Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants

ABSTRACT We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects β-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA’s extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

Lines 122 -123: "no differential accessory genome" -different from what? Line 134: Isolate A1 was susceptible to erythromycin, and the rest were resistant. Was there a change in the rRNA sequences? How was it determined that all three copies of the rRNA gene were the same (assembly of short-read sequences would be hard to distinguish between two and three copies)?

How was it decided what was supplementary material?
What I assume to be Figure S1 was difficult to decipher. Tiny print cannot be read without zooming then you can't see much of the total picture. When you say "boxes," do you mean the labels at the top of each chart or the bars that make up the charts. I do not see a distinction between the red, green, and black bar legend -why are multiple colors used. What is a "main in-length variable polyN tract"?
Line 290: Table S1 is list of polyNtracts -what does that have to do with MICs as stated in this line? Lines 194 -195: References from 2007 and2004 probably do not represent current practices on antimicrobial usage for prophylactics in animal husbandry. There have been dramatic shifts in those practices in the last decade, at least in developed countries.
Lines 313 -314: In what sense is the phrase "accessory genome" being used? The ordinarily understood sense would be relative to the universal population, in which case there is no bacterial genome that doesn't have some accessory genome. The statement could be correct if it is just referring to the 12 isolates in the study. Clarification is needed.
References are incomplete: 12,13,14,15,16,18,19,21,24,25,26,29,30,31,32,34,38,43,44,49 Reviewer #2 (Comments for the Author): This study from Nunes et al. is investigating in vivo development of antimicrobial resistance from two independant infection with Campylobacter jejuni strain. The authors demonstrated the potential involved mechanisms as well as the complete caracterisation of strains. This study is well conducted and well written. The data showed support adequately the conclusions. I have no major comments to address. Is the authors have verified if the observed phenomenon should be reversible after numerous plating of the bacteria on standard media? Also, bacterial name should be italicized, particularly in the introduction section.
Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.

Spectrum01070-23
We would like to thank the reviewers for the comments that have improved the quality of the manuscript. Please find below the point-by-point responses to the concerns highlighted by the reviewers.

Reviewer #1 (Comments for the Author):
Nunes et al. describe two sets of Campylobacter jejuni isolates, each set from an immunocompromised pa ent that had long-term infec ons that became resistant to an microbials. Resistance to fluoroquinolones, tetracycline and ampicillin were as expected based on the gene c markers. However, resistance to carbapenems had a previously undescribed associa on with changes in the porA gene. The associa on was very strong, but it should s ll be recognized that that this is circumstan al evidence.
Line 121: Although a ribu on score data are not shown, there should be a reference here that shows how that score was developed.
R: Yes, we agree with this comment. The references for material (REF 46) and method (REF 45) for that specific analysis is described in materials and methods sec on.
To be more specific: for molecular source a ribu on, we used a method and a subset of 15 host-segrega ng genes that have been iden fied in a previous study by Thepault et al.,(46). Alleles for each isolate were extracted using Blastn command line tool and were assigned to a number. Using a training dataset of 583 isolates from chicken, ruminant and environment reservoirs, STRUCTURE sta s cal analysis (45) was conducted in order to a ribute each alleles number combina on to its poten al source.
In order to include these 2 references in the results, the text was adjusted as follows: "Using STRUCTURE so ware (45) and C. jejuni host-segrega ng loci as previously described by Thepault et al.,(46), the en re set of isolates was a ributed to the chicken popula on with a ribu on scores of 100% (data not shown)." Lines 122 -123: "no differen al accessory genome" -different from what?
Reply: For each clinical case, we found no differences among isolates, regarding the first isolate collected. We clarified in the text: Although some phage remnant sequences were found (data not shown), no differen al accessory genome was observed within either group of isolates, regarding the first isolate collected from each clinical case.
Line 134: Isolate A1 was suscep ble to erythromycin, and the rest were resistant. Was there a change in the rRNA sequences? How was it determined that all three copies of the rRNA gene were the same (assembly of short-read sequences would be hard to dis nguish between two and three copies)?
Reply: there was a change in the 23S rRNA sequences, with a 100% the replacement of an A to a G in posi on 2075 in all resistant isolates. In detail, when we mapped the reads of these resistant isolates against isolate A1, we observed a 100% fixed muta on in this posi on, with an increase depth coverage that corresponds to 3X the coverage of the genome median coverage. In addi on, in order to confirm the existence of the same muta on in the 3 copies, we mapped the reads against individual copies of the 23S rRNA (Cjr02, Cjr05, Cjr08) from a reference genome available at Genbank (NCTC11168). This informa on is detailed in Figure 1.

How was it decided what was supplementary material?
Reply: that is a very per nent comment. Phase varia on is a major mechanism of crea ng heterogeneity for host adapta on in C. jejuni, and the two described cases of long-term Campylobacter infec ons nicely illustrated the contribu on of these phenomena for the in vivo evolu on of the bacteria. On the other hand, the main relevance of this study is the development of high-level resistance to carbapenems and the new puta ve mechanisms associated. Therefore, to not lose the focus on the resistance, authors have decided to place the phase varia on sec ons in supplementary material. However, with the reviewer's comment, we feel that some parts of the material and methods, regarding the bacterial culture, genome assembly and SNP-based microevolu onary analysis, should be placed in the manuscript, in order to make it easier for the readers to follow the experiments and analyses performed. Therefore, these modifica ons were performed.
What I assume to be Figure S1 was difficult to decipher. Tiny print cannot be read without zooming then you can't see much of the total picture. When you say "boxes," do you mean the labels at the top of each chart or the bars that make up the charts. I do not see a dis nc on between the red, green, and black bar legend -why are mul ple colors used. What is a "main in-length variable polyN tract"?
Reply: As stated in the respec ve legend, boxes are the colored (blue, yellow and black) labels on the top of each chart. The blue (case A) and yellow (case B) boxes represent all phase variable polyN regions with gene c heterogeneity among isolates, while black boxes (from both cases) represent polyN regions where the main in-length variable polyN tract was found to be conserved among isolates.
We understand the need for an explana on of the colours, and we add the following in Figure  S1 legend: For all polyG/C regions, the green bars represent the "ON" expression status, the red bars represent the "OFF" expression status and the black bars refers to non-coding regions. The grey bars refers to polyA/T tracts that fall in non-coding regions.
The "main in-length variable polyN tract" refers to the predominant tract from a polyN region. For instance, in case B, for locus Cj0318, 9G/C is the main tract for all isolates, except for isolate B1, which shows mixture alleles, in which the 9G/C comprises 50% of the tracts observed.
Line 290: Table S1 is list of polyNtracts -what does that have to do with MICs as stated in this line?
Reply: this was a mistake, we removed including Table S1 from this sentence: more details are provided in supplementary materials and methods. Lines 194 -195: References from 2007 and2004 probably do not represent current prac ces on an microbial usage for prophylac cs in animal husbandry. There have been drama c shi s in those prac ces in the last decade, at least in developed countries.
Reply: we fully agree with the reviewer concerning current prac ces on the an microbial use in the food animal sector. However, our aim was to emphasize the importance of an bio c exposure to the development of an microbial resistance. In the case of Campylobacter, the best model to illustrate this adapta ve response is the development of macrolide-resistant mutants that involves a mul step process and requires prolonged exposure to the an bio c. therefore, and in agreement with the reviewer, we rephrased the sentence, sta ng that the use of an microbials is not a current prac ce, but instead a former prac ce.
Lines 313 -314: In what sense is the phrase "accessory genome" being used? The ordinarily understood sense would be rela ve to the universal popula on, in which case there is no bacterial genome that doesn't have some accessory genome. The statement could be correct if it is just referring to the 12 isolates in the study. Clarifica on is needed.
Reply: Yes, we agree with the reviewer, and we have modified the sentence accordingly: For each clinical case, assemblies were aligned using the progressive algorithm of MAUVE so ware version 2.3.1 (h p://darlinglab.org/mauve/mauve.html) to inspect the accessory genome among isolates, and addi onally queried for the existence of phages, mobile gene c elements (MGEs) and plasmids.
All the references listed were reviewed in accordance with the journal's citation rules. The updated references were added using the EndNote output style for ASM Journals provided in the website: https://endnote.com/style_download/american-society-for-microbiology-asmjournals-2/ Reviewer #2 (Comments for the Author): This study from Nunes et al. is inves ga ng in vivo development of an microbial resistance from two independant infec on with Campylobacter jejuni strain. The authors demonstrated the poten al involved mechanisms as well as the complete caracterisa on of strains. This study is well conducted and well wri en. The data showed support adequately the conclusions. I have no major comments to address. Is the authors have verified if the observed phenomenon should be reversible a er numerous pla ng of the bacteria on standard media? Also, bacterial name should be italicized, par cularly in the introduc on sec on.
Reply: we thank the positive comments regarding our work. We did not evaluate the reversibility of the porA in-frame insertion. Editor's note: It seems you have uploaded the marked up version of the manuscript with the changes, but not the clean version with the changes. The manuscript that was uploaded was the original version (version submitted prior to review). Please upload both the marked up version and clean version of the revised manuscript.
Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • 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 For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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Spectrum01070-23
We would like to thank the reviewers for the comments that have improved the quality of the manuscript. Please find below the point-by-point responses to the concerns highlighted by the reviewers.

