Evaluation of chlorogenic acid and carnosol for anti-efflux pump and anti-biofilm activities against extensively drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa

ABSTRACT Efflux pumps and biofilm play significant roles in bacterial antibiotic resistance. This study investigates the potential of chlorogenic acid (CGA) and carnosol (CL), as phenolic and diterpene compounds, respectively, for their inhibitory effects on efflux pumps. Among the 12 multidrug-resistant (MDR) strains of Staphylococcus aureus and Pseudomonas aeruginosa isolated from nosocomial skin infections, eight strains were identified as extensively drug resistant (XDR) using the disc diffusion method. The presence of efflux pumps in MDR strains of S. aureus and P. aeruginosa was screened using carbonyl cyanide-m-chlorophenylhydrazone. Between the 12 MDR strains of S. aureus and P. aeruginosa, 80% (4 out of 5) of the S. aureus strains and 85.7% (6 out of 7) of the P. aeruginosa strains exhibited active efflux pumps associated with gentamicin resistance. The checkerboard assay results, in combination with gentamicin, demonstrated that CGA exhibited a reduction in the minimum inhibitory concentration (MIC) for XDR S. aureus strain. Similarly, CL showed a synergistic effect and reduced the MIC for both XDR strains of S. aureus and P. aeruginosa. Flow cytometry was used to examine efflux pump activity at sub-MIC concentrations of 1/8, 1/4, and 1/2 MIC in comparison to the control. In XDR S. aureus, CGA demonstrated 39%, 70%, and 19% inhibition, while CL exhibited 74%, 73.5%, and 62% suppression. In XDR P. aeruginosa, CL exhibited inhibition rates of 25%, 10%, and 15%. The inhibition of biofilm formation was assessed using the microtiter plate method, resulting in successful inhibition of biofilm formation. Finally, the MTT assay was conducted, and it confirmed minimal cytotoxicity. Given the significant reduction in efflux pump activity and biofilm formation observed with CGA and CL in this study, these compounds can be considered as potential inhibitors of efflux pumps and biofilm formation, offering potential strategies to overcome antimicrobial resistance. IMPORTANCE In summary, CGA and CL demonstrated promising potentiating antimicrobial effects against XDR strains of Staphylococcus aureus and Pseudomonas aeruginosa, suggesting their probably potential as candidates for addressing nosocomial pathogens. They exhibited significant suppression of efflux pump activity, indicating a possible successful inhibition of this mechanism. Moreover, all substances effectively inhibited biofilm formation, while showing minimal cytotoxicity. However, further advancement to clinical trials is needed to evaluate the feasibility of utilizing CGA and CL for reversing bacterial XDR efflux and determining their efficacy against biofilms. These trials will provide valuable insights into the practical applications of these compounds in combating drug-resistant infections.

• Upload 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 • Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version • Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file • Any supplemental material intended for posting by ASM should be uploaded separate from the main manuscript; you can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files, with all associated legends included For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
Data availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide Spectrum production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact production staff (Spectrum@asmusa.org)immediately with the expected release date.
Publication Fees: For information on publication fees and which article types are subject to charges, visit our website.If your manuscript is accepted for publication and any fees apply, you will be contacted separately about payment during the production process; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.
ASM Membership: 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.
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 submitting your paper to Spectrum.

Sincerely, Krisztina Papp-Wallace Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): In this study, the authors tried to evaluate the ant-efflux and antibiofilm activity of chlorogenic acid and carnosol, two plant derived compounds, against drug-resistant S. aureus and P. aeruginosa clinical strains.A series of experiments were conducted in selected strains of XDR S. aureus and P. aeruginosa which include antibiotic potentiating effects, efflux pump inhibition, biofilm inhibition and cell viability testing to identify the activity of these two phytochemicals in combination with gentamicin.
The study could have been improved by testing a greater number of resistant isolates instead of testing single selected isolates, at least for checkerboard assay and biofilm inhibition assays, which are easy to perform.Confidently attributing the results of a single strain to all clinical isolates is challenging, given the variations in resistance mechanisms observed among different clinical isolates.Though we could observe some antibiotic potentiating effect in the 2 selected isolates tested, even when combined with carnosol and chlorogenic acid, the gentamicin's MIC remains significantly higher than the cutoff value.Similarly, minimal inhibition in biofilm formation was observed for S. aureus which may also could vary between different strains.Despite the well-done study, the author may need to address the following concerns.

Reviewer comments
In this study, the authors tried to evaluate the ant-efflux and antibiofilm activity of chlorogenic acid and carnosol, two plant derived compounds, against drug-resistant S. aureus and P. aeruginosa clinical strains.A series of experiments were conducted in selected strains of XDR S. aureus and P. aeruginosa which include antibiotic potentiating effects, efflux pump inhibition, biofilm inhibition and cell viability testing to identify the activity of these two phytochemicals in combination with gentamicin.
The study could have been improved by testing a greater number of resistant isolates instead of testing single selected isolates, at least for checkerboard assay and biofilm inhibition assays, which are easy to perform.Confidently attributing the results of a single strain to all clinical isolates is challenging, given the variations in resistance mechanisms observed among different clinical isolates.Though we could observe some antibiotic potentiating effect in the 2 selected isolates tested, even when combined with carnosol and chlorogenic acid, the gentamicin's MIC remains significantly higher than the cutoff value.Similarly, minimal inhibition in biofilm formation was observed for S. aureus which may also could vary between different strains.
Despite the well-done study, the author may need to address the following concerns.

Activities against Clinical Extensively Drug-Resistant (XDR) Strains of Staphylococcus aureus and Pseudomonas aeruginosa
The manuscript titled " Evaluation of Chlorogenic Acid and Carnosol for Anti-Efflux Pump and Anti-Biofilm Activities against Clinical Extensively Drug-Resistant (XDR) Strains of Staphylococcus aureus and Pseudomonas aeruginosa" presents an interesting and relevant study in the field of microbiology and antibiotic resistance.
The study design, contents and information of the manuscript are needed to improve in scientific level.All part should be revised and rearranged.
Following requests are necessary to be addressed to improve quality of the study.

Title:
-" clinically" bit confuse, the tested bacterial strain is in vitro resist to wide range of antibiotic not in vivo or resist to antimicrobial therapy during infection. Abstract: -Line 13, need to explain on which based you selected these two compounds (previous study or documented action on the other bacteria rather than staphylococcus and pseudomonas).-Line, did you detect the MIC of CCCP against staph.And pseudomonas -Line 18, why you choose gentamycin, many different antimicrobial agent is more important as anti-staph.Infection.You need to show that study deal with staphylococcus strains that cause skin infection -In this section you need to suggest a possible mechanism for inhibition and is this compound inhibit specific type of efflux pump protein or not.

Introduction:
-In this section you need to paragraph described the family of efflux pump system in staphylococcus and pseudomonas and those that efflux gentamycin.-Line 57, if you use expression profile (with qPCR or NGS) for selected efflux protein it will good proof for overexpression on down expression of the efflux pump that results from the tested efflux inhibitor -Line 67, need to explain role of efflux pump in virulence and aquarium sensing in bacteria to show how EPIs are important

:
-Line 90, you need to mention details about isolated strain, is it from skin or other part, type of infection, host (human or animal).
-Line 102, basically all EPIs has antimicrobial activity for example Reserpine which well documented as A potent EPIs.Just double check your statement that chromogenic acid and carnosol have no antibacterial activity.-Line 112, you need to detect the MIC of CCCP also explain why use this inhibitor with gentamicin.-Line 123, need to mention some details about isolated strain (host, type of infection etc.) -Line 125, you need reference " CGA, and CL that did 124 not impact cell viability " -Line139, P. aeruginosa this isolated associated with human infection or not did host receive antibiotic (please provide information about isolates) -Line 162, at least mention that you use cell to check viability with some details Discussion: In this section you need to -Explain why use two assumed EPIs -Relationship between biofilm and Efflux pump and how EPIs are important -Suggested the source of EPIs (plant source or active chemical agent -Suggest possible mechanisms for both RPIs that used -Is the tested EPIs have selected action or not?-Explain limitation of the study, didn't do expression profile of efflux pump to prof inhibition action of EPIs, used on type of antibiotic Methods: -Line 305, " from patients with 304 wound infection and burns" need to show how isolated the bacteria -Line 308, need to mention stage of bacterial growth that used in sensitivity assay. -Line 318, it is perfect to used agar based method to confirm MIC -Finally, all above, the manuscript is minor revision Manuscript (#Spectrum03934-23)

Response to Reviewers
Here is a point-by-point response to the reviewers' comments and concerns:

Comments from Reviewer 1
 Comment 1: Line 16 -It is mentioned that, a total of 12 XDR strains were studied.
Certain strains seem to be MDR pathogens rather than XDR ones, as they are sensitive to many drugs.Only disc diffusion method is used to identify the sensitivity of antibiotics and all the antibiotics active against S. aureus and P. aeruginosa were not tested/shown (Table 1 and 2).This needs clarification.Also, Gentamicin is not used as a monotherapy and is not the treatment of choice for XDR S. aureus.
Response: Thanks for your kind reminders.In response to your guidance, amendments were made to the manuscript on page 1/lines 14-20.Furthermore, the use of the disc diffusion method for antibiotic sensitivity testing is a recognized and validated approach in microbiology.As you know, extensively drug resistant (XDR) was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories).
However, we ideally believe that a broader array of antibiotic discs would have been preferable, but, readily accessible antibiotic discs were employed due to financial constraints limiting the ability to expand the disc selection.Despite the infrequent use of gentamicin as monotherapy for treating XDR S. aureus infections, it is commonly employed in the management of post-burn infections caused by this pathogen.In addition, one of the purposes of this study is to investigate the combined effect of chlorogenic acid and carnosol in association with gentamicin.
 Comment 2: Line 103-It is mentioned that MIC values of chlorogenic acid and carnosol are equal/above 1024μg/ml.Are MIC values assessed for all clinical isolates, or is the testing limited to specific individual isolates of S. aureus and P. aeruginosa for chlorogenic acid and carnosol?Similarly, in efflux pump inhibition assay and biofilm inhibition assay it is mentioned that ½ MIC values for S. aureus -3 and P. aeruginosa -5 strain as 64 μg/ml and 256 μg/ml respectively (figure 4 and 5), whereas actual MIC for these strains is 512 μg/ml and 1024 μg/ml as shown in Table 1 and 2. Discrepancies should be corrected.
 Response: Thanks for your question.In response to your query, yes, the MIC values of chlorogenic acid and carnosol were assessed for all the clinical isolates under investigation.And as mentioned in the text of the manuscript, "Gentamicin MIC value was also determined for all clinical strain using microplate broth microdilution method.
Clinical strains had different gentamicin MIC values that ranged from 8 to 512 for S. aureus and 2 to 1024 µg/ml for P. aeruginosa, respectively."Page 3/lines 97-99.It is also mentioned in the rest of this section that "The most resistant XDR strain of each bacterium was used for subsequent steps (S. aureus-3, and P. aeruginosa-5)."Page 4/lines 101-102.The actual gentamicin MIC for S. aureus-3, and P. aeruginosa-5 are 512 μg/ml and 1024 μg/ml, respectively, as mentioned by the respected reviewer above.
Finally, the results obtained from the checkerboard assay, as described thoroughly on page 4/lines 103-113, were utilized in the efflux pump inhibition assay and inhibition of biofilm formation.For further clarification, Table 3 of the manuscript can be consulted, as it is also referenced below. Response: I appreciate the reminder.According to the Fractional inhibitory concentration (FIC) index interpretation, an FICI between 0.50 and 1.00 represents an additive effect.
In this case, CGA had an additive effect on the selected XDR P. aeruginosa-5 strain (FICI = 0.75).Table 3 was revised accordingly.
 Comment 4: Line 165for both XDR strains which selected wasshould be rephrased  Response: Thank you for your comment.In response to your suggestion, the phrase "for both XDR stains which selected was" revised to "the maximum MIC values for CGA and CL in combination with gentamicin were 64 μg/ml for XDR strains of S. aureus-3 and P.

