Genetic basis of clarithromycin resistance in Bacillus anthracis

ABSTRACT The high-consequence pathogen Bacillus anthracis causes human anthrax and often results in lethal infections without the rapid administration of effective antimicrobial treatment. Antimicrobial resistance profiling is therefore critical to inform post-exposure prophylaxis and treatment decisions, especially during emergencies such as outbreaks or where intentional release is suspected. Whole-genome sequencing using a rapid long-read sequencer can uncover antimicrobial resistance patterns if genetic markers of resistance are known. To identify genomic markers associated with antimicrobial resistance, we isolated B. anthracis derived from the avirulent Sterne strain with elevated minimal inhibitory concentrations to clarithromycin. Mutants were characterized both phenotypically through broth microdilution susceptibility testing and observations during culturing, as well as genotypically with whole-genome sequencing. We identified two different in-frame insertions in the L22 ribosomal protein-encoding gene rplV, which were subsequently confirmed to be involved in clarithromycin resistance through the reversion of the mutant gene to the parent (drug-susceptible) sequence. Detection of the rplV insertions was possible with rapid long-read sequencing, with a time-to-answer within 3 h. The mutations associated with clarithromycin resistance described here will be used in conjunction with known genetic markers of resistance for other antimicrobials to strengthen the prediction of antimicrobial resistance in B. anthracis. IMPORTANCE The disease anthrax, caused by the pathogen Bacillus anthracis, is extremely deadly if not treated quickly and appropriately. Clarithromycin is an antibiotic recommended for the treatment and post-exposure prophylaxis of anthrax by the Centers for Disease Control and Prevention; however, little is known about the ability of B. anthracis to develop resistance to clarithromycin or the mechanism of that resistance. The characterization of clarithromycin-resistant isolates presented here provides valuable information for researchers and clinicians in the event of a release of the resistant strain. Additionally, knowledge of the genetic basis of resistance provides a foundation for susceptibility prediction through rapid genome sequencing to inform timely treatment decisions.

1st Editorial Decision Re: Spectrum04180-23 (Genetic basis of clarithromycin resistance in Bacillus anthracis) Dear Dr. David Sue: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
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Additional comments:
Thank you for submitting your work on the genetic basis of clarithromycin resistance in Bacillus anthracis.Although this pathogen is an unusual cause of human infections, the consequences of disease are severe and your work will be of use to practitioners in this space, particularly as molecular diagnostics becomes increasingly accessible.Overall, the manuscript is well-written but would benefit from the suggestions recommended by the reviewers.Minor changes are recommended as follows: • Background, lines 79-82: The authors could also state that genotypic methods of detecting resistance will prevent unnecessary culture manipulation, which is relevant in reducing laboratory personnel risk when dealing with this hazardous pathogen.
• Methods, lines 238 and 243 and throughout the manuscript: Please provide the company name and headquarters for the companies mentioned.• Methods, line 264 and line 271: SBA is used for the first time as an abbreviation, please write out in full.The same applies to AL. • Methods, line 329: Replace "until dry" with "and left to dry".• Methods, line 406: Write out BMD in full when it is first mentioned.
Reviewer #1 (Comments for the Author): The study reported by Maxon et al. describes the genotypic variation in L22 ribosomal protein encoding gene rplv of Bacillus anthracis strains as a reason for the elevated minimal inhibitory concentration (MIC) against clarithromycin.Since the strains show reduction in MIC upon genetic reversal, the authors suggest that rplv can serve as a flag for likely clarithromycin resistance.Further, they have formulated a genotypic resistance detection assay for rplv insertion which can be extended to the near-neighbor B cereus group strains since rplv in both the species shares high identify.
Following are my concerns: -Figure 1. Are the sequences corresponding to the isolates given here deposited to NCBI? -Explain POX1 and POX2 in page #1.Details come only in page #5 -Why CLR1-2, CLR-12 and CLR1-32 are isolated from independent selection and CLR1-11,   Summary: Maxson et al. provide a manuscript entitled "Genetic basis of clarithromycin resistance in Bacillus anthracis".The authors were able to detect and characterize rplV mutations in four independent B. anthracis serial passage experiments using clarithromycin (CLR) as a selective pressure.They were able to confirm that insertions in the rplV gene, which encodes the L22 ribosomal encoding protein, play a role in the development of CLR resistance in the avirulent Sterne strain genetic background through CRISPR-mediated reverted strains.The authors provide a compelling study of how CLR resistance could potentially develop in B. anthracis and how ONT long-read sequencing could be employed to detect potential AMR signatures of CLR-R.Furthermore, their methods section provides excellent detail for reproducibility.After reading the manuscript, it's not clear to me the functional implications of these insertions beyond they are associated with CLR MIC increases.Thus, it may be difficult to use genotype/phenotype correlations to predict CLR-R with B. anthracis.It's quite surprising that these mutations don't appear to incur any fitness cost.However, their results do signify a functional role of L22 ribosomal encoding protein mutations conferring CLR-R as has been observed in other bacterial organisms.I only have a few comments listed below.

