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The Strange Case of Prevotella copri: Dr. Jekyll or Mr. Hyde?
Cell Host & Microbe
Previews The Strange Case of Prevotella copri: Dr. Jekyll or Mr. Hyde? Sandrine P. Claus1,* 1LNC Therapeutics, 17 place de la Bourse, 33076 Bordeaux, France *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.10.020
This issue of Cell Host & Microbe highlights the opposite faces of Prevotella copri (P. copri). Tett et al. (2019) and Fehlner-Peach et al. (2019) unravel the complexity of gut dwelling P. copri, whereas Rolhion et al. (2019) explore the P. copri dark side with its ability to potentiate Listeria monocytogenes pathogenicity. The human gut microbiota of Westernized populations is dominated by two bacterial phyla, the Firmicutes and the Bacteroidetes, the latter being mostly dominated by either Bacteroides or Prevotella species. Both are highly saccharolytic bacteria, but they tend to mutually exclude one another, and therefore have been proposed to form two distinct gut enteroytpes (Costea et al., 2018). Prevotelladominated microbiomes have been shown to occur in populations fed a fiber-rich diet, such as rural African children (De Filippo et al., 2010), where these bacteria were also associated with a reduction of Enterobacteriaceae, a bacterial family comprising a number of opportunistic pathogens. Prevotella increase has also been associated with improvement in glucose metabolism and has been suggested as a potentially beneficial group of bacteria (Kovatcheva-Datchary et al., 2015). Yet, a number of studies have also reported increased Prevotella spp. levels in patients suffering from hypertension (Li et al., 2017) and of new onset rheumatoid arthritis (NORA) (Scher et al., 2013), which have led to suspicions that Prevotella spp. potentiate a pro-inflammatory status. These seemingly contradictory visions of Prevotella’s role on human physiology is likely due to a poor understanding of their diversity. Indeed, despite their widespread prevalence, little is known about the role of Prevotella spp. in human health because most of our current knowledge has been derived from the study of the two type strains isolated from intestinal samples: P. copri and P. stercorea (Hayashi et al., 2007). In 2017, Truong et al. observed that P. copri species are highly diverse and are characterized by an important inter-
individual strain specificity. More recently, De Filippis et al. (2019) analyzed the pangenome of P. copri recovered from nearly 50 Italian subjects and demonstrated species-specific diet associations. In this issue of Cell Host & Microbe, Tett et al. (2019) investigated the diversity of human-gut-associated P. copri by using a large-scale cross-continent metaanalysis of reconstructed P. copri genomes. From an initial dataset comprising > 6,800 metagenomes of Westernized and non-Westernized humans, they recovered 1,023 high quality P. copri genomes for population analysis. This genome catalog was then used to identify population structure and revealed that P. copri genomes clustered within 4 distinct clades (A, B, C, and D) that were defined on the basis of average nucleotide identity (ANI) scores. Intra-clade variation was low (ANI < 5%) but inter-clade distances was very high (ANI range from 13% to 21.4%), which according to previous large-scale metagenome studies would be considered as different species (Pasolli et al., 2019). Yet, the authors argue that the clade distinction is supported by core genome single-nucleotide distance and gene content distance and propose the naming ‘‘Prevotella copri complex’’ to encompass these 4 clades. In a parallel study, Fehlner-Peach et al. (2019) newly isolated 83 clones of P. copri from 11 donors. All fell within one of the four clades identified by Tett et al. (2019), although clades B and D had only one representing clone each. Most clones clustered within clade A. The authors further characterized all isolates by biochemical and genomic methods to confirm membership to the P. copri complex despite some noticeable variations in genomes and enzyme activities. Further-
more, they confirmed a high genomic diversity within the P. copri complex that was illustrated by a relatively modest core genome of 1,750 genes, whereas the accessory genome ranged from 1,250 to up to 2,250 genes in some strains. Because Prevotella spp. are well known for their highly saccharolytic activity, the authors hypothesized that genes involved in carbohydrate metabolism would likely reflect a broad range of saccharolytic functions and thus focused on studying the genomic diversity of well characterized genes encoding polysaccharide catabolism enzymes that are commonly grouped in polysaccharide utilization loci (PULs). They then used this information to predict growth on specific polysaccharides, which was further validated in vitro. This revealed that some polysaccharide utilizing abilities were clade specific. For example, all