Quantitative Microbial Risk Assessment of North Carolina Type 2 Reclaimed Water for Agricultural Reuse

Round 1

Reviewer 1 Report

The document presents an ordered structure and uses different mathematical expressions that clearly explain the analysis of the data for the subsequent interpretation of the results. Another element that provides solidity to this work is the statistical analysis, which is of special relevance, since these analyzes generally have a great need, since it implies a triangular biological scenario; a) the improved genetics of the microorganism, b) the immune response of the recipient and c) the transfer process.

Regarding the statement described in lines 124 and 125, "2) penetration of microorganisms through the root system or another pathway into a plants' internal parts", it is pertinent to indicate that a phenomenon of penetration must be understood as a change in the qualitative composition of the rhizosphere associated with the plant being cultivated. From the perspective of Microbial Ecology, it is a phenomenon that must be treated as part of the process of competition of microorganisms for the ecological niche of an autotrophic organism, where there is potentially a source of resources, and that is the reason why give competition phenomena. Treating this scenario under the concept of "contaminating agent", despite the fact that it is understood by the focus of the work, maintains a clinical perspective, which does not contemplate that these microorganisms under this ecosystem niche can express a non-pathogenic phenotype. This observation is interesting, since the results that the work later discusses assume that these organisms are intrinsically pathogenic, however, there are elements during transit from their presence in the soil and food to the receiving organisms where factors and virulence genes, can be lost and gained, either by mechanisms of intrinsic variability (due to mutation and phenotypic plasticity) or by exogenous mechanisms such as horizontal gene transfer. These two elements are not considered in the analysis and are present in the system, which could affect the reproducibility of this type of analysis, whose strength is based on statistical analysis. In this sense, I consider it pertinent that the authors include a clarification of this aspect in the discussion of the work.

Finally, in line 239, the reasons that contribute to measuring the potential risk of water use are listed, however, in my opinion and despite the fact that it is not weighted in the work, I think it would be convenient to add that the time of year It is also a factor to consider since the environmental temperature range in turn conditions microbial growth, which determines to a certain extent the number of bacteria that come into contact with the receiving organism.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

The research conducted a quantitative microbial risk assessment using microbial data collected from reclaimed waters, which have been proposed as supplemental irrigation water. The topic of the article is interesting. I recommend adjusting the points below for publication:

-Please, briefly explain the protocols used to detect target microorganisms.

-Please, explain clearly, why equation 1 was chosen to calculate microbial concentrations (It is not expected that all microorganisms have the same behavior).

- Please, include a "Conclusions" item in the document

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf