fosA11, a novel chromosomal-encoded fosfomycin resistance gene identified in Providencia rettgeri

ABSTRACT This study investigated resistance genes corresponding to the fosfomycin resistance phenotype in clinical isolate Providencia rettgeri W986, as well as characterizing the enzymatic activity of FosA11 and the genetic environment. Antimicrobial susceptibility testing was performed using the agar microdilution method based on the Clinical and Laboratory Standards Institute guidelines. The whole genomic sequence of Providencia rettgeri W986 was obtained using Illumina sequencing and the PacBio platform. The fosA-11 gene was amplified by PCR and cloned into the pUCP20 vector. The recombinant strain pCold1-fosA11-BL21 was expressed to extract the target protein, and absorbance photometry was applied for enzymatic parameter determination. Minimal inhibitory concentration (MIC) tests showed that W986 conferred fosfomycin resistance and was inhibited by phosphonoformate, thereby indicating the presence of a FosA protein. A novel resistance gene designated as fosA11 was identified by whole-genome sequencing and bioinformatics analysis, and it shared 54.41%–64.23% amino acid identity with known FosA proteins. Cloning fosA11 into Escherichia coli obtained a significant increase (32-fold) in the MIC with fosfomycin. Determination of the enzyme kinetics showed that FosA11 had a high catalytic effect on fosfomycin, with Km = 18 ± 4 and Kcat = 56.1 ± 3.2. We also found that fosA11 was located on the chromosome, but the difference in the GC content between the chromosome and fosA11 was dubious, and thus further investigation is required. In this study, we identified and characterized a novel fosfomycin inactivation enzyme called FosA11. The origin and prevalence of the fosA11 gene in other bacteria require further investigation. IMPORTANCE Fosfomycin is an effective antimicrobial agent against Enterobacterales strains. However, the resistance rate of fosfomycin is increasing year by year. Therefore, it is necessary to study the deep molecular mechanism of bacterial resistance to fosfomycin. We identified a novel chromosomal fosfomycin glutathione S-transferase, FosA11 from Providencia rettgeri, which shares a very low identity (54.41%–64.23%) with the previously known FosA and exhibits highly efficient catalytic ability against fosfomycin. Analysis of the genetic context and origin of fosA11 displays that the gene and its surrounding environments are widely conserved in Providencia and no mobile elements are discovered, implying that FosA11 may be broadly important in the natural resistance to fosfomycin of Providencia species.

Reviewer #2 (Comments for the Author): In this work , Lu et al provide novel insights into fosfomycin resistance in Providencia rettgeri.Specifically, using a UTI P. rettgeri isolate, P. rettgeri W986, the authors present work on a novel fos resistance gene, which they name fosA11.They provide evidence of catalytic activity against fosfomycin, although FosA11's affinity for fosfomycin is lower than the characterized FosA3 from E. coli.This study reports that this novel gene, fosA11 is chromosomally-encoded and prevalent among Providencia species, which could explain intrinsic resistance to fosfomycin in such isolates.Overall the study is linearly performed and provides new insight into the field.However, there are areas that lack rigor, which need to be addressed.1) There are no statistical analyses for the MICs and more extensive description of how experiments were performed would be useful.
2) Critical information shown in the figures and tables is oftentimes not discussed in the main text.For example, it is not mentioned in the text that G6P is not required for uptake of fosfomycin by Providencia.However this is demonstrated in Table 3.Does Providencia not have the Uhp system described in E. coli?This would be good to discuss, given that Providencia is understudied and the authors have performed whole genome sequencing 3) There is no description of how the candidate fosfomycin resistance genes were identified (Lines297-300).As mentioned above, it would be more useful to provide a table of the genes that are putatively associated with fosfomycin or other drug resistance in the main text rather than the supplement.
4) There should be some genetic validation with mutagenesis of the catalytic pocket of FosA11 to validate that this mutant stops imparting fosfomycin resistance to E. coli.
5) The authors state that there are numerous Providencia species that have the fosA11.Since the authors have run the analyses, they should be quantitative here: "out of xx sequenced genomes, Y number of genomes code for fosA11".

Minor
The article needs editing for English.In some cases, like in lines 238-241 it is hard to understand what the authors meant to state.Please make sure that the references are separated by a space between the reference and the last word in a sentence.
The MICs for the other drugs for strain W986 should be given in the form of a table instead of described in the text.(lines 241-248) In materials and methods, did you mean 10 micromolar or 10 millimolar IPTG?

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Spectrum02542-23 (fosA11, a Novel Chromosomal Encoded Fosfomycin Resistance Gene Identified in Providencia rettgeri)
Editor: Silvia Cardona >>RESPONSE: Thank you for your favorable evaluation of our manuscript, and we thank the reviewer for his/her comments and feedback.We have studied the comments carefully and have made revisions accordingly which we hope meet with approval.Changes are highlighted in yellow using the highlight tool in the revised manuscript to make the revisions notable, and point-by-point responses to the reviewers' comments are listed below.

Comments of the referees:
Referee: 1 (Comments to the Author): >>COMMENT: In this study, authors first identified and characterized a novel Fosfomycin inactivate enzyme, named FosA11.The function has been confirmed using a cloning experiment and Kinetic assay was conducted.The genetic structure has been presented.In fact, some studied has indicated that chromosomal FosA is widely distributed among Gram negative pathogenic species including Providencia stuartii and contributes to intrinsic fosfomycin resistance (PMID: 28851843).I wonder whether this gene is inherent and whether the name fosA11 is proper.

