Genomic investigation of the emergence of vanD vancomycin-resistant Enterococcus faecium
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Vancomycin-resistant Enterococcus (VRE) is an increasingly identified cause of human disease, with most infections resulting from the vanA and vanB genotypes; less is known about other clinically relevant genotypes. Here we report a genomic exploration of a vanD VRE faecium (VREfm), which arose de novo during a single infectious episode. The genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm were subjected to long-read sequencing and closed, with whole-genome alignments, cross-mapping and orthologue clustering used to identify genomic variation. Three key differences were identified. (i) The VREfm chromosome gained a 142.6 kb integrative conjugative element (ICE) harbouring the vanD locus. (ii) The native ligase ( ddl ) was disrupted by an IS Efm1 insertion. (iii) A large 1.74 Mb chromosomal inversion of unknown consequence occurred. Alignment and phylogenetic-based comparisons of the VREfm with a global collection of vanD -harbouring genomes identified strong similarities in the 120–160 kb genomic region surrounding vanD, suggestive of a common mobile element and integration site, irrespective of the diverse taxonomic, geographical and host origins of the isolates. This isolate diversity revealed that this putative ICE (and its source) is globally disseminated and is capable of being acquired by different genera. Although the incidence of vanD VREfm is low, understanding its emergence and potential for spread is crucial for the ongoing efforts to reduce antimicrobial resistance.
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Thank you very much for submitting this revised manuscript to Access Microbiology and addressing the reviewer suggestions. I am pleased to inform you that your manuscript is now accepted for publication. Congratulations to all authors!
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Comments to Author
This is an article comparing 2 Enterococcus faecium genomes, from strains isolated from the same patient, 2 months apart. In the second isolate they find that an ICE sequence has been inserted, which confers resistance to vancomycin. This is relevant and is a remarkable contribution, which helps to understand how antibiotic resistance spreads between species. The methodology is adequate to analyze this sequence and present its expansion. I would just like to see the complete comparison between both isolates: genes present and absent in the second isolate, and total number of variants between both genomes. I also add a number of points to be addressed: 185: Does it refer to the whole genome or only to plasmids? Two variants seems really few. Fig. 1: Which identity threshold has been used? Red means …
Comments to Author
This is an article comparing 2 Enterococcus faecium genomes, from strains isolated from the same patient, 2 months apart. In the second isolate they find that an ICE sequence has been inserted, which confers resistance to vancomycin. This is relevant and is a remarkable contribution, which helps to understand how antibiotic resistance spreads between species. The methodology is adequate to analyze this sequence and present its expansion. I would just like to see the complete comparison between both isolates: genes present and absent in the second isolate, and total number of variants between both genomes. I also add a number of points to be addressed: 185: Does it refer to the whole genome or only to plasmids? Two variants seems really few. Fig. 1: Which identity threshold has been used? Red means passing that threshold and blue the same but inverted? In any case, the number of variants found throughout the genome should be indicated in the results to better estimate the divergence between the two genomes. Fig. 2: Do the numbers in parentheses represent the year of isolation? The fact that ICE appears in different genres should be highlighted more. For example, making a Blast of its sequence, it is found in Clostridia or Blautia, at 96% identity. Is it expected to be the same ICE? I am missing more information on what the vancomycin resistance genotypes are based on. Data not shown should be given as Supplementary Figures, to confirm results obtained.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
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?
Yes
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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, with whom I agree that this is indeed a valuable contribution to understand the spread of antibiotic resistance. Please find below the reviewer comments and suggestions to improve your manuscript. Please pay special attention to those concerning the addition of references, a deeper comparison between the isolates, the addition of information on the vancomycin resistance genotypes and the suggestion to highlight the ICE. With respect to the data not shown, as per the open data policy of Access Microbiology, I will kindly ask you to provide them as supplementary material.
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Comments to Author
I found this work to be very thoroughly conducted, presented in a clear and precise manner, with clear figures and text. My only point of contention is minor - there are some sentences that I feel should contain references to back up the statements made. I have included those statements below, as well as a few other minor comments. Otherwise this is a great piece of research which I recommend for publication. L67 - Enterococcus represents a genus of human pathogenic bacteria, associated with a diverse range of disease manifestations and high mortality when nosocomially acquired. L71 - The large majority of human enterococcal disease is caused by E. faecalis and E. faecium; vancomycin resistance in these lineages predominantly represented by the vanA or vanB genotypes. L114- The minimum inhibitory …
Comments to Author
I found this work to be very thoroughly conducted, presented in a clear and precise manner, with clear figures and text. My only point of contention is minor - there are some sentences that I feel should contain references to back up the statements made. I have included those statements below, as well as a few other minor comments. Otherwise this is a great piece of research which I recommend for publication. L67 - Enterococcus represents a genus of human pathogenic bacteria, associated with a diverse range of disease manifestations and high mortality when nosocomially acquired. L71 - The large majority of human enterococcal disease is caused by E. faecalis and E. faecium; vancomycin resistance in these lineages predominantly represented by the vanA or vanB genotypes. L114- The minimum inhibitory concentrations (MICs), determined by Etest at the time of isolation were 1 mg/L and ≥ 256 mg/L for vancomycin, and 2 mg/L and ≥ 256 mg/L for teicoplanin, for the VSEfm and VREfm respectively. L129 - "All contigs were checked manually for assembly errors"; what is the criteria for the manual assessment of assembly errors? L188 - "three large structural genomic difference"; spelling, differenceS
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
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?
Yes
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