A multifaceted interplay between virulence, drug resistance, and the phylogeographic landscape of Mycobacterium tuberculosis

ABSTRACT Latin-American Mediterranean (LAM) family is one of the most significant and global genotypes of Mycobacterium tuberculosis. Here, we used the murine model to study the virulence and lethality of the genetically and epidemiologically distinct LAM strains. The pathobiological characteristics of the four LAM strains (three drug resistant and one drug susceptible) and the susceptible reference strain H37Rv were studied in the C57BL/6 mouse model. The whole-genome sequencing was performed using the HiSeq Illumina platform, followed by bioinformatics and phylogenetic analysis. The susceptible strain H37Rv showed the highest virulence. Drug-susceptible LAM strain (spoligotype SIT264) was more virulent than three multidrug-resistant (MDR) strains (SIT252, SIT254, and SIT266). All three MDR isolates were low lethal, while the susceptible isolate and H37Rv were moderately/highly lethal. Putting the genomic, phenotypic, and virulence features of the LAM strains/spoligotypes in the context of their dynamic phylogeography over 20 years reveals three types of relationships between virulence, resistance, and transmission. First, the most virulent and more lethal drug-susceptible SIT264 increased its circulation in parts of Russia. Second, moderately virulent and pre-XDR SIT266 was prevalent in Belarus and continues to be visible in North-West Russia. Third, the low virulent and MDR strain SIT252 previously considered as emerging has disappeared from the population. These findings suggest that strain virulence impacts the transmission, irrespective of drug resistance properties. The increasing circulation of susceptible but more virulent and lethal strains implies that personalized TB treatment should consider not only resistance but also the virulence of the infecting M. tuberculosis strains. IMPORTANCE The study is multidisciplinary and investigates the epidemically/clinically important and global lineage of Mycobacterium tuberculosis, named Latin-American-Mediterranean (LAM), yet insufficiently studied with regard to its pathobiology. We studied different LAM strains (epidemic vs endemic and resistant vs susceptible) in the murine model and using whole-genome analysis. We also collected long-term, 20-year data on their prevalence in Eurasia. The findings are both expected and unexpected. (i) We observe that a drug-susceptible but highly virulent strain increased its prevalence. (ii) By contrast, the multidrug-resistant (MDR) but low-virulent, low-lethal strain (that we considered as emerging 15 years ago) has almost disappeared. (iii) Finally, an intermediate case is the MDR strain with moderate virulence that continues to circulate. We conclude that (i) the former and latter strains are the most hazardous and require close epidemiological monitoring, and (ii) personalized TB treatment should consider not only drug resistance but also the virulence of the infecting strains and development of anti-virulence drugs is warranted.

of focusing on virulence and not solely on resistance.In addition, focusing on a relatively small relatively undersequenced sublineage, it allows to formulate new hypotheses regarding the evolution of this sublineage (LAM-RUS).The methods are globally appropriate and the gathered data are clearly presented.Despite the global relevance of the study, some points needs to be improved before publication: Major points : -distribution between results and discussion is sometimes irrelevant: *Results related to SNPs specific of different strains (table S15) should belong to results section, even if the significance of these results may be low.Accordingly, corresponding methods should be added in the methods section.*The same is true for results related to Fig. 6.Regarding these data, it is important to specify on what collections they rely and whether these collection could be biased.*In contrast to these two subpoints part of which should move from discussion to results section, limitations paragraph (currently in results section) belongs to discussion.
-the phylogenetic analysis is not performed in a way that allows to assess correctly the relative distance between strains: the tree of Fig. 1 lacks an outgroup to ensure proper rooting.Please rebuild the tree including an outgroup and perform bootstrap analyses.Also, basic information should be added in the legend: number of SNPs in the matrix used to build the tree, tree scale, bootstrap support of the main branches at least.It is important to mention whether the tree is derived from SAM-TB or Phyresse SNPs inference.Also, please specify whether resistance loci where included or not in the building of the tree.

Minor points:
-please provide deposit SRA onto a public platform and present accession number -l.137 : please specify the time and place of sampling -l.206: specify what was taken as a reference for weight coefficient and mention the range of weights for the reference point.-l.251: rephrase "drug resistance properties of their spoligotypes": a spoligotype has no resistance properties per se (but you may mention correlations between these features as specified shortly after) -l.267: specify what difference was found significant: day14 as compared to day7?And refer to strains not to group numbers or use numbers in association to infecting strains in the figure and in the first mention in the text.-l.372 and following: some info on the severity index used could be transferred to methods and values should be shown in a supplemental table or all provided in the text.-l.403 to 409, discussion on evolution pathways of SITs is of little interest to the study and could be reduced -l.425 More number of resistance mutations -> change for more resistance mutations -Fig.2A, correct colour code (red line in the legend for 4542 strain) -p.i.upon first use, specify abbreviation

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Spectrum01392-23 A multifaceted interplay between virulence, drug resistance and phylogeographic landscape of Mycobacterium tuberculosis
Igor Mokrousov et al.

ANSWERS TO REVIEWER
Dear Reviewer, thank you very much for comments and critiques.We addressed all your comments in the revised version of our manuscript.The detailed answers are below.We also provide a manuscript with marked-up changes as a supporting file (for review only).
Reviewer #1 (Comments for the Author): The manuscript by Mokrousov et al deals with the assessment of virulence of Mycobacterium tuberculosis strains.It combines the use of the animal model of C57BL/6 mice to the exploration of disease dynamics in humans.The choice of strains to be studied further was done according to a phylogenetic study using WGS data.Distant strains were chosen to maximize the chance for showing different phenotypes by selecting multidrug resistant isolates and one susceptible.The study deals with a very low number of strains from a specific lineage of M. tuberculosis strains impeding large conclusions on whether the animal model can reliably help characterize the virulence of pathogenic strains, but it interestingly draws back attention to the necessity of focusing on virulence and not solely on resistance.In addition, focusing on a relatively small relatively undersequenced sublineage, it allows to formulate new hypotheses regarding the evolution of this sublineage (LAM-RUS).The methods are globally appropriate and the gathered data are clearly presented.Despite the global relevance of the study, some points needs to be improved before publication: Major points: -distribution between results and discussion is sometimes irrelevant: *Results related to SNPs specific of different strains (table S15) should belong to results section, even if the significance of these results may be low.Accordingly, corresponding methods should be added in the methods section.

ANSWER:
Only first part of this paragraph is suitable for Results.Otherwise, Table S15 and further text present a discussion based on the published studies (Page 19).
Accordingly, we moved part of this section to Results (Page 16, last para), and also added briefly to Methods that "As potentially biologically meaningful mutations, we defined those with significant SIFT P value or short indels.We also considered mutations in the putative promoter regions."(Page 7, lines 168-170).Otherwise, SNPs were detected using SAM-TB pipeline that we cited.
*The same is true for results related to Fig. 6.Regarding these data, it is important to specify on what collections they rely and whether these collection could be biased.
*In contrast to these two subpoints part of which should move from discussion to results section, limitations paragraph (currently in results section) belongs to discussion.
-the phylogenetic analysis is not performed in a way that allows to assess correctly the relative distance between strains: the tree of Fig. 1 lacks an outgroup to ensure proper rooting.Please rebuild the tree including an outgroup and perform bootstrap analyses.Also, basic information should be added in the legend: number of SNPs in the matrix used to build the tree, tree scale, bootstrap support of the main branches at least.It is important to mention whether the tree is derived from SAM-TB or Phyresse SNPs inference.Also, please specify whether resistance loci where included or not in the building of the tree.
ANSWER: we added an outgroup strain of the other and distant LAM sublineage (RD-Rio) and rebuilt the ML tree (see revised Figure 1, with scale and bootstrap values).Yes, we used only SAM-TB online tool, and under its pipeline, PE/PPE and resistance genes are excluded from phylogenetic analysis (this is added to revised Methods section) (Page 6, lines 150-151).We also provide a distance matrix for LAM-RUS strains as new supplementary Figure S2.
Minor points: -please provide deposit SRA onto a public platform and present accession number ANSWER: PRJNA was provided in M&M (lines 152-153 in the original ms): "Data for the M.
-l.137 : please specify the time and place of sampling ANSWER: Information on strain, origin or source was shown in Table S2 and is available under SRA deposited data for all strains.In revised paper, we added year of isolation in Table S2.
-l. 206: specify what was taken as a reference for weight coefficient and mention the range of weights for the reference point.

ANSWER:
In M&M, we provided a detailed description on this calculation (Page 9, lines 210-212).In Results, Figure 2, we show these values for lung and spleen weight coefficients as means with SD (error bars).
-l.251: rephrase "drug resistance properties of their spoligotypes": a spoligotype has no resistance properties per se (but you may mention correlations between these features as specified shortly after) ANSWER: this sentence was rephrased for clarity and references were added: "The isolates also differed in the drug resistance profile (see this and other information on strains in Table S2) that altogether correlated with drug resistance patterns commonly observed in strains of these spoligotypes [3][4][5][6][11][12][13][14]21].In particular, SIT264 is known to be mainly drugsusceptible, SIT266 is XDR-associated, SIT252 and SIT254 are MDR-associated."(Page 11, lines 259-260).
-l. 267: specify what difference was found significant: day14 as compared to day7?And refer to strains not to group numbers or use numbers in association to infecting strains in the figure and in the first mention in the text.
ANSWER: yes compared to day 7.The sentence is revised (Page 12, lines 275-276).Regarding providing strain numbers along with SIT, they are shown in all figures except for this technical omission in Fig. 2A.We corrected.
-l. 372 and following: some info on the severity index used could be transferred to methods and values should be shown in a supplemental table or all provided in the text.
ANSWER: this paragraph was revised as you suggested (Page 16,.Its first sentences were moved to Methods (Page 10, lines 233-237).Table 1 was moved to Supplement, as new Table S15.
-l. 403 to 409, discussion on evolution pathways of SITs is of little interest to the study and could be reduced
-l. 425 More number of resistance mutations -> change for more resistance mutations ANSWER: I am sorry but not sure I understood correctly.An assumption suggested long ago, is that more changes in drug resistance genes (some encode for the key proteins, e.g rpoB -for RNA polymerase beta-subunit) would counteract drug action but would reduce strain fitness hence transmission.Multiple such mutations will theoretically reduce fitness (and virulence) -as we speculatively show for strain 7074 / SIT252.However, some successful strains are both MDR and sufficiently virulent, as we see on the example of strain 4542 / SIT266.Which is why we write: "More number of resistance mutations is in negative correlation with virulence in SIT252 but the situation was more complex with strain SIT266 which is both the most virulent and bears 10 drug resistance mutations." -Fig.2A, correct colour code (red line in the legend for 4542 strain) ANSWER: corrected.
-p.i.upon first use, specify abbreviation ANSWER: done (section Animal study design in M&M).Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.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, Alexandra Aubry Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • 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