Nanopore sequencing elucidates in vivo development of meropenem resistance by insertion of a mobile genetic element in the porin gene ompC in E. coli
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An ESBL-producing E. coli isolate recovered from a patient undergoing long-term treatment developed resistance to meropenem without acquiring carbapenem-hydrolysing enzymes. We performed Nanopore and Illumina sequencing and subsequent full hybrid genome assembly of this isolate and the meropenem-susceptible isolate recovered almost 8 weeks prior. Whole genome MLST patterns did not differ between isolates. However, we found the insertion of an IS5-like element in the sequence of the ompC gene and an increase in the number of copies of the CTX-M-15 gene in the resistant isolate. These results show that E. coli can develop meropenem resistance under antibiotic pressure by mutations in ompC genes and increasing the copy number of ESBL genes, and the value of next generation sequencing to reveal resistance mechanisms not detected by conventional PCR.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The author has investigated an unusual pattern of resistance and has detected the presence of insertion sequence in the OmpC gene which may have resulted in the increased MIC of meropenem drug. The manuscript needs a little amendments and minor revision. Here are a few comments regarding the manuscript. 1. Isolate B has been isolated from the patient described with the medical condition, however the origin of isolate A is unclear. Was this isolated from blood cultures of the same patient or different. It would be better to describe it. Although the aim of this study is not to investigate the transmission path of the isolate, but it would be interesting to know if there is any link between the isolate A isolate B. 2. Line no. 19 of the abstract state that "E. coli can develop resistance under …
Comments to Author
The author has investigated an unusual pattern of resistance and has detected the presence of insertion sequence in the OmpC gene which may have resulted in the increased MIC of meropenem drug. The manuscript needs a little amendments and minor revision. Here are a few comments regarding the manuscript. 1. Isolate B has been isolated from the patient described with the medical condition, however the origin of isolate A is unclear. Was this isolated from blood cultures of the same patient or different. It would be better to describe it. Although the aim of this study is not to investigate the transmission path of the isolate, but it would be interesting to know if there is any link between the isolate A isolate B. 2. Line no. 19 of the abstract state that "E. coli can develop resistance under antibiotic pressure by mutations in ompC genes and increasing the copy number of ESBL genes" however, it is not clear which type of resistance or resistance to which antibiotic being referred here? Cephalosporins/carbapenem? 3. Line no. 75 is unclear. It should be explained which β-lactamase gene amplification it is referring to? 4. Line 79 states that previous report"s" showed that disruptions in the sequence of the ompC gene by insertion of IS elements. However, the author has provided only one reference. It would be better to add more references if it has been reported in multiple studies. 5. Line no. 40 states that author did not detect any additional resistance genes or other (point) mutations known to 41 confer carbapenem resistance such as polymorphisms in genes gyrA, parC or others Were these not tested, or they were not found while analyzing the WGS data on ResFinder? Moreover, in the conclusion author state that "No resistance determinant was detected by our qPCR method." Have they screened the resistance determinants by WGS data? Or qPCR? Author(s) in here
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially 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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Comments to Author
The short communication/letter "Nanopore sequencing elucidates in-vivo development of meropenem resistance by insertion of a mobile genetic element in the porin gene ompC in E. coli" is a good example of IS-mediated beta-lactamase resistance developed in vivo. The communication is written well and concise. The hypothesis in the antimicrobial resistance mechanism is a 1200 bp insertion in a non-specific outermembrane protein protein that is supposed to playing a major role in meropenem activity. Based on some reference, this is definitive likely. Nevertheless, the insertion alone is not an infinite prove of diminished meropenem activity. This finding raises a lot of questions for further research which would be valuable to add some of these in the discussion paragraph of the manuscript: - Would cloning …
Comments to Author
The short communication/letter "Nanopore sequencing elucidates in-vivo development of meropenem resistance by insertion of a mobile genetic element in the porin gene ompC in E. coli" is a good example of IS-mediated beta-lactamase resistance developed in vivo. The communication is written well and concise. The hypothesis in the antimicrobial resistance mechanism is a 1200 bp insertion in a non-specific outermembrane protein protein that is supposed to playing a major role in meropenem activity. Based on some reference, this is definitive likely. Nevertheless, the insertion alone is not an infinite prove of diminished meropenem activity. This finding raises a lot of questions for further research which would be valuable to add some of these in the discussion paragraph of the manuscript: - Would cloning of an ompC (or other other ompA/ompF) into a ΔompC clone leading to meropenem susceptibility? And can this be demolished by an insertion element like the one described in this manuscript? - The ompC protein has several adherence, tranport, and other functions. What is the - suggested - fitness of isolate B as these functions will be loosened. - Is it possible to determine the function and level of activity of meropenem of the ompC in isolate A and B by varying osmolarity of the environment in culture under meropenem pressure? Of course, such questions are relevant to be addressed in a full paper, it would be valuable to add these questions to abet further research.
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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