using an ion sprayer (5) and then observed with SEM (GeminiSEM 300)." The concentration and exposure time used in the transcriptome sequencing experiment was determined two-fold. On the one hand, we referred to previous studies (3,8,9). On the other hand, the time-kill curve indicated that after 8 hours of incubation, growth characteristics of bacterial populations tended to be stabilized, 4 h as midpoint time, and the bacterial loads of combined treatment were found to be approximately 3 orders of magnitude lower than other groups. We have added the information required as explained above. "A. baumannii AB145 was grown in MHB till the early exponential phase; next, bacterial cells were treated with 64 µg/mL doxycycline, 125 µg/mL baicalein alone, or their combination for 4 h (9)." (Page 18, line 398-400).
Thank the reviewer for the constructive comments and suggestions. In the revised version, the new text is highlighted in yellow, and the underline indicates the revised text to identify better the changes made to the previous version. Our point-to-point response is provided below in red text. We hope these changes improve the clarity and accuracy of the presentation.
In what follows, we would like to answer the questions you mentioned and give a detailed account of the changes made to the original manuscript.

Comment 1: Move tables S1 and S2 into the main text.
Response: Thank you for this suggestion. We merged supplementary Table 1 and   supplementary Table 2 into Table 2.  (10), a checkerboard assay showed that baicalein could act as a synergistic bacteriostatic effect (FICI ≤ 0.5) with most of the conventional antibiotics (10/14, 71.43%), which included doxycycline (FICI = 0.1875) ( Table 1). A similar phenomenon was found in the sensitive strain AB43 (Table S1)." (Page 6, line 95-100). Fig 1C and Table S2 show the same data for the same ten isolates of A baumannii, but in a different order. Please present both in the same order, perhaps numerical by strain ID.

Comment 3:
Response: Thank you for your reminder. We performed sequential adjustments based  As the reviewer points out, it is an excellent suggestion to elevate MICs for other antibiotics in doxycycline-resistant mutants. "Antibiotic resistance is a significant challenge in the treatment of bacterial infections. Serial passaging for 31 days in the presence of doxycycline increased MIC for A. baumannii AB145 and K. pneumoniae KP1 by 32 and 64 folds, respectively. In contrast, the combination treatment did not allow resistance to develop ( Figure 2G-H). It is worth noting that the per-generation doxycycline-resistant mutants exhibited cross-resistance (12) to multiple different classes of antibiotics, and the expression levels of tetA were significantly increased in some high-level doxycycline-resistant mutants ( Figure S1, Table S2). Furthermore, all of the doxycycline-resistant mutants had multiple nonsynonymous mutations in the tetA genes (Table S3). Baicalein was found to be effective in inhibiting doxycycline resistance in tetA-positive MDR A. baumannii and K. pneumoniae KP1" (Page 6, line 108-118).  (3,6,8,9,11,(13)(14)(15)(16).  fig S6 into the main text, merging it with fig 9. Response: As the reviewer suggested, it is a good idea, and we have merged Figure   S6 with Figure 9. We have changed "We found that the exogenous addition of K + , Ca 2+ , Mg 2+ , Cu 2+ , and Zn 2+ inhibited the activity of baicalein, and Mg 2+ strongly inhibited the synergistic antibacterial effects, while Fe 3+ , Mn 2+ , and Na + had no significant impact on the action ( Figure 4C-E)." to " We found that the exogenous addition of K + , Ca 2+ , Mg 2+ , Cu 2+ , and Zn 2+ inhibited the activity of baicalein, and Mg 2+ strongly inhibited the antibacterial effects of baicalein combined doxycycline, while Fe 3+ , Mn 2+ , and Na + had no significant impact on the action ( Figure 4C-E)." (Page 7, line 136-139).

Comment 11: Move
We have changed "The MICs of baicalein are higher with the exogenous addition of phospholipids, and PG blocks the synergy between baicalein and antibiotics ( Figure   5). These data suggest that baicalein-mediated membrane perturbation is vital for its synergistic effect with doxycycline." to " The MICs of baicalein are higher with the exogenous addition of phospholipids, and PG blocks the antibacterial effects of baicalein combined with antibiotics ( Figure 5). These data suggest that baicaleinmediated membrane perturbation is vital to promote the antibacterial efficacy of doxycycline." (Page 8, line 145-148).
We have revised the text to address your concerns and hope it is more evident. We have made the change. "Baicalein disrupts the cell membrane of gram-negative bacteria." to "Baicalein disrupts the inner and outer membranes of gram-negative bacteria." (Page 7, line 119-120).
"In bacteria, the cell membrane is the first line of defense against antibiotics (18)." to "In bacteria, the cell envelope is the first line of defense against antibiotics (19)." (Page 7, line 122-123).
"The SEM images revealed that after the combination treatment, the cell surface was depressed, shrunk, collapsed, and lysed compared with no treatment, indicating that baicalein's effect on bacteria was possibly associated with the rapidly disrupted cell wall and cell membrane." to "The SEM images revealed that after the combination treatment, the cell surface was depressed, shrunk, collapsed, and lysed compared with no treatment, indicating that baicalein's effect on bacteria was possibly associated with the rapidly disrupted cell wall and the cell's inner and outer membranes." (Page 7, line 125-129).
"The bacterial cell membrane is fluid, which is critical for bacterial proliferation and survival (20)." to "The bacterial outer membrane is fluid, critical for bacterial proliferation and survival (20)." (Page 9, line 176-177). We feel sorry for the inconvenience brought to the reviewer. We have adjusted the references. "In bacteria, the cell envelope is the first line of defense against antibiotics (19)." (Page 7, line 122-123).

Comment 15: The writing is mostly clear and grammatical but does include some errors. Suggest editing for English.
Response: This manuscript has been revised extensively according to the reviewers' constructive suggestions. In addition, the expression of the manuscript has been improved with the help of a native English speaker. The certificate of English language editing has been uploaded to the system along with the revised manuscript. (Fig 1, fig 2,

etc) and by adding the legends at the bottom of each figure (similar to the supplemental material).
Response: Thank you for pointing this out. We have made corrections.

Comment 17: Reference list: Inclusion of doi numbers is much appreciated!
Response: Thank you for pointing this out. We have corrected it according to the reviewer's comments.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.
And here, we did not list the changes but marked them in red in the revised paper.
We appreciate for Editors/Reviewers' warm work earnestly and hope that the correction will meet with approval.
We look forward to hearing from you regarding our submission and responding to any further questions and comments you may have.
Once again, thank you very much for your comments and suggestions. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Thank you for better filling out the methods and legends by including more explanation of how experiments were done. I agree with your decision to retain the mechanistic experiments. While they are not necessarily conclusive with regard to baicalein mechanism of action, we need to understand how bacteria and metazoans respond to potential antimicrobials and adjuvants to develop new approaches to therapy.
Since the original submission, several experiments have been repeated, and the manuscript has been reorganized and edited. Most of the concerns described in the previous reviews have been addressed, at least in part. Some specific comments follow.
1. Mechanistic studies. In responses to concerns by both previous reviewers, most of these experiments have been repeated with much shorter incubation times. In some cases, added controls were added (polymyxin or colistin to propidium iodide and NPN staining; baicalein-only to transcriptomics). It would have been preferable to include antibiotic controls also in other assays, such as ATP, ROS, NADH, PMF, etc. However, the shorter incubation times make these studies much more interpretable and less likely to simply reflect reduced bacterial growth -as would happen with any effective antibiotic.
2. Animal infection. The lung infection study is nice and should be included. I continue to believe that the Galleria and mouse peritonitis studies have little value and should omitted. My reasoning is as follows. The goal of a mouse efficacy study is to see whether the drug can travel through the bloodstream to the site of infection and have a beneficial physiologic effect. If the drug is administered directly to the site of infection (both injected IP), very little information is obtained. Almost any compound (or combination) with an MIC will appear to be efficacious. Thus the IP/IP model is not helpful in determining whether a compound justifies further effort. Pharmaceutical antibiotic-discovery researchers usually prefer animal models with a quantitative readout (such as cfu in lung or another organ). See examples below of papers describing early animal studies for several antibacterial compounds that were found to be effective for treating human infections. Note that these include experiments monitoring survival following IP infection, but in these cases the drug was administered either IV or subcutaneously.
The authors' response letter provided nine references, from five or so academic research groups around the world, illustrating that this use of mice as "furry test tubes" is a common error.
With regard to Galleria: Invertebrate infection models are a useful alternative to in-vitro susceptibility testing if you are seeking inhibitors of virulence mechanisms that may not be essential in standard broth media. These models might also be useful as an alternative to mice if compound supply is extremely limited, or if you are testing so many compounds that mouse testing is not feasible.
3. Serum -The previous review asked about the effect of serum on antibacterial activity. The response letter indicated the the serum in cell culture medium had been heat-inactivated. This misses the point. The question is whether baicalein binds to serum proteins. It is very common to think a compound lacks cytotoxicity, only to find that the compound has been sequestered by serum in the culture medium. The simplest way to evaluate this possibility is to add serum to the bacterial culture medium and see if the MIC is shifted upward. 4. Methods. Suggest careful re-reading of this section to be sure that it accurately reflects the procedures. Line 314, "bacterium solution" should be "bacterial suspension" Line 315, did you actually culture supernatants (after centrifugation), or did you take aliquots of the bacterial culture for dilution and plating? 5. Check for inconsistencies. One example noted: Time-kill curves are described as indicating bacteriostatic activity on line 104, but bactericidal on line 756. Also note the checkerboard MIC assay is described (line 97) as showing bacteriostatic activity. Suggest referring here to antibacterial activity, as an MIC determination cannot distinguish static from cidal activity.

