Multiple Mechanisms Synergistically Induce Pseudomonas Aeruginosa Multiple Drug Resistance
Round 1
Reviewer 1 Report
The authors presented a study in which carbapenemase-resistant strains of P. aeruginosa isolated in hospitals were analyzed. The results show that these strains are resistant by other mechanisms besides the presence of carbapenamases, but the types of carbapenemases found are not described. This part should be explained in the discussion.
There are some errors in Materials and Methods, correct the formatting of Table 1, the R primer of OprD gene is on two lines. Also correct the pre denaturation temperature which is written incorrectly.
Uniform the writing multi-drug resistant or extensively resistant P. aeruginosa. Sometimes it is written MDR-PA or XDM-PA and sometimes MDPA or XDPA.
In the Carbapenemase production chapter change the phrase "strains were detected by" to "strains were analyzed by" and put the percentage of strains positive for the Carbe NP test.
In Gene and expression analysis chapter add RT- PCR to the sentence “determined by semi-quantitative “.
Put all gene names and P. aeruginosa in italics.
Author Response
1.The authors presented a study in which carbapenemase-resistant strains of P. aeruginosa isolated in hospitals were analyzed. The results show that these strains are resistant by other mechanisms besides the presence of carbapenamases, but the types of carbapenemases found are not described. This part should be explained in the discussion.
Response: The presence of carbapenamases was confirmed by Antimicrobial susceptibility testing and the CARBE NP test. The types of carbapenemases were not further identified.
.2.There are some errors in Materials and Methods, correct the formatting of Table 1, the R primer of OprD gene is on two lines. Also correct the pre denaturation temperature which is written incorrectly.
Response: Revised.
3.Uniform the writing multi-drug resistant or extensively resistant P. aeruginosa. Sometimes it is written MDR-PA or XDM-PA and sometimes MDPA or XDPA.
Response: Revised.
4.In the Carbapenemase production chapter change the phrase "strains were detected by" to "strains were analyzed by" and put the percentage of strains positive for the Carbe NP test.
Response: Revised.
5.In Gene and expression analysis chapter add RT- PCR to the sentence “determined by semi-quantitative “.
Response: Revised.
6.Put all gene names and P. aeruginosa in italics.
Response: Revised.
Reviewer 2 Report
In this manuscript, the authors have investigated different resistant mechanisms in 34 strains of clinically isolated Pseudomonas aeruginosa. The findings highlight the contribution of efflux pumps, outer membrane proteins and degradative enzymes that render the bacteria multi or extensively drug resistant.
6th line in Introduction needs to be rephrased, "resistant" is redundant.
In the methodology of RT-qPCR, " transcriptional levels were diploid higher", needs to be rephrased. Transcription was up two fold or two times upregulated.
3.1 Results section needs to be rephrased. Consistency is required when you are writing a number; digits vs spelling it out. It would be helpful to have this data in tabular form.
3.3: semi quantitative RT PCR. Need to elaborate and refer about how Mex is involved in drug efflux.
Rephrase: Compared to the control group, OprD expression was significantly downregulated to different magnitudes.
Mention in section 3.3, about the overlap among strains that produce carbapenemases and had upregulated efflux genes.
In Figure 1: Graphs don't have any error bars. Were these experiments done in duplicate/triplicate?
Y-axis spell check " Relative mRNA)
Explain what is depicted by X-axis.
Avoid the use of "diploid". In the figure legend it should be either clinical isolates of Pseudomonas aeruginosa or cinically isolated Pseudomonas aeruginosa.
3.4 Format all Pseudomonas aeruginosa to italics.
The whole section needs rephrasing. The sentence describing gene cassettes needs to be broken down in two sentences. Unclear what is being said.
Leads to resistance
Decreased Sensitivity
3.5: In Fig. 3A, For the CRPA strains, did they not produce carbapenemases? Was decreased expression of OprD the only mechanism of resistance? Fig. 3c, graph key is missing.
Overall, each result section needs rationale of investigating the pertinent gene etc., and needs to be phrased more clearly.
Author Response
1.6th line in Introduction needs to be rephrased, "resistant" is redundant.
Response: Revised.
2. In the methodology of RT-qPCR, " transcriptional levels were diploid higher", needs to be rephrased. Transcription was up two fold or two times upregulated.
Response: We changed it into two times higher.
3.Results section needs to be rephrased. Consistency is required when you are writing a number; digits vs spelling it out. It would be helpful to have this data in tabular form.
Response: Revised.
