Two Screening Assays to Detect Vancomycin-Resistant Enterococcus spp.

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Dear Reviewer, please find enclosed our replies.

Thank you for helping us improving the paper,

Beniamino Cenci Goga

Author Response File: Author Response.pdf

Reviewer 2 Report

The study by Beniamino Cenci Goga et al. showed 2 screening assays for detection of vancomycin resistance in enterococci, Barnes Basal Medium Agar (Ba) and Brain Hearth Infusion broth with added 1% TTC (BHI). It is an interesting and meaningful research. The work would benefit with further recision to help strengthen the main conclusions and to better understand.

Some errors in the figures presented, such as in fig3 and 4, should be “0.7” but not “0,7” et al.

In the whole text, “cfu” or “cfus”should be “CFU” or “CFUs”

There are many grammatical errors in the article, and even some simple errors that lack of disconnection between sentences. In addition, the logic of introduction is a little chaotic, with no progression or turning between paragraphs, which makes the article difficult to read.

Line29, “broth with added 1% TTC (BHI) with”, “with…with…” should be revised.

Line37-39, “Antibiotic-resistant enterococci represent an impending danger, due both to the high mortality associated with the infections they sustain and to the possible spread of resistance to other bacterial species”. This sentence is too long and too complex. “due both to” not common used.

Line40, “in nosocomial infections from difficult to treat enterococci” “from”?

Line44, “This is in part explained by the resistance of some of these bacteria to most antibiotics”, this sentence is difficult to comprehend.

Line46, “According to Werner, Coque, Franz, Grohmann, Hegstad, Jensen, van Schaik and Weaver”, only the main author to be mentioned here, such as Werner et al.

Line51, “Gram-positive bacteria It appears also that” should be “Gram-positive bacteria. It also appears that”.

Line54, “On the other hand, they have been ascribed a beneficial or detrimental role in foods”, what does “they” refer to?

Author Response

Dear Reviewer, please find enclosed our replies.

Thank you for helping us improving the paper,

Beniamino Cenci Goga

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Line 62- Use  VRE for vancomycin-resistant enterococci. 

Line 96- Make VanC as italic.

Please check the manuscript for these types of minor errors. Check for uniformity, italic, extra space, and etc. 

Reviewer 2 Report

The manuscript has been carefully revised and all the suggestions of the reviewers conveniently addressed.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In this manuscript Cenci-Coga et al. describe the application of different growth media to evaluate vancomycin resistance among enterococci. To do so a general medium (MH), a differential medium (BHI-TTC) and a selective and differential medium (Ba) are used to measure growth inhibition using different concentrations of vancomycin and the results are compared to standard MIC protocols.

Whereas the objective of the work can be very useful for quick assessment of antibiotic resistance spread, the data presented here do not substantiate that this has been achieved. It is unclear whether the applicability of this method still requires isolation of the enterococcal strains or can be extended to complex samples.

Overall, the work reads well although some typos and strange expressions (eg. Line 59 is grammatically incorrect; Line 46, formazzano; line in this thesis; line 94 whit; line 107 R is not specified; species names are not displayed in italics in the text and references).

The methods section is not sufficiently described. Moreover, the sterilization protocol applied is disregarded for Ba medium according to the provider, who clearly indicates that autoclaving must be avoided. Additionally, using just one strain (with only one of the many types of vancomycin resistance described) to develop a method that aims at broad application does not seem sufficient to prove the utility of the method. Some other issues:

-The term antibiotic activity used in the formula (line 71) is not clearly described.

-The authors indicate the use of TTC in the media for enterococci identification but it is not used in MH. Moreover, other microorganisms that might be in a sample can reduce TTC as well.

-The volume of medium used to pour MH plates is not indicated, thus the ratio cells:antibiotic molecules can vary.

-The title indicates that solid media are being used, but three media with different inoculation techniques and one of them liquid is used as a sort of MPN method (line 113).

-Lines 137-148 are more appropriate for a discussion

 

The results are presented in a repetitive manner. Thus, for each medium tables and graphics using the same data in a different way are provided and this could be simplified. Statistical analysis is not indicated and standard deviations are not presented. Considering the experimental variations that MIC determinations can have, a 2-fold variation falls within the expected error. VanA resistance in literature typically refers to levels above 64 mg/L vancomycin, far above that of the reference strain used.

Reviewer 2 Report

I'm very sorry, but this article do not provide any advance on the topic and in general it is wrote in a non scientific way.

In the work titled "An agar screen plate-based method for the detection of vancomycin-resistant Enterococcus spp" the authors describe a "new" agar-based screening method to identify Enterococcus vancomycin-resistant strains. For that, the authors did agar dilution assays adding different CFU/ml of cells and using three different culture medium. The authors claim that this methodology is innovative, but it is not, and they are not providing any advantage with respect to the current methodologies. In fact, agar dilution methods for the detection of the MIC (minimal inhibitory concentration) are generally used for antibiotic resistance, and the CFU/mL used in such assays to be comparable clearly described. Perhaps, the only difference concerning the current method is that the authors are adding the bacteria to the medium directly (if I understood correctly). The introduction is insufficient and the objective of the work is not established, the benefits of the proposed methodology in the current ones are not indicated. Material and method are extremely descriptives and impossible to understand clearly. The strains selected for the assays was Enterococcus durans, indicating that it is vancomycin-resistant. However and given the results, it is not, since the observed MIC for vancomycin in the presented results was 2mg/L or lower and the breaking point for Enterococcus and vancomycin according to ECOFF is 4mg/L. So it is sensitive. Next, they used three different mediums for the assay, between them Barnes agar. The authors indicate that the culture mediums were autoclaved while this medium is not autoclavable. About the medium inoculation, the author inoculated the different agar medium (un unknown volume) with 1ml of cells at 10e4 to 10e9. Even if they diluted 10 times into the medium, the obtained plates will have uncountable colonies. This is the case of MH medium and Ba medium, although in this last case the authors also claim for another way, spreading the cells at different concentration on the surface of the plate. Anyway, the counting of bacteria is not possible using this methodology. In the case of BHI medium, the authors did it in liquid, diluting the bacteria 10 times and looking for the change in TTC colour as an indication of inhibition. In all of the cases, the incubation time was 48h which is an excessive time for an antimicrobial test against this kind of bacteria according to the currently accepted methodologies. Overall for material and method, the authors are extremely descriptives but they don’t provide useful information. Besides, some methodological approaches used are not correct or unclearly described. About results and discussion points, the first is that discussion is missing. The authors just list the results. For the proposed MH ''new'' approach the author claims just one antibiotic dilution of difference respect the reference method, but what reference method? How can the authors compare different kinds of methodologies? It is absolutely clear that if you use different methodologies for MIC determination the result is not the same. What CFU/mL is comparing to the reference method? How the author count 7 log CFU/mL without serial dilutions? I have the same concerns for the rest of the work. Neither error bars are indicated in the figures. Finally, in conclusions, the authors claim for something not related to the results and even they comment about results obtained in other different media not described in the work (Peptone water).