Reviewer #1 (Comments for the Author):
Nunes et al. describe two sets of Campylobacter jejuni isolates, each set from an immunocompromised pa ent that had long-term infec ons that became resistant to an microbials. Resistance to fluoroquinolones, tetracycline and ampicillin were as expected based on the gene c markers. However, resistance to carbapenems had a previously undescribed associa on with changes in the porA gene. The associa on was very strong, but it should s ll be recognized that that this is circumstan al evidence.
Line 121: Although a ribu on score data are not shown, there should be a reference here that shows how that score was developed.
R: Yes, we agree with this comment. The references for material (REF 46) and method (REF 45) for that specific analysis is described in materials and methods sec on.
To be more specific: for molecular source a ribu on, we used a method and a subset of 15 host-segrega ng genes that have been iden fied in a previous study by Thepault et al.,(46). Alleles for each isolate were extracted using Blastn command line tool and were assigned to a number. Using a training dataset of 583 isolates from chicken, ruminant and environment reservoirs, STRUCTURE sta s cal analysis (45) was conducted in order to a ribute each alleles number combina on to its poten al source.
In order to include these 2 references in the results, the text was adjusted as follows: "Using STRUCTURE so ware (45) and C. jejuni host-segrega ng loci as previously described by Thepault et al.,(46), the en re set of isolates was a ributed to the chicken popula on with a ribu on scores of 100% (data not shown)." Lines 122 -123: "no differen al accessory genome" -different from what?
Reply: For each clinical case, we found no differences among isolates, regarding the first isolate collected. We clarified in the text: Although some phage remnant sequences were found (data not shown), no differen al accessory genome was observed within either group of isolates, regarding the first isolate collected from each clinical case.
Line 134: Isolate A1 was suscep ble to erythromycin, and the rest were resistant. Was there a change in the rRNA sequences? How was it determined that all three copies of the rRNA gene were the same (assembly of short-read sequences would be hard to dis nguish between two and three copies)?
Reply: there was a change in the 23S rRNA sequences, with a 100% the replacement of an A to a G in posi on 2075 in all resistant isolates. In detail, when we mapped the reads of these resistant isolates against isolate A1, we observed a 100% fixed muta on in this posi on, with an increase depth coverage that corresponds to 3X the coverage of the genome median coverage. In addi on, in order to confirm the existence of the same muta on in the 3 copies, we mapped the reads against individual copies of the 23S rRNA (Cjr02, Cjr05, Cjr08) from a reference genome available at Genbank (NCTC11168). This informa on is detailed in Figure 1.