Comments from Reviewer 2
Title:  Comment 1: "clinically" bit confuse, the tested bacterial strain is in vitro resist to wide range of antibiotic not in vivo or resist to antimicrobial therapy during infection.
 Response: Thank you for pointing this out.The term "clinical" was used to emphasize that the strains investigated in this study were isolated from nosocomial infections, as well as from patients with wound and burn infections that were resistant to antibiotic treatment.The title has been revised in accordance with your suggestion (Page 1/ line 2).

Abstract:
 Comment 1: Line 13, need to explain on which based you selected these two compounds (previous study or documented action on the other bacteria rather than staphylococcus and pseudomonas).
 Response: Thanks for your comment.However, we believe that addressing the importance of these compounds, which somehow influenced the selection and study of their potential in this study would be more appropriate in the introduction section because the word limit in the abstract section does not allow that this issue should be addressed well.A background regarding this matter was outlined on page 3/lines 77-84. 

Introduction:
 Comment 1: In this section you need to paragraph described the family of efflux pump system in staphylococcus and pseudomonas and those that efflux gentamycin.
 Response: Thanks for your comment.In P. aeruginosa, intrinsic low-level resistance to aminoglycosides is mediated by the expression of the Mex XY-OprM system.However, as previously mentioned, the objective of our study was to investigate the inhibition of efflux pump activity by evaluating and comparing dye accumulation within the cell using flow cytometry.While it is beneficial to address the issue of which efflux pumps may be involved in gentamicin resistance, incorporating this topic in the introduction may set up expectations for it to be specifically addressed in the results and discussion section, which seems slightly out of the scope of our study.

 Comment 2: Line 57, if you use expression profile (with qPCR or NGS) for selected efflux protein it will good proof for overexpression on down expression of the efflux pump that results from the tested efflux inhibitor
 Response: Thank you for the suggestion.Line 57 of the manuscript provides readers insight into the background of the subject being studied, by referencing earlier research.
While the use of techniques such as NGS or qPCR could have offered interesting insights into our study, it was not feasible for us to explore this aspect, because our aim in this study was to investigate efflux pump inhibition using flow cytometry.We used flow cytometry to measure the intracellular accumulation of a fluorescent dye that is a substrate for the efflux pump, which involved comparing the percentage of cells located in the second quadrant or the gated cells of the test and control.NGS is not readily accessible to everyone in Iran, and taking advantage of its benefits would come at a high cost.While modern techniques offer ease of operation and increased study accuracy, we are unable to utilize them due to the associated high costs.
 Comment 3: Line 67, need to explain role of efflux pump in virulence and aquarium sensing in bacteria to show how EPIs are important  Response: Thank you for the nice reminder.The introduction section has been revised accordingly on page 2/lines 61-62.

Results:
 Comment 1: Line 90, you need to mention details about isolated strain, is it from skin or other part, type of infection, host (human or animal).
 Response: Thank you for pointing this out.As mentioned by the reviewer, the revision was made on page 3/ line 93.