>>RESPONSE:
Thanks for the reviewer's insightful comments.This is a very important point regarding the nomenclature of fosA gene and gene transferable properties.The drug resistance gene was named according to the order of the latest reported resistance gene fosA10 (PMID: 32431524), and several previously reported fosA genes present on the chromosome were also named by this method.Such as fosA2 located in chromosomes of Enterobacter cloacae (PMID: 21392044) and fosA7 founded in chromosomes of Salmonella (PMID: 28533247).The gene is indeed located on the chromosome of Providencia, and the current sequencing analysis found that no known mobile elements were found in the upstream and downstream of the gene; moreover, search throughout the NCBI database and the analysis of Providencia collected in our laboratory it can be seen that this gene only exists on the chromosomes of a small part of Providencia, and most Providencia have not found this gene, so it can be called inherent drug resistance gene in small part of Providencia.

>>COMMENT:
The minor comment is that the Table 1 could be transferred to supplement materials.

>>RESPONSE:
Thanks for the reviewer's insightful comments.the Table 1 has been transferred to supplement materials and merged with Table S1.(See Supplement Tables S1) Referee: 2 (Comments to the Author): >>COMMENT: In this work, Lu et al provide novel insights into fosfomycin resistance in Providencia rettgeri.Specifically, using a UTI P. rettgeri isolate, P. rettgeri W986, the authors present work on a novel fos resistance gene, which they name fosA11.They provide evidence of catalytic activity against fosfomycin, although FosA11's affinity for fosfomycin is lower than the characterized FosA3 from E. coli.This study reports that this novel gene, fosA11 is chromosomally-encoded and prevalent among Providencia species, which could explain intrinsic resistance to fosfomycin in such isolates.
Over all the study is linearly performed and provides new insight into the field.However, there are areas that lack rigor, which need to be addressed.
>>COMMENT: 1) There are no statistical analyses for the MICs and more extensive description of how experiments were performed would be useful.

>>RESPONSE:
Thanks for the reviewer's careful reading and constructive suggestions, which has significantly improved the presentation of our manuscript.>>COMMENT: 2) Critical information shown in the figures and tables is oftentimes not discussed in the main text.For example, it is not mentioned in the text that G6P is not required for uptake of fosfomycin by Providencia.However, this is demonstrated in Table 3.Does Providencia not have the Uhp system described in E. coli?This would be good to discuss, given that Providencia is understudied and the authors have performed whole genome sequencing.

>>RESPONSE:
Thanks for the reviewer's meaningful comments.We have added discussion contents of the table to the main text.(See Lines 308-314) >>COMMENT: 3) There is no description of how the candidate fosfomycin resistance genes were identified (Lines297-300).As mentioned above, it would be more useful to provide a table of the genes that are putatively associated with fosfomycin or other drug resistance in the main text rather than the supplement.>>RESPONSE: Thanks for the reviewer's constructive suggestions.We agree with the reviewer's suggestion that we have described in more detail on the process of how potential fosfomycin resistance genes were discovered.(See Lines 303-307) Moreover, we have transferred the table of the genes that are putatively associated with fosfomycin or other drug resistance from the supplementary material to the main text.(See Table 5) >>COMMENT: 4) There should be some genetic validation with mutagenesis of the catalytic pocket of FosA11 to validate that this mutant stops imparting fosfomycin resistance to E. coli.

>>RESPONSE:
Thanks for the reviewer's insightful comments.based on reviewer's suggestion we have completed the mutagenesis experiments and according to the experiment results, the corresponding part of the papper has been improved.(See Lines 181-182 and Lines 369-392 and Table 1) >>COMMENT: 5) The authors state that there are numerous Providencia species that have the fosA11.Since the authors have run the analyses, they should be quantitative here: "out of xx sequenced genomes, Y number of genomes code for fosA11".>>RESPONSE: Thanks for the reviewer's valuable comments.We have revised the detailed expression of the text on analyses results of numerous Providencia species that have the fosA11 according to reviewer's detailed suggestion.(See Lines 328-329) Minor >>COMMENT: The article needs editing for English.In some cases, like in lines 238-241 it is hard to understand what the authors meant to state.

>>RESPONSE:
Thanks for the reviewer's useful advice.The revised paper has been sent to professional English staff for language polishing.>>COMMENT: Please make sure that the references are separated by a space between the reference and the last word in a sentence.

>>RESPONSE:
Thanks for the reviewer's careful advice.We have revised the citation format throughout the manuscript according to reviewer's detailed suggestion.

>>COMMENT:
The MICs for the other drugs for strain W986 should be given in the form of a table instead of described in the text.(lines 241-248) >>RESPONSE: Thanks for the reviewer's pertinent advice.We've made the appropriate changes to the papper.(See Supplement TablesS4 and Lines 250-253) >>COMMENT: In materials and methods, did you mean 10 micromolar or 10 millimolar IPTG? >>RESPONSE: Thanks for the reviewer's insightful comments.It is 10 millimolar, and has made corresponding changes in the paper.(See Lines 201) Again, we would like to extend our gratitude to you and the reviewers for bringing these issues to our attention.My co-authors and I agree that the changes made to the manuscript have greatly improved this manuscript.We really appreciate it.
Regards, Shenghai Wu wu_shai@163.com • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred