The Effect of Cell-Free Nontuberculous Mycobacterium Supernatants on Antibiotic Resistance and Biofilm Formation of Opportunistic Pathogens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this article, Shchuplova et. al conducted a study on the effects of nontuberculous mycobacterium supernatants on antibiotic resistance and biofilm formation of four opportunistic pathogens, namely E. coli IKVS S1, E. coli IKVS S18, S. epidermidis IKVS S7 and S. epidermidis IKVS S13. The authors observed that the cell-free supernatants can enhance the biofilm formation and reduce antibiotic susceptibility of the investigated pathogenic strains, which are consistent with previous studies and hypotheses.

 

The study is generally well-designed to support the conclusion, and the article is well-written in conveying the study design and scientific findings. The background information adequately highlights the importance and significance of the study. However, there are a few improvements needed to meet the publishing standards of the journal.

 

1.     In Result 2.1, the main finding is that the cell-free supernatant of M. iranicum can stimulate the biofilm formation of the four pathogenic strains. However, Figure 1 directly presents the calculated enhancement of biofilm formation and compares different pathogens, which may not be the most direct way to support this conclusion. I suggest the authors add another figure to display the raw OD450 data, including control samples, and indicate the statistical significance level between control and supernatant-treated samples.

 

2.     In Result 2.1, the author notes that the enhancement of biofilm formation varies among different pathogenic strains and mycobacterium strain supernatants. However, the potential biological significance of this observation is not provided. What may be causing this difference? What clinical implications does this difference hold? Explaining this observation with a sentence or two can enhance the impact and significance of the study.

 

3.     Please specify the number of replicates in this experiment, either in the figure or in the Method section.

 

4.     Tables 2-5 should be numbered as Tables 1-4.

 

5.     Please specify the number of replicates in Tables 1-4, either in the table footnotes or in the Method section.

 

6.     In lines 99-103, please add a citation or provide data to support this argument.

 

7.     In line 100, this is the first mention of "anti-hemoglobin" in the main text. Please annotate the abbreviation accordingly and provide a brief background explanation of the function of anti-hemoglobin and its relevance to this study for the reader.

Comments on the Quality of English Language

- Lines 99-103: this sentence contains two instances of "compare." To make the sentence more concise and clearer, please rephrase it.

- Line112: S. epidermidis IKVS 7 [and] 13. “and” is replaced by “u” in the original text.

Author Response

Dear reviewer! Thank you for your careful reading of our work and for your suggestions for improvement.

1. In Result 2.1, the main finding is that the cell-free supernatant of M. iranicum can stimulate the biofilm formation of the four pathogenic strains. However, Figure 1 directly presents the calculated enhancement of biofilm formation and compares different pathogens, which may not be the most direct way to support this conclusion. I suggest the authors add another figure to display the raw OD450 data, including control samples, and indicate the statistical significance level between control and supernatant-treated samples.

For better understanding, the graphics have been divided. The ability of biofilm formation was calculated using the given formula relative to the optical density of solutions in (%). The average OD values are presented in the table.

E. coli IKVS 1
M. iranicum IKVS S1	ОП 0,190±0,003	M. iranicum IKVS S 2	ОП 0,178±0,003
Control	0,144±0,002	Control	0,153±0,002
E. coli IKVS 18
M. iranicum IKVS S1	ОП 0,177±0,003	M. iranicum IKVS S 2	ОП 0,163±0,003
Control	0,147±0,002	Control	0,149±0,002
S. epidermidis IKVS 7
M. iranicum IKVS S1	ОП 0,223±0,004	M. iranicum IKVS S2	ОП 0,219±0,004
Control	0,177±0,003	Control	0,213±0,003
S. epidermidis IKVS 13
M. iranicum IKVS S1	ОП 0,273±0,005	M. iranicum IKVS S2	ОП 0,269±0,005
Control	0,212±0,003	Control	0,250±0,004

 

 

2. In Result 2.1, the author notes that the enhancement of biofilm formation varies among different pathogenic strains and mycobacterium strain supernatants. However, the potential biological significance of this observation is not provided. What may be causing this difference? What clinical implications does this difference hold? Explaining this observation with a sentence or two can enhance the impact and significance of the study.