How was it decided what was supplementary material?
Reply: that is a very per nent comment. Phase varia on is a major mechanism of crea ng heterogeneity for host adapta on in C. jejuni, and the two described cases of long-term Campylobacter infec ons nicely illustrated the contribu on of these phenomena for the in vivo evolu on of the bacteria. On the other hand, the main relevance of this study is the development of high-level resistance to carbapenems and the new puta ve mechanisms associated. Therefore, to not lose the focus on the resistance, authors have decided to place the phase varia on sec ons in supplementary material. However, with the reviewer's comment, we feel that some parts of the material and methods, regarding the bacterial culture, genome assembly and SNP-based microevolu onary analysis, should be placed in the manuscript, in order to make it easier for the readers to follow the experiments and analyses performed. Therefore, these modifica ons were performed.
What I assume to be Figure S1 was difficult to decipher. Tiny print cannot be read without zooming then you can't see much of the total picture. When you say "boxes," do you mean the labels at the top of each chart or the bars that make up the charts. I do not see a dis nc on between the red, green, and black bar legend -why are mul ple colors used. What is a "main in-length variable polyN tract"?
Reply: As stated in the respec ve legend, boxes are the colored (blue, yellow and black) labels on the top of each chart. The blue (case A) and yellow (case B) boxes represent all phase variable polyN regions with gene c heterogeneity among isolates, while black boxes (from both cases) represent polyN regions where the main in-length variable polyN tract was found to be conserved among isolates.
We understand the need for an explana on of the colours, and we add the following in Figure  S1 legend: For all polyG/C regions, the green bars represent the "ON" expression status, the red bars represent the "OFF" expression status and the black bars refers to non-coding regions. The grey bars refers to polyA/T tracts that fall in non-coding regions.
The "main in-length variable polyN tract" refers to the predominant tract from a polyN region. For instance, in case B, for locus Cj0318, 9G/C is the main tract for all isolates, except for isolate B1, which shows mixture alleles, in which the 9G/C comprises 50% of the tracts observed.
Line 290: Table S1 is list of polyNtracts -what does that have to do with MICs as stated in this line?
Reply: this was a mistake, we removed including Table S1 from this sentence: more details are provided in supplementary materials and methods. Lines 194 -195: References from 2007 and2004 probably do not represent current prac ces on an microbial usage for prophylac cs in animal husbandry. There have been drama c shi s in those prac ces in the last decade, at least in developed countries.
Reply: we fully agree with the reviewer concerning current prac ces on the an microbial use in the food animal sector. However, our aim was to emphasize the importance of an bio c exposure to the development of an microbial resistance. In the case of Campylobacter, the best model to illustrate this adapta ve response is the development of macrolide-resistant mutants that involves a mul step process and requires prolonged exposure to the an bio c. therefore, and in agreement with the reviewer, we rephrased the sentence, sta ng that the use of an microbials is not a current prac ce, but instead a former prac ce.
Lines 313 -314: In what sense is the phrase "accessory genome" being used? The ordinarily understood sense would be rela ve to the universal popula on, in which case there is no bacterial genome that doesn't have some accessory genome. The statement could be correct if it is just referring to the 12 isolates in the study. Clarifica on is needed.
Reply: Yes, we agree with the reviewer, and we have modified the sentence accordingly: For each clinical case, assemblies were aligned using the progressive algorithm of MAUVE so ware version 2.3.1 (h p://darlinglab.org/mauve/mauve.html) to inspect the accessory genome among isolates, and addi onally queried for the existence of phages, mobile gene c elements (MGEs) and plasmids.
All the references listed were reviewed in accordance with the journal's citation rules. The updated references were added using the EndNote output style for ASM Journals provided in the website: https://endnote.com/style_download/american-society-for-microbiology-asmjournals-2/ Reviewer #2 (Comments for the Author): This study from Nunes et al. is inves ga ng in vivo development of an microbial resistance from two independant infec on with Campylobacter jejuni strain. The authors demonstrated the poten al involved mechanisms as well as the complete caracterisa on of strains. This study is well conducted and well wri en. The data showed support adequately the conclusions. I have no major comments to address. Is the authors have verified if the observed phenomenon should be reversible a er numerous pla ng of the bacteria on standard media? Also, bacterial name should be italicized, par cularly in the introduc on sec on.
Reply: we thank the positive comments regarding our work. We did not evaluate the reversibility of the porA in-frame insertion.