 Comment 2: Line 102, basically all EPIs has antimicrobial activity for example
Reserpine which well documented as A potent EPIs.Just double check your statement that chromogenic acid and carnosol have no antibacterial activity.
 Response: Thanks for your comment.As was mentioned in the manuscript, on page 4/lines 104-106: "The antibacterial potentiating effect of chlorogenic acid and carnosol was evaluated.None of the substances exhibited clinically applicable antibacterial activity, as MIC values were above or equal to 1024 µg/ml."The antimicrobial effect of the studied compounds in higher concentrations was not evaluated, because of the presence of possible cytotoxicity.
 Comment 3: Line 112, you need to detect the MIC of CCCP also explain why use this inhibitor with gentamicin.
 Response: Thank you very much for pointing this out.The manuscript was revised accordingly.We have made the following changes:  The determination of the MIC of CCCP has been included in the Materials and Methods section on page 10/lines 322 and 326.The results of this determination have been added to the Results section on page 4/lines 115-117.
 An explanation for using CCCP has been added to the Materials and Methods section on page 11/lines 354-355.
 Comment 4: Line 123, need to mention some details about isolated strain (host, type of infection etc.)  Response: Thank you for your comment.Line 123, entitled "a) Flow cytometric analysis of efflux pump inhibition on the XDR S. aureus-3" serves as a subheading for the efflux pump inhibition assay within the results section.It may not be suitable to discuss the issue raised by the respected reviewer within this particular section.We previously discussed these details on page 3/line 93.
 Comment 5: Line 125, you need reference " CGA, and CL that did 124 not impact cell viability "  Response: Thanks for your comment.In line 125, it was mentioned that "All experiments conducted in this study utilized concentrations of gentamicin, CGA, and CL that did not impact cell viability" which further points out that sub-inhibitory concentrations of these compounds have been used to continue the study process, "… to investigate the efflux pump activity, subinhibitory concentrations (1/8, 1/4 and 1/2 MIC) of gentamicin in combination with sub-MIC concentrations of CGA (16, 32, and 64 µg/ml along with 8, 16, and 32 µg/ml) and CL (4, 8, and 16 µg/ml along with 8, 16, and 32 µg/ml) were evaluated, respectively…"  Comment 6: Line139, P. aeruginosa this isolated associated with human infection or not did host receive antibiotic (please provide information about isolates)  Response: Thank you.Line 139, entitled "b) Flow cytometric analysis of efflux pump inhibition on the XDR P. aeruginosa-5" serves as a subheading for the efflux pump inhibition assay within the results section.Only the name of the isolate was mentioned in this part, and the results related to it were discussed further.It may not be suitable to discuss the issue raised by the respected reviewer within this particular section.We previously discussed these details on page 3/line 93.Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.Data Availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact ASM production staff immediately with the expected release date.
Publication Fees: For information on publication fees and which article types have charges, please visit our website.We have partnered with Copyright Clearance Center (CCC) to collect author charges.If fees apply to your paper, you will receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact CCC at ASM_Support@copyright.com or toll free at +1-877-622-5543.CCC makes every attempt to respond to all emails within 24 hours.
ASM Membership: 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.
PubMed Central: ASM deposits all Spectrum articles in PubMed Central and international PubMed Central-like repositories immediately after publication.Thus, your article is automatically in compliance with the NIH access mandate.If your work was supported by a funding agency that has public access requirements like those of the NIH (e.g., the Wellcome Trust), you may post your article in a similar public access site, but we ask that you specify that the release date be no earlier than the date of publication on the Spectrum website.

Embargo Policy:
A press release may be issued as soon as the manuscript is posted on the Spectrum Latest Articles webpage.The corresponding author will receive an email with the subject line "ASM Journals Author Services Notification" when the article is available online.
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 submitting your paper to Spectrum.

Table 3 :
Assessment of gentamicin antimicrobial activity in combination with CGA and CL by checkerboard assay.