Major comments:
Lines 130 -132: I think it would be helpful for the reader to have an overall understanding of the genetic distance between ancestor and progeny strains.While they mention no changes in virulence factors were involved, it may be helpful to provide a clarification in terms of pairwise SNP differences and if possible, a variant table if only a few are present.Additionally, it's not clear what effect these insertions have in terms of rplV activity.I think it would help to describe where these mutations occur within the L22 protein to provide the reader some context on the predicted functional result of these mutations as well as maybe provide some speculative discussion on to why resistance may occur in these mutants within the discussion.

Minor comments:
Lines 114 -116: Quantification of sporulation/germination was mentioned in the methods, so was curious to know if there were any absolute/relative differences in the mutant's capacity to sporulate/germinate compared to the ancestral Sterne strain.Lines 116 -119: A supplementary table that presents the MICs of each of these antibiotics may be helpful to the reader.I found it interesting that clindamycin was equally susceptible in the mutant and ancestral background given that macrolides and lincosamides have similar mechanisms of action.Lines 124 -129: It could help to include the orientation of the mutations relative to the coding sequence of RplV to better understand the effect of these insertions.The authors mention that these are in-frame mutations, but this is not entirely clear from the publicly available annotated genome of the 'Ames ancestor' strain.
Lines 170-172: It's not clear which panel of Fig S3 corresponds to this experiment.An independent supplemental figure that presents the workflow may be useful.Lines 301 -304: Is this a variant quality score?Should be clarified.Lines 428: Should have a data availability section with WGS data available through a public repository.

Summary:
Maxson et al. provide a manuscript entitled "Genetic basis of clarithromycin resistance in Bacillus anthracis".The authors were able to detect and characterize rplV mutations in four independent B. anthracis serial passage experiments using clarithromycin (CLR) as a selective pressure.They were able to confirm that insertions in the rplV gene, which encodes the L22 ribosomal encoding protein, play a role in the development of CLR resistance in the avirulent Sterne strain genetic background through CRISPR-mediated reverted strains.The authors provide a compelling study of how CLR resistance could potentially develop in B. anthracis and how ONT long-read sequencing could be employed to detect potential AMR signatures of CLR-R.Furthermore, their methods section provides excellent detail for reproducibility.After reading the manuscript, it's not clear to me the functional implications of these insertions beyond they are associated with CLR MIC increases.Thus, it may be difficult to use genotype/phenotype correlations to predict CLR-R with B. anthracis.It's quite surprising that these mutations don't appear to incur any fitness cost.However, their results do signify a functional role of L22 ribosomal encoding protein mutations conferring CLR-R as has been observed in other bacterial organisms.I only have a few comments listed below.