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Thank you for submitting your paper to Microbiology Spectrum. In vivo studies were conducted using a neutropenic mouse lung infection model, we found that "Following the addition of baicalein, the bacterial loads in the bronchoalveolar lavage fluid and mouse organs were the lowest in the combination treatment group (Figure 9). Additionally, the levels of four inflammatory cytokines (IL-1β, IFN-γ, TNF-α, and IL-8) in serum were considerably lower after 48 h of pulmonary infection in the combination treatment group than that in other groups, which was confirmed by the alleviated pulmonary pathology ( Figure S7). These results highlighted that baicalein combined with doxycycline can inhibit bacterial invasion into host cells and reduce the host inflammatory response to these infections in vivo." (Page 11, line 216-224)

Comments of Reviewer 2
General comments: Since the original submission, several experiments have been repeated, and the manuscript has been reorganized and edited. Most of the concerns described in the previous reviews have been addressed, at least in part. Some specific comments follow.
Overall response to Reviewer 2: Thank you for reviewing the manuscript and for your positive and constructive comments. In the revised version, the new text is highlighted in yellow, and the underline indicates the revised text to identify better the changes made to the previous version. Our point-to-point response is provided below.
We hope these changes improve the clarity and accuracy of the presentation.
In what follows, we would like to answer the questions you mentioned and give a detailed account of the changes made to the original manuscript.  Response: Thank you for the comment, with which we fully agree. We have removed the Galleria mellonella and mouse peritonitis studies, and revised the text accordingly.  "Tested antibiotics were added in triplicate into flat-bottomed 96-well plate (Corning) wells, followed by bacterial inoculum supplementation (0.5 × 10 6 CFUs/mL)." to "Tested antibiotics were added into triplicate wells of 96-well flat-bottomed tissue culture plates (Corning), and two-fold serial dilution was performed, followed by the addition of the prepared bacterial inoculum (0.5 × 10 6 CFUs/mL)." (Page 14, line 310-312).
"When required, HCl or NaOH was supplemented to the medium to adjust the pH to 5-9." to "HCl or NaOH was added to the medium to adjust the pH to 5-9." (Page 15, The LIVE/DEAD BacLight bacterial viability kits (Molecular Probes) containing SYTO9 and PI were used to distinguish between live and dead bacteria. The samples were visualized using a Zeiss Axio Scope A1 upright microscope, and the images were processed to merge using the ImageJ software (6)." (Page 15, line 330-337). Response: Thank you so much for your careful check. We have made correction according to the reviewer's comments. We've revised "a checkerboard assay showed that baicalein could act as a synergistic bacteriostatic effect (FICI ≤ 0.5)" to "a checkerboard assay showed that baicalein can act as a synergistic antibacterial activity (FICI ≤ 0.5)" (Page 6, line 96-98).  Response: Thanks for your constructive suggestion, which is highly appreciated. We have carefully scrutinized the manuscript, and made corresponding revisions including some typos, grammatical errors and long sentences, ect. In addition, the manuscript has been polished by an English language editing company. The certificate of English language editing has been uploaded to the system along with the revised manuscript.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.