Minimum Inhibitory Concentrations (MICs) of … Checkerboard Results based on interpretation of FIC index GEN
Line 106-CGA had an additive effect on P. aeruginosa strain.In table 3 it is mentioned indifference.Discrepancy should be corrected.
GEN: Gentamicin, CGA: Chlorogenic acid, CL: Carnosol, FIC: Fractional Inhibitory Concentration, XDR: Extensively Drug Resistant.a synergistic effect on XDR P. aeruginosa, investigation of efflux pump activity was conducted using subinhibitory concentrations (1/8, 1/4 and 1/2 MIC) of gentamicin (64,  Comment 3: Thank you for the nice reminder.As you indicated, gentamicin is commonly employed in the control of post-burn infections and skin infections caused by S. aureus.Thank you for this suggestion.It would have been interesting to explore this aspect further.However, in the case of our study, it appears to be slightly out of scope as the effects of CGA and CL on efflux pump activity were evaluated phenotypically, and Comment 2: Line, did you detect the MIC of CCCP against staph.And pseudomonas  Response: Thank you for your question.In response, a dilutions series of CCCP were prepared ranging from a concentration of 2-1024 μg/ml using MIC microdilution broth before conducting the efflux pump screening.The growth of S. aureus and P. aeruginosa strains was examined in this series of dilutions.It was observed that the growth of both S. aureus and P. aeruginosa strains remained unaffected until the concentration of 1024 μg/ml that was investigated.Finally, to examine efflux pump activity, CCCP at a final concentration of 25 µg/ml was added to each microtiter plate well containing 2-1024 µg/ml of gentamicin. Comment 3: Line 18, why you choose gentamycin, many different antimicrobial agent is more important as anti-staph.Infection.You need to show that study deal with staphylococcus strains that cause skin infection  Response:  Comment 4: In this section you need to suggest a possible mechanism for inhibition and is this compound inhibit specific type of efflux pump protein or not. Response: Comment 7: Line 162, at least mention that you use cell to check viability with some details  Response: Thank you for your comment.Line 162, entitled "Analysis of Cell Viability by the MTT Assay" serves as a heading within the results section, with the associated results elaborated on subsequently.The details of the MTT assay, including the cell line that was used (NIH/3T3), have been extensively detailed in the Materials and Methods on page 12/lines 396-420.Thank you for your comment.In this study, CGA was commercially prepared as an active chemical agent (Merck, Germany), but carnosol was isolated and identified from Salvia abrotanoides by Ghanadian et al. and then used.Previously mentioned in materials and methods, page 9/line 300.Thank you for your suggestion.Our main focus was to understand the overall impact of CGA and CL as potential efflux pump inhibitors on antibiotic efficacy, rather than delving into the specifics of efflux pump expression profiles.As mentioned in the Evaluation of Chlorogenic Acid and Carnosol for Anti-Efflux Pump and Anti-Biofilm Activities against Extensively Drug-Resistant (XDR) Strains of Staphylococcus aureus and Pseudomonas aeruginosa)  Comment 3: Suggested the source of EPIs (plant source or active chemical agent  Response:  Comment 5: Is the tested EPIs have selected action or not? Response: Thank you for your question.Further research is needed to provide more insight into this issue.Studies should focus on various bacterial isolates and strains, each demonstrating varying resistance to distinct antimicrobial compounds.Nonetheless, the current study demonstrated that CGA and CL potentially exhibit inhibitory effects on XDR S. aureus and XDR P. aeruginosa isolates, consequently leading to the suppression of efflux pump activity. Comment 6: Explain limitation of the study, didn't do expression profile of efflux pump to prof inhibition action of EPIs, used on type of antibiotic  Response:  Comment 3: Line 318, it is perfect to used agar-based method to confirm MIC  Response: Thank you for your comment.Both agar-based and MIC microdilution methods have their unique strengths and limitations.The MIC microdilution method is widely accepted and standardized, making it a reliable and reproducible technique for determining MIC values.It also allows for high-throughput screening of antimicrobial agents.