Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage
, Abimael Gomes da Silva 1, Tamiris Yoshie Kiyama de Oliveira 1, Tatiane Carla Silva 1, Fabio Gomes Assis Junior 1, Loane Dantas Krug 1, Adriano Augusto de Paiva Custódio 2, Rui Pereira Leite Júnior 2, Paulo Eduardo Teodoro 3, Bart Fraaije 4 and Paulo Cezar Ceresini 1,*Round 1
Reviewer 1 Report
In this manuscript, the authors are devoted to analyzing the correlation between efflux pumps and the multidrug resistance of PoTl. The authors determined the sensitivity of several Po strains to four efflux pump substrates and performed a Pearson’s correlation analysis between their data and the previous drug resistance data. This work analyzed a large number of publications on this topic. The paper is very well written, but I have only one significant concern. The authors claimed in the abstract that “the main objective of this study was to test the hypothesis that QoI-, DMI- and/or SDHI-resistant isolates of PoTl overexpress efflux pumps.” However, there was no direct experiment to solve this question in this manuscript. Although the four chemicals were often used as the antifungal efflux pump substrates, other factors leading to the sensitivity changes can not be excluded. Systemic expression analysis of efflux pumps in Pol after drug treatment needs to be conducted to further support the conclusion (qPCR or RNA-seq may be a choice).
Other minor comments:
1. Line 163, which method was used in the standardization?
2. Line 196, the word “the” was duplicated.
3. Line 212, 213, 215: the citation format should keep consistent with the main text.
4. In Figure 1, the meaning of the bar should be given in the legend. Is the standard deviation?
5. Many scientific names in titles are not italic in the reference list, but I am not sure whether some editor would correct them.
Author Response
We thank the reviewer very much for the suggestions made to improve the final version of the manuscript.
Addressing the first comment: "...but I have only one significant concern. The authors claimed in the abstract that “the main objective of this study was to test the hypothesis that QoI-, DMI- and/or SDHI-resistant isolates of PoTl overexpress efflux pumps.” However, there was no direct experiment to solve this question in this manuscript. Although the four chemicals were often used as the antifungal efflux pump substrates, other factors leading to the sensitivity changes can not be excluded. Systemic expression analysis of efflux pumps in PoTl after drug treatment needs to be conducted to further support the conclusion (qPCR or RNA-seq may be a choice)":
The reviwer comment is sound but our goal was not to test hypothesis on efflux pump mechanisms by looking at systemic expression analyses after drug treatment. Instead, we looked at revealing efflux pump mediated MDR by phenotipically characterizing the sensitivity of constrasting PoTl isolates to efflux pump substrates. To comply with these objectives, we adjusted the phrase on the hypothesis testing, so it reads:
"Therefore, the main objective of this study was to test the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to antifungal efflux pump substrates. "
Other minor comments:
1. Line 163, which method was used in the standardization? We replaced the word standardization with "normalization", which gives better understanding of what we have done. The new phrase now reads: "Resistance factors (RF) were determined to estimate the relative fold-inhibition of all the strains tested based on their EC50 values normalized by the lowest EC50 for each substrate."
2. Line 196, the word “the” was duplicated. Removed as suggested.
3. Line 212, 213, 215: the citation format should keep consistent with the main text. Thanks for noticing those citations. All three were adjusted as needed.
4. In Figure 1, the meaning of the bar should be given in the legend. Is the standard deviation? No, it is not standard deviation. Bars contain the sample median and outer quantiles at 95% confidence interval (the 2.5th and the 97.5th percentiles as the lower and the upper confidence limits). Data points are distributed along the bar. We added this phrase in the legend as suggested.
5. Many scientific names in titles are not italic in the reference list, but I am not sure whether some editor would correct them. Thanks for noticing those missing italics in the references. There were plenty, in fact. We fixed them all.
Reviewer 2 Report
The manuscript written in good format and contains high quality data
Author Response
Thanks a lot for your positiveness towards our study.
Reviewer 3 Report
The study characterized the QoI, DMI, and SDHI resistant Pyricularia oryzae Triticum lineage isolates by their substrates to see whether those isolates were MDR. Four antifungal substrates were tested in the study. Overall, the paper were in good shape. The main question I got was why don’t you include the sensitive PoTl isolates in your study, to test their sensitivity to four substrates. Thus, you can compare the RF of the resistant isolates with the sensitive isolates. Besides, Table 1 need to be modified, it is not self-explained. Also Table 2 can be improved, seems some lines were missing.
Author Response
Thanks a lot for your consideration towards our study.
Addressing the reviewer´s main concern: "The main question I got was why haven´t you included the sensitive PoTl isolates in your study, to test their sensitivity to the four substrates."Unfortunately, we do believe that this information went unnoticed by the reviewer because sensitive isolates for all three fungicides were included as controls. Please check Table 2: a) for QoI: isolates 12.1.015, 12.1.165, 12.1.130 and four PoO isolates are all QoI sensitive; b) for DMI: PoO 656, 421, 674 and 704, Pg 363 are all sensitive to DMI; in this case, in particular, our population study with more than 170 isolates indicated no sensitivity to DMI in PoTl (reference: Poloni et al. [9]), which demanded us to include sensitive isolates from the syster species PoO (P. Oryzae Oryza lineage); for SDHI: isolates PoTl 12.1.005, 12.1.165 and 18PRH9, PoO 656 and 421, and Pg 363 are all sensitive to SDHI.
Another concern from reviewer 3: "Besides, Table 1 need to be modified, it is not self-explained. Also Table 2 can be improved, seems some lines were missing." We do believe that Tables 1 and 2 are self explanatory, containing extensive legends, and therefore there is no need for adjustment.