In strains with high levels of AntiHbA (except for S. epidermidis IKVS 7), increased biofilm formation was observed under the influence of supernatants. We consider antihemoglobin activity as one of the pathogenicity factors. Strains characterized by a high level of this property contribute to the development of an infectious-inflammatory process, as shown in the works of [T.M. Pashkova, N.V. Morozova, M.D. Kuzmin, O.L. Kartashova, L.P. Popova Characteristics of the pathogenic potential of Escherichia coli isolated from patients with calculous pyelonephritis Urology. 2021. No. 4. pp. 19-24. DOI: https://dx.doi.org/10.18565/urology.2021.4.19-24] It was previously shown (data not presented) that strains with high levels of AntiHbA are characterized by the presence of other pathogenicity factors (hemolytic, gelatinase, adhesive activity, etc.) [Shchuplova E.A., Herzen N.V., Fadeev S.B., Valyshev A.V. Antihemoglobin activity of enterococci // Bulletin of OSU 2014, No. 13. P. 139-142.]. This study revealed increased biofilm formation in strains with high levels of AntiHbA (with the exception of S. epidermidis IKVS 7), the authors suggest that opportunistic microorganisms in the body can also lead to the development of an infectious process when influenced by NTM.

3. Please specify the number of replicates in this experiment, either in the figure or in the Method section.

All experiments were performed in triplicate, and the results are expressed as the mean ± SD. Student's test was performed to determine the significance of the results obtained.

The necessary corrections have been made to the text of the article.

4. Tables 2-5 should be numbered as Tables 1-4.

The table numbering has been corrected.

5. Please specify the number of replicates in Tables 1-4, either in the table footnotes or in the Method section.
6. In lines 99-103, please add a citation or provide data to support this argument.

This proposal reflects the experimental results obtained by the authors and are shown in Table 3. The generalization we made in this proposal is not correct and has been corrected. In lines 170-175

7. In line 100, this is the first mention of "anti-hemoglobin" in the main text. Please annotate the abbreviation accordingly and provide a brief background explanation of the function of anti-hemoglobin and its relevance to this study for the reader.

Comments on the Quality of English Language

- Lines 99-103: this sentence contains two instances of "compare." To make the sentence more concise and clearer, please rephrase it.

The necessary changes have been made.

It should be noted, it can be noted that under the effect of the M. iranicum IKVS S1 supernatant, the strain E. coli with the higher anti-HbA values showed a lesser decrease in the susceptibility to antibiotics of the group of β-lactams, aminoglycosides, fluoroquinolones and azalides compared to the strain E. coli characterized by the lower anti-HbA values.

- Line112: S. epidermidis IKVS 7 [and] 13. “and” is replaced by “u” in the original text.

The necessary changes have been made.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors evaluated the effect of cell-free M. iranicum on the biofilm formation and antibiotic resistance of E. coli and S. epidermidis. The results are clearly presented. My comments/suggestions are as follows:

1.     In Figure 1, the author used the cell free supernatant from two strains of M. iranicum to test their effect on E. coli and S. epidermidis, I was wondering why didn’t show the data of control? The X-axis represents Biofilm formation (%), I suggest that using the data directly showing the biofilm formation. Moreover, the label of significant analysis is not so clear, the labels should align with the columns while they are not.

2.     All the table numbers in this manuscript were not right.

3.     What does “IKVS” mean? Why do all the three bacterial strains use this word? The author should give its full name when you mention it for the first time.

4.     M. iranicum IKVS S1 induces more biofilm formation compared to M. iranicum IKVS S2, but why in some cases the cells show decreased susceptibility when treated by M. iranicum IKVS S2 but not by M. iranicum IKVS S1? e.g. E. coli IKVS 1 to cefepime and gentamicin; S. epidermidis IKVS 7 to ciprofloxacin.

5.     I don’t understand why the authors describe the characters of NTM strains in the Materials and Methods part, this information belongs to the background of this research and should be in the introduction.

 

6.     Why did the author use different times in testing the biofilm formation intensity and the bacteria antibiotic susceptibility? Moreover, when testing bacteria antibiotic susceptibility, it seems the cells to be checked are free cells, I didn’t see they had some relationship with biofilm.

Author Response

please find them attached 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for addressing my questions.

 

I have no more questions.