Synergistic Inhibitory Effect of Polymyxin B in Combination with Ceftazidime against Robust Biofilm Formed by Acinetobacter baumannii with Genetic Deficiency in AbaI/AbaR Quorum Sensing

ABSTRACT Carbapenem resistance of Acinetobacter baumannii poses challenges to public health. Biofilm contributes to the persistence of A. baumannii cells. This study was designed to investigate the genetic relationships among carbapenem resistance, polymyxin resistance, multidrug resistance, biofilm formation, and surface-associated motility and evaluate the antibiofilm effect of polymyxin in combination with other antibiotics. A total of 103 clinical A. baumannii strains were used to determine antibiotic susceptibility, biofilm formation capacity, and motility. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting was used to determine the genetic variation among strains. The distribution of 17 genes related to the resistance-nodulation-cell division (RND)-type efflux, autoinducer-receptor (AbaI/AbaR) quorum sensing, oxacillinases (OXA)-23, and insertion sequence of ISAba1 element was investigated. The representative strains were chosen to evaluate the gene transcription and the antibiofilm activity by polymyxin B (PB) in combination with merapenem, levofloxacin, and ceftazidime, respectively. ERIC-PCR-dependent fingerprints were found to be associated with carbapenem resistance and multidrug resistance. The presence of blaOXA-23 was found to correlate with genes involved in ISAba1 insertion, AbaI/AbaR quorum sensing, and AdeABC efflux. Carbapenem resistance was observed to be negatively correlated with biofilm formation and positively correlated with motility. PB in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with deficiency in AbaI/AbaR quorum sensing. Our results not only clarify the genetic correlation among carbapenem resistance, biofilm formation, and pathogenicity in a certain level but also provide a theoretical basis for clinical applications of polymyxin-based combination of antibiotics in antibiofilm therapy. IMPORTANCE Deeper explorations of molecular correlation among antibiotic resistance, biofilm formation, and pathogenicity could provide novel insights that would facilitate the development of therapeutics and prevention against A. baumannii biofilm-related infections. The major finding that polymyxin B in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with genetic deficiency in AbaI/AbaR quorum sensing further provides a theoretical basis for clinical applications of antibiotics in combination with quorum quenching in antibiofilm therapy.

Line 357 "This is again proof of a finding as mentioned above that biofilm formation more virulent A. baumannii strains tend to be more susceptible to certain antibiotics". Is the intent to say that virulence and ability to form biofilm are related? I don't think that biofilm formation necessarily equates to virulence. Biofilm associated infections tend to involve foreign bodies and result in more of a chronic infection issue.
Overexpression of blaOXA51 is associated with ISAba1 insertion gene. Oxa-51 was not assessed in this study; however, given that it is a major carbapenemase, the limitations of not detecting this beta-lactamase should be discussed.   The "inhibition" is graded from 0 to 100, but what is meant by inhibition. I gather that these are 6 different strains of A. baumannii. It would be important to know if RBFI within an isolate is related to FIC yet the X and Y axis for panel D appears to be raw concentration. For some of those isolates, it appears that there are very narrow conditions required to promote biofilm formation. If inhibition is achieved, biofilm is suppressed. However, antibiotics at marginal subinhibitory concentrations promote the most biofilm formation.
Reviewer #2 (Comments for the Author): Here authors analyse the drug resistant phenotypes of 103 clinical isolates of Acinetobacter baumannii and correlated these phenotypes to the presence of other genes and biofilm formation and motility. This is an interesting manuscript to read, and authors have performed an impressive amount of work. My main critique is that overall, the manuscript is lacking detail, particularly in the methods and description of the figures. My specific comments are below.
Major comments 1. Not all the figures or figure panels are referred to in the text. 2. There is insufficient detail provided in the methods section. For example a. 'Bacterial collection and identification'. Please include a supplemental table listing the 103 strains. Include information on the source of each strain and any known genetic markers. b. L126. What was the negative control? c. L133. Was the media solidified with 0.5% agar? Or was the media diluted to 0.5%? This is not clear.
d. Indicate the number of biological and technical replicates for each assay. e. L141 -142. No DNA extraction method is indicated. f. L149. What does the abbreviation CRGV-PCR refer to? Has this method been published before? If so include a reference. If not provide details of this fingerprinting method. g. L170. Was 200 uL the final volume in the wells or the inoculum volume? h. L174 -181. Provide a reference for the FIC calculation. Also in more recent publications I have seen the additive range as 0.5 -4, and antagonism > 4.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Responses to Reviewers
Re: Spectrum01768-21 (Balanced evolution of carbapenem resistance versus biofilm formation, and synergistic anti-biofilm effect of polymyxin B in combination with caftazidime against Acinetobacter baumannii) Authors: Yinyue Li, et al.
We sincerely thank all Reviewers for your valuable comments and giving us the chance to revise our manuscript. We have finished the point-by-point responses for this manuscript, and you will find the changes in the Marked Up Manuscript up-loaded.