4.semi quantitative RT PCR. Need to elaborate and refer about how Mex is involved in drug efflux.
Response: MexA, a membrane fusion protein, attached to the inner membrane with a lipid anchor, found in the periplasmic space, that is essential for in vivo transport and pump assembly. and OprD, a channel from the outer membrane factor family located in the outer membrane that allows the extrusion of antibiotics. These are the two main mechanisms of P. aeruginosa resistance. We added it in Results section.
5.Rephrase: Compared to the control group, OprD expression was significantly downregulated to different magnitudes.
Response: Revised.
6.Mention in section 3.3, about the overlap among strains that produce carbapenemases and had upregulated efflux genes.
Response: Revised.
7.In Figure 1: Graphs don't have any error bars. Were these experiments done in duplicate/triplicate?
Response: The experiment was repeated three times.We have revised it.
- axis spell check " Relative mRNA
Response: Revised.
Explain what is depicted by X-axis.
Response: Revised.
Avoid the use of "diploid". In the figure legend it should be either clinical isolates of Pseudomonas aeruginosa or cinically isolated Pseudomonas aeruginosa.
Response: Revised.
8.Format all Pseudomonas aeruginosa to italics.
Response: Revised.
9.Avoid the use of "diploid". In the figure legend it should be either clinical isolates of Pseudomonas aeruginosa or cinically isolated Pseudomonas aeruginosa.
Response: Revised.
10.The whole section needs rephrasing. The sentence describing gene cassettes needs to be broken down in two sentences. Unclear what is being said.
Leads to resistance
Decreased Sensitivity
Response: Revised.
11.In Fig. 3A, For the CRPA strains, did they not produce carbapenemases? Was decreased expression of OprD the only mechanism of resistance? Fig. 3c, graph key is missing.
Overall, each result section needs rationale of investigating the pertinent gene etc., and needs to be phrased more clearly.
Response: We deleted Figure 3. We have revised the grammar of the full text.
Reviewer 3 Report
1. The authors mentioned different synergistic mechanisms for the induction of multidrug resistance in Pseudomonas aeruginosa. But it's disorganized. These mechanisms need to be described and substantiated in more detail.
2. There are some statements that need to be reconfirmed. And recheck the full manuscript.
Line 224: "pathogens in the first place of [11]."
3. More experiments are needed to support the conclusions.
Author Response
- The study is an epidemiological investigation. After exploring the major mechanisms of drug resistance, further investigation of one of these mechanisms will be carried out. Current work is focused on screening for major resistance mechanisms.
- We corrected some grammar mistakes.
- We add a description of Study Limitation in the discussion section.
Reviewer 4 Report
This paper reports the drug resistance of carbapenem-resistant Pseudomonas aeruginosa (CRPA). 34 CRPA strains were isolated and their resistance to antibiotics was detected. Carbapenemase production and the expression of MexA and OprD were employed to evaluate the drug resistance. These experiments design is rational and reliable. This study allows researchers know more about the Pseudomonas aeruginosa resistance. It may provide some useful information for the clinical treatment for CRPA. The shortcomings of this study are that it only gives detection data for the resistance and makes some discussion. The authors did not attempt to develop drug formula to solve the resistance problem. This paper only tells readers what happens, but does not provide solution.
Some questions, see below.
Page 6, line 146, <and OprD> should be <And OprD>.
Page 6, line 147, < These are the two main mechanisms of P. aeruginosa resistance> I do not think this expression is not accurate enough. <MexA and OprD are the two main genes that lead to P. aeruginosa resistance.>may be more appropriate.
Page 11, line 224, in the first place of…, missing words?
Page 11, lines 225-236, resistance rate was discussed. Please give the calculation method for the resistance rate in the section of materials and methods.
Author Response
- The study is an epidemiological investigation. After exploring the major mechanisms of drug resistance, further investigation of one of these mechanisms will be carried out. The attempt to develop drug to solve the resistance problem will be carried out too. Current work is focused on screening for major resistance mechanisms.
- We corrected some grammar mistakes. The calculation of resistance rate was added.
Round 2
Reviewer 2 Report
The authors have revised the manuscript based on my comments. They have now provided sufficient explanation in the results section and have corrected the grammar and formatting, through out. i am satisfied with the author's response.
Author Response
Thank you very much.
Reviewer 3 Report
Some modifications were made by the authors. But there are still some errors in the manuscript.
Such as Line 150 "that lead to P. aeruginosa resistance..". Remove redundant punctuation.