Major comments:
Lines 130 -132: I think it would be helpful for the reader to have an overall understanding of the genetic distance between ancestor and progeny strains.While they mention no changes in virulence factors were involved, it may be helpful to provide a clarification in terms of pairwise SNP differences and if possible, a variant table if only a few are present.Additionally, it's not clear what effect these insertions have in terms of rplV activity.I think it would help to describe where these mutations occur within the L22 protein to provide the reader some context on the predicted functional result of these mutations as well as maybe provide some speculative discussion on to why resistance may occur in these mutants within the discussion.

Minor comments:
Lines 114 -116: Quantification of sporulation/germination was mentioned in the methods, so was curious to know if there were any absolute/relative differences in the mutant's capacity to sporulate/germinate compared to the ancestral Sterne strain.Lines 116 -119: A supplementary table that presents the MICs of each of these antibiotics may be helpful to the reader.I found it interesting that clindamycin was equally susceptible in the mutant and ancestral background given that macrolides and lincosamides have similar mechanisms of action.Lines 124 -129: It could help to include the orientation of the mutations relative to the coding sequence of RplV to better understand the effect of these insertions.The authors mention that these are in-frame mutations, but this is not entirely clear from the publicly available annotated genome of the 'Ames ancestor' strain.Lines 170-172: It's not clear which panel of Fig S3 corresponds to this experiment.An independent supplemental figure that presents the workflow may be useful.Lines 301 -304: Is this a variant quality score?Should be clarified.• Background, lines 79-82: The authors could also state that genotypic methods of detecting resistance will prevent unnecessary culture manipulation, which is relevant in reducing laboratory personnel risk when dealing with this hazardous pathogen.
• Added statement to intro (line 84): "Genotypic resistance prediction can additionally minimize manipulation of live culture, providing a relevant reduction in risk for laboratory personnel working with dangerous pathogens such as B. anthracis."• Methods, lines 238 and 243 and throughout the manuscript: Please provide the company name and headquarters for the companies mentioned.
• Added company headquarters info for first instance of mention throughout document.• Methods, line 264 and line 271: SBA is used for the first time as an abbreviation, please write out in full.The same applies to AL.
• Spelled out SBA at first usage.Buffer AL is the full name of the solution provided by Qiagen -the company does not provide what the abbreviation stands for.Added that the buffer AL is provided in the extraction kit.• Methods, line 329: Replace "until dry" with "and left to dry".
• Changed wording as requested.• Methods, line 406: Write out BMD in full when it is first mentioned.
• Spelled out BMD at first usage.
Reviewer #1: • Figure 1.Are the sequences corresponding to the isolates given here deposited to NCBI?
• All sequencing data has been uploaded to NCBI under BioProject PRJNA1086569.The Bioproject will be publicly released upon acceptance of the manuscript and can be shared with reviewers or editors if requested.Added the Bioproject number to the methods in on line 322.• Explain POX1 and POX2 in page #1.Details come only in page #5 • Added an explanation that we are working with an avirulent strain lacking pXO2 at the end of the introduction.Given that the virulence plasmids (pXO1 and pXO2) are not involved in antimicrobial resistance and are thus tangential to the work at hand, we cite the best references that explore these anthrax plasmids (references 1-3).• Why CLR1-2, CLR-12 and CLR1-32 are isolated from independent selection and CLR1-11, CLR-13 and CLR1-46 from same selection experiments?
• While most of our selection experiments resulted in a single resistant colony, three resistant colonies were observed in one selection experiment.We decided to characterize all three despite knowing they may have arisen from a single parent cell dividing in the liquid culture step and thus likely be clonal.To make this clear in the text; we have revised the language explaining this on line 104.• Added the accession number to table 1.Also corrected the accession number in the methods, as there was a transcription error.• Figure S2.Any comments on why the OD is lower for CLR1-12 compared to the others • Added a sentence to clarify that the differing growth had an unknown cause.

