Selection and maintenance of mobile linezolid-resistance genes and plasmids carrying them in the presence of florfenicol, an animal-specific antimicrobial
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Mobile linezolid-resistance genes (optrA, poxtA, and cfr) that confere resistance to linezolid and florfenicol have been detected globally in various sources. Linezolid is a last-resort antimicrobial used in human clinical settings, and florfenicol is commonly used in veterinary clinical settings. The present study sought to evaluate the potential of florfenicol in veterinary use to select for linezolid-resistant bacteria. The growth and fitness of linezolid-resistant bacteria harboring mobile linezolid-resistance genes were assessed in the presence and absence of florfenicol using Enterococcus faecalis and E. faecium, respectively. The bacterial strains harbored wild and cloning plasmids carrying mobile linezolid-resistance genes, which reduced their susceptibility to linezolid and florfenicol. The acquisition of plasmids carrying mobile linezolid-resistance genes improved bacterial growth in the presence of florfenicol, and conferred fitness costs in its absence. Florfenicol imposes a selection pressure on bacteria harboring plasmids carrying mobile linezolid-resistance genes. Hence, the appropriate use of florfenicol in veterinary clinical settings is important to control the dissemination of mobile linezolid-resistance genes and to ensure the sustained effectiveness of linezolid against multidrug-resistant bacteria, including vancomycin-resistant enterococci in human clinical settings.
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Editor's Comments to Author Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments are attached at the bottom of this email. Although they agree that the study presented is of interest, they have a number of concerns and multiple corrections suggested that will strengthen the manuscript. Please provide a revised version of the manuscript (including a tracked changes document), along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
1. In Figure 1, authors examined ODs under the absence or presence of florfenicol. Why did authors measure bacterial growth fitness until 16 hours? It seems to be too short to confirm the bacterial fitness and to reflect the real situation for bacterial evolution in environment. In addition, if it keeps measuring over 16 hours, the other mutants (TF_K6D_FA and TF_L6E_FA) could reach to a close level or catch up that of TF_L6D_FA strain. 2. In Table 1, authors generated transformants and transconjugants to evaluate the bacterial growth in absence or presence of florfenicol selective pressure. However, the E. faecium strain carrying the pAM_poxtA_BM vector did not exhibit increased resistance to florfenicol, compared to the strain having the poxtA gene, which might be derived from an identical origin of …
Comments to Author
1. In Figure 1, authors examined ODs under the absence or presence of florfenicol. Why did authors measure bacterial growth fitness until 16 hours? It seems to be too short to confirm the bacterial fitness and to reflect the real situation for bacterial evolution in environment. In addition, if it keeps measuring over 16 hours, the other mutants (TF_K6D_FA and TF_L6E_FA) could reach to a close level or catch up that of TF_L6D_FA strain. 2. In Table 1, authors generated transformants and transconjugants to evaluate the bacterial growth in absence or presence of florfenicol selective pressure. However, the E. faecium strain carrying the pAM_poxtA_BM vector did not exhibit increased resistance to florfenicol, compared to the strain having the poxtA gene, which might be derived from an identical origin of the poxtA gene to make these clones. Is there specific reason about this event? 3. Authors mentioned that the isolates carrying both linezolid-resistant genes and fexA or fexB take advantage of maintaining viability in the presence of selective pressure of florfenicol. In light with this, however, the results shown in Figure 3 does not be consistent with this mention. More specifically, if the mutant strains possessing both linezolid-resistant genes and fexA or fexB (e.g. TF_K6D_FA or TF_L6E_FA) should be much higher, compared to those carrying only one gene, but it was opposite to your opinion. Collectively, this article tried to explain the association between the use of florfenicol in veterinary settings and the emergence and selection of the isolates harboring genetic determinants for linezolid resistance in animals, but the data did not fully support your address and also there was no novel results and conclusion (to have beneficial for bacterial survival and fitness under selective pressure of antimicrobials in the bacterial isolates carrying those resistance genes is so natural event).
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Not at all
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Fukuda and co-workers investigated on the bacterial growth and possible fitness cost of linezolid-resistant Enterococcus faecium and Enterococcus faecalis harboring mobile linezolid-resistance genes (cfr, optrA and poxtA) in the presence and absence of florfenicol. Furthermore, the authors cloned the 3 aforementioned linezolid resistance genes in plasmid vectors in order to study their actual contribution to the permanence and fitness cost in the enterococcal background. This is an interesting study regarding the analysis of the fitness costs of the mobile cfr, optrA and poxtA resistance genes in enterococcal background. The results confirming that presence of florfenicol is significant for the growth of bacteria harboring plasmid borne linezolid-resistance genes, despite some of them contribute a …
Comments to Author
Fukuda and co-workers investigated on the bacterial growth and possible fitness cost of linezolid-resistant Enterococcus faecium and Enterococcus faecalis harboring mobile linezolid-resistance genes (cfr, optrA and poxtA) in the presence and absence of florfenicol. Furthermore, the authors cloned the 3 aforementioned linezolid resistance genes in plasmid vectors in order to study their actual contribution to the permanence and fitness cost in the enterococcal background. This is an interesting study regarding the analysis of the fitness costs of the mobile cfr, optrA and poxtA resistance genes in enterococcal background. The results confirming that presence of florfenicol is significant for the growth of bacteria harboring plasmid borne linezolid-resistance genes, despite some of them contribute a fitness cost. However, some issues have to be addressed. Please see below: Line 46: Gram-positive. Line 50: also ribosomal proteins L3 and L4. Lines 56: please remove "active efflux". The OptrA and PoxtA are ribosomal protecion proteins. Please see the citation #3 and #4. Line 60: please indicate the resistance mechanism for the fex genes (phenicols efflux proteins). Lines 71-78: This paragraph refers to a previous work of the authors (reference #5). However, not all readers of the journal may have read that paper, therefore the authors should rewrite this paragraph more clearly as it represents the rationale and starting point of the current study. Lines 80-97: Please briefly explain to the reader the rationale of this paragraph. Since the plasmids listed in the "Bacterial strains" section co-harbouring linezolid and florfenicol genes, I think that these cloning assays were performed because the authors want to create plasmids containing only linezolid resistance genes and then verify only their contribution to plasmids persistence and to a possible fitness cost in the presence or absence of florfenicol. Lines 109-111: This sentence is not clear. Please rephrase. Lines 118-127: Since in competition experiments (transconjugant/transformant mixes with recipients) there is a possibility that plasmids can transfer to the recipient in the broth culture (in the citation #5 the authors found conjugative plasmids), the authors have to indicate that they performed all the controls that this phenomenon does not occur. Line 171-192: This paragraph seems a bit confusing to me, I have trouble following it. Also, the condition of figure 3a (E. faecalis FA2-2) is not described. Please rewrite the paragraph so that the readers of the journal can follow more fluently and clearly.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: Not applicable
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