Responses to Reviewer 1
Line 87 -The authors talk about polymyxin combination therapy and reduction in nephrotoxicity. This is only true if the dose of polymyxin is reduced. I would recommend that that this statement be removed to further focus the article. For checkerboard assay, actually we created a 6 × 6 matrix in a 8 × 12 well plate.
Compared with 8 × 8 matrix, the advantage of 6 × 6 matrix is that the dilution preparation of the two drugs can be done on the same plate. For easy to understand, relevant changes have been made at method part. (Line 182-183, 204-205) Line 203 -The manuscript would be easier to read if too many significant figures were avoided. For some of the percent values are 2, 3, or 4 significant digits with no apparent difference in precision. Three should be adequate.
 As suggested, the significant digit of all percent values has been changed to 3 in the text.
Statistics -There are several uses of the Spearman Rank Correlation test. Given the data involved this simplifies to a 2 X 2 Chi square test. I wonder if readers would more likely understand the application of a chi square test.
 As suggested, a chi-square test was applied in data analysis, and relevant changes have been made at data analysis part. (Line 214) Line 248 -The correlation between the two fingerprinting methods is not intuitive.
ERIC PCR was completed and allowed clustering of the organisms into 5 groups.
Separately, the organism were subjected to CRGV-PCR, and organisms were clustered into 6 groups. Since the assay is looking at different genes why would the cluster designation be correlated between the methods. This analysis would also indicate some sort of hierarchy to the order of clusters and some reason to suspect that the cluster hierarchy is expected to be related in some way. Aren't these two methods distinct ways of grouping organisms?
 ERIC-PCR fingerprinting has been demonstrated to be a reliable technique for discriminating intraspecific variations. CRGV-PCR method attempts to analyze the genetic differences in terms of the distribution of 17 genes among 103 strains. The original intention of the correlation analysis between ERIC-PCR and CRGV-PCR was to illustrate the correlation between intraspecific variations and the distribution of 17 genes. Given the fact that this method of analysis is difficult to understand and has never been used before, it has been abandoned in this study. Instead, a direct description of the distribution of genes in different genotype strains was used. Indeed, for isolates with higher MIC values for antibiotics including meropenem, achieving a concentration of 1/2 MIC is hard to sustain due to dosing limitations. Therefore, the representative strains used in the checkerboard test were susceptible strains or strains with lower levels of resistance to these antibiotics. A statement has been provided. (L173-180) Line 357 "This is again proof of a finding as mentioned above that biofilm formation more virulent A. baumannii strains tend to be more susceptible to certain antibiotics".
Is the intent to say that virulence and ability to form biofilm are related? I don't think that biofilm formation necessarily equates to virulence. Biofilm associated infections tend to involve foreign bodies and result in more of a chronic infection issue.
 We agree with the reviewer's point of view. This sentence has been deleted.
Overexpression of blaOXA51 is associated with ISAba1 insertion gene. Oxa-51 was not assessed in this study; however, given that it is a major carbapenemase, the limitations of not detecting this beta-lactamase should be discussed.     d. Indicate the number of biological and technical replicates for each assay.  Considering that the CRGV-PCR typing method has been abandoned in this study, this problem will no longer exist. 7. Figure 5. Numbers on the plots are too small. What does the bracket indicate?
Provide more detail in the figure legend.
 This figure has been modified and numbered as Figure 2 in revised manuscript.
Font size of numbers has been enlarged. As suggested, a detail description has been provided in figure legend. (L666, Figure 2) 8. L281-286. This section is unclear.
 The sentence has been changed for easy understanding. (L329-331) 9. Figure 3. Provide more detail in the legend. Text in the figure is too small.
 As suggested, this figure has been modified and numbered as Figure S3  14. Figure 4 is not referenced in the text. Provide more detail in the figure legend, there is no mention of the individual panels. What do the arrows mean?
 Considering original Figure 4 showed no significant differences between groups, it has been deleted in revised manuscript.
15. Figure 6. Provide a rational for why these specific strains were selected. Why are heat maps only shown for select antibiotic combinations for select strains? Figure 6A -assuming this is for planktonic cells? Provide more detail in the figure legend.
 As suggested, the rationale for selecting these strains has been described in the method part. The statement for strain selecting has been provided. (L173-180, 200-

202, 354-356)
 Original Figure 6A showed the antimicrobial effect for planktonic cells. This  Table 2 is referenced in the text before Table 1.  Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines the reviewer has recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised 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. Detailed instructions on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
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. Sincerely,

William Lainhart
Editor, Microbiology Spectrum Reviewer comments: Reviewer #2 (Comments for the Author): Overall the manuscript is much improved. However the figure legends are still lacking sufficient detail.These should enable interpretation of the figures separately from having to read the methods and results. Specific comments are below - Figure 1 is still confusing. More detail needs to be provided in the legend to clarify. It is unclear what the differences are between B and D. What do the arrows indicate? Figure 3 and 4. How is the data presented? mean +/-SD? I don't see any error bars in Figure 3? - Figure 5 legend. Indicate what the differences indicated by * are compared to. Assuming it is to the untreated control, but this is not indicated. -L227-229. This is still not clear. Is the definition of a group that it contains more than three strains?
Once these changes have been made, I do not need to re-review this manuscript.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.  Figure 5 legend. Indicate what the differences indicated by * are compared to.

Responses to Reviewers
Assuming it is to the untreated control, but this is not indicated.
 As suggested, more detailed information have been provided in legend. Box with orange border refers to control without antibiotic treatment. All data were presented as means of three biological replicates. The significances were determined by nonparametric one-way ANOVA. * indicates significant increase at a P value of 0.05 when compared to control. * indicates significant decrease at a P value of 0.05 when compared to control. (Line 703-707) -L227-229. This is still not clear. Is the definition of a group that it contains more than three strains?
 The definition of a group is that it contains more than three strains. A clear description has been made at Line 227.