• Line #196 expand CLSI
• Spelled out CLSI as first usage • Line #348 expand SBA • Spelled out SBA at first usage • Page #19: Explain the parameters used in BLAST -what is the E value cut-off, substitution matrix etc. used?
• Added additional details of the Blast analysis in the relevant methods section.• Lines 130 -132: I think it would be helpful for the reader to have an overall understanding of the genetic distance between ancestor and progeny strains.While they mention no changes in virulence factors were involved, it may be helpful to provide a clarification in terms of pairwise SNP differences and if possible, a variant table if only a few are present.Additionally, it's not clear what effect these insertions have in terms of rplV activity.I think it would help to describe where these mutations occur within the L22 protein to provide the reader some context on the predicted functional result of these mutations as well as maybe provide some speculative discussion on to why resistance may occur in these mutants within the discussion.
• Added a variant table for all isolated strains relative to the parent Sterne strain to the supplemental and added a sentence to the results to indicate this inclusion.• Added information on the predicted effects of the insertions in rplV on the structure of the L22 protein and how that is known to effect drug binding in other species, along with 2 new references to support the proposed effects (Wekselman et al. and Zaman et al.) Reviewer #2: • Lines 114 -116: Quantification of sporulation/germination was mentioned in the methods, so was curious to know if there were any absolute/relative differences in the mutant's capacity to sporulate/germinate compared to the ancestral Sterne strain.
• Specified that sporulation frequencies of the mutants were similar to that of the parent Sterne strain.• Lines 116 -119: A supplementary table that presents the MICs of each of these antibiotics may be helpful to the reader.I found it interesting that clindamycin was equally susceptible in the mutant and ancestral background given that macrolides and lincosamides have similar mechanisms of action.
• Added a table with MIC values for all drugs tested for all strains to the supplemental.• Lines 124 -129: It could help to include the orientation of the mutations relative to the coding sequence of RplV to better understand the effect of these insertions.The authors mention that these are in-frame mutations, but this is not entirely clear from the publicly available annotated genome of the 'Ames ancestor' strain.
• Added an SI figure with the full sequence of rplV along with the translated sequence and indications of where the insertions are.Also modified the alignment given in figure 1 the insertions are highly repetitive and can be aligned in different positions; the positions the insert were aligned to in the figure did not show them as in-frame so they were re-aligned to show that they can be aligned to an in-frame position.The inserts are both multiples of 3, and thus do not change the frame of the transcript.• Lines 170-172: It's not clear which panel of Fig S3 corresponds to this experiment.An independent supplemental figure that presents the workflow may be useful.
• An additional figure (Figure S6) was included that shows the time involved for each step of the rapid workflow and the resulting visualizations of the genome assembly.• Lines 301 -304: Is this a variant quality score?Should be clarified.
• Added the qualifier of "variant" before quality score to make clear it is a variant quality score.• Lines 428: Should have a data availability section with WGS data available through a public repository.
• All sequencing data has been uploaded to NCBI under BioProject PRJNA1086569.The Bioproject will be publicly released upon acceptance of the manuscript and can be shared with reviewers or editors if requested.Added the Bioproject number to the methods in on line 322.Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Thank you for submitting your paper to Spectrum.

Sincerely, Kessendri Reddy Editor Microbiology Spectrum
Fig S3: Paragraph indicators are included in your labels.

Lines 428 :
Should have a data availability section with WGS data available through a public repository.Fig S3: Paragraph indicators are included in your labels.point-by-point responses to the issues raised by the reviewers: Editorial comments:

•
Ames ref. location in table 1: Give the NCBI ref.ID in table 1 (given in page #19)

•
Fig S3: Paragraph indicators are included in your labels.•We believe the review may have had view paragraph indicators turned on, and as the supplemental is a word document, those labels may have shown the paragraph indicators as the labels are in independent text boxes.We do not see paragraph indicators when we open the file -converting to a PDF would presumably resolve the issue if needed.