ESBL/AmpC-Producing Enterobacteriaceae Fecal Colonization in Dogs after Elective Surgery

Round 1

Reviewer 1 Report

In the manuscript by Belas et.al, authors evaluate the presence and load of ESBL/AmpC-producing Enterobacteriaceae faecal carriage in healthy dogs before and after surgery.

However, there are several points that need to be addressed/improved:

Line 40: please be more specific when describing the resistance conferred by ESBLs e.g most beta-lactam antibiotics, including penicillins, cephalosporins, and the monobactam aztreonam.

Lines 42-46: The sentence needs to be completely revised.

 “ESBLs and plasmid-mediated cephamycinases (pAmpC), involve mutations in genes targeted by the antimicrobial…” is not clear at all, especially since both ESBLs and AmpCs are enzymes capable of hydrolyzing the beta-lactam ring of beta-lactam antibiotics before they reach their target, so which mutations are authors referring to?

Also “the transfer of resistance determinants borne on plasmids, and other mobile genetic elements carrying resistance genes, such as plasmid-encoding β-lactamases and transposons can easily be transmitted by conjugation to other bacteria even across species”

1. Determinants are CARRIED by plasmids and other MGEs not borne, and that includes transposons
2. Perhaps authors mean plasmid ENCODED ß-lactamases?
3. Only plasmids are disseminated (not transmitted) by conjugation, transposons are contained with plasmids, they do not conjugate on their own.

Also, please refer to pAmpCs as plasmid encoded AmpC beta-lactamases.

Line 46. Please consider rearranging to “the most frequent ESBL producers are…

Line 91: Can authors please provide the rationale for using 2ug/mL of cefotaxime considering CLSI's breakpoint for Enteobacteriaceae is 1 ug/mL?

Line 108: Can authors confirm that CLSI 2018 document was in fact used for interpretation, and what is the rationale for not using the latest document? Also please provide exact reference.

Line 114: ß-lactamase resistance genes

Line 120: Due to the low number of samples collected a risk factor analysis is not recommended, however if authors conducted a sample calculation or any other statistical validation, please include it. Otherwise, it would be advisable to just remove this part.

Line 124: perform

Lines 148-149: please rearrange for clarity “In this study, 20.0% (n=5/25) of the dogs were colonized with ESBL-producing Enterobacteriaceae at the time of hospital admission (BS).

Also, what species were found? All E.coli?

Line 151: 2 of the 5? Or two of the 3 with the described phenotype. Please clarify

Lines 153-156: please clarify if more than a single species was found in any of the samples, and if so in how many.

Please consider removing “surgery group” throughout the manuscript since it is redundant and confusing due to the fact that all animals included in the study were subject to surgery.

Table 1: Can authors please confirm if FMVS3a, b, c, d   were obtained from the same fecal sample? If so, were All 3 K.pneumoniae (b, c, d) really morphologically different on the MacConkey plate?

Lines 187 -190: The results of these analyses need to be shown, otherwise “none of the variables” (when the variables analyzed are not even clearly mentioned anywhere in the text) has no real meaning. Please refer to comment on Line 120 above.

Lines 208-212: I suggest removing or rearranging this sentence since authors themselves argue in the very next sentence that in most cases 3CG resistance in E.cloacae is caused in most cases by overproduction of “AmpCs”, more specifically, their own constitutive (and most importantly INDUCIBLE) AmpC.  

Discussion: Please be careful with the chosen language to avoid overstatements.

e.g. Line 227: “significantly increased DURING” … as stated in the text, evaluation points were before surgery and one week after surgery.

Can authors confirm how long were dogs treated with the mentioned antibiotics? Specifically, were BS samples collected before initiation for prophylactic treatment? and were dogs still being administered antibiotics when AS were samples collected ????

e.g. Line 251: What do authors mean by higher diversity? In the BS surgery E.coli and K.pneumoniae were found, vs. E.coli, K.pneumoniae and E.cloacae in AS, one more species can hardly be qualified as “higher diversity”

Lines 263-270: since this particular study does not deal with microbiome, these paragraphs although interesting don’t seem to fit with the overall narrative of the manuscript. Consider removing them.

Line 277: consider revising in line with previously raised issues about sample size.

Author Response

We thank Reviewer 1 for stating the importance of the subject studied in this manuscript. In the next answers we have approached the Reviewer 1 comments point-by-point and made changes to improve the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors report findings from a small study that are relevant specifically to veterinary practice and more broadly to human and global health. Comments:

1) Variables, line 80 forward: Diet is long known to affect fecal bacteria flora.

https://rupress.org/jem/article/115/6/1161/3074/THE-EFFECT-OF-DIET-ON-THE-FECAL-BACTERIAL-FLORA-OF

https://onlinelibrary.wiley.com/doi/full/10.1111/jsap.13000

Comment on why these data were not collected. In the Discussion, list and explain this as a limitation.

2) Medications other than anti-microbials modify the gut microbiota.

https://www.nature.com/articles/s41467-019-14177-z

As above, comment on why these data were not collected. In the Discussion, list and explain this as a limitation. 

3) Results, lines 133-147: Report these descriptive statistics in a table.

4) Conclusions, line 280-: What are current guidelines for prophylactic ABx in Europe, the US and elsewhere? Do none exist?

Author Response

We thank Reviewer 2 for stating the importance of the subject studied in this manuscript. In the next answers we have approached the Reviewer 2 comments and made changes to improve the manuscript.

Author Response File: Author Response.pdf