isolates except the only isolate in clade B, were able to grow on arabinoxylan and arabinan. On the contrary, only the clade B isolate was able to degrade heparin. These observations prompt the conclusion that P. copri strain selection must be driven by diet, especially plant-based food. Unfortunately, the authors did not include any diet record along these investigations, which would have been a nice addition to demonstrate P. copri strain selection by the type of fiber intake. Interestingly, Tett et al. (2019) also questioned the ability of P. copri to degrade plant-derived carbohydrates but focused on carbohydrate active enzymes (CAZymes). They discovered that even if all clades had the potential to degrade plant-derived carbohydrates, some CAZy families were only found in specific clades. This observation might explain how several close P. copri strains
Cell Host & Microbe 26, November 13, 2019 ª 2019 Elsevier Inc. 577
Cell Host & Microbe
Previews of different clades might co-inhabit the same host, given that they likely use complementary enzymatic activities to extract usable carbons from complex plantderived polysaccharides. Tett et al. (2019) further investigated how these four clades are geographically distributed but little geographical stratification could be identified among the 22 countries investigated, indicating that the P. copri complex is widely distributed. An interesting feature of the distribution of these four clades is their distribution among non-Westernized populations. Indeed, the authors were able to identify P. copri in nearly all investigated samples (95.4% prevalence compared with only 29.6% prevalence in Westernized populations). This high prevalence of Prevotella bacteria, and of P. copri in particular, had already been reported by previous studies (De Filippo et al., 2010) whose datasets were included in the present analysis. Still, the increased diversity of the P. copri complex among non-Westernized populations was striking. Most Westernized microbiomes were colonized with a single clade, whereas most of the non-Westernized microbiomes hosted at least two or three clades and 61.6% of them contained genomes from the four clades. On the contrary, only 4.6% of Westernized individuals contained the four P. copri complex clades, indicating an impoverishment of Westernized P. copri diversity. Consistent with Fehlner-Peach et al. (2019), this translated into a much larger P. copri functional potential in non-Westernized humans. The authors further questioned whether the high P. copri prevalence and co-presence of the four clades in non-Westernized populations was a pattern similarly observed in ancient human civilizations. For this purpose, they obtained metagenomic data from the O¨tzi Iceman who lived 5,300 years ago in the Southern Alps, and from 3 Mexican coprolites extracted from a Mexican cave that was inhabited by pre-Columbian humans about 1,300 B.P. Examining the metagenomes from these samples revealed that the P. copri complex distribution was very similar to this of non-Westernized individuals, both in terms of high prevalence and co-presence of more than one clade within the same individual. On the basis of these observations, one could expect that some clades might therefore be associated with specific dis-
eases or clinical markers. On the contrary, there was no evidence in this work that any of the four clades were associated with a disease among the five investigated conditions (i.e., colorectal cancer, type 2 diabetes, hypertension, liver cirrhosis, and inflammatory bowel diseases), nor did they associate with body mass index (BMI) or age. The authors went deeper into the analysis of sub-clades associations with these disorders but could not identify any significant association either. Yet, such association with a disease was suggested by findings of Rolhion et al. (2019). In this fascinating work, they discovered that P. copri bacteria potentiate growth of the pandemic pathogen Listeria monocytogenes. Using the L. monocytogenes strain EGD-e, a Lineage II strain, they demonstrated that Listeria produce a bacteriocin, Lmo2776, which impedes L. monocytogenes infection potential by targeting Prevotella spp. both in vivo and in vitro. Remarkably, the Lmo2776 seemed specific to P. copri strains, given that seven other Prevotella spp. were tested but remained unaffected. Finally, P. copri was found to degrade the mucus layer in vivo, which facilitated access of L. monocytogenes to the epithelium and was associated with an increased in lipocalin-2, a marker of intestinal inflammation. Although these latter findings support a pro-inflammatory role of P. copri in the context of gastrointestinal disorders, and in light of the newly discovered functional heterogeneity in the P. copri complex, it remains difficult to conclude about the potentially beneficial or detrimental influence of P. copri on human health. Altogether, these studies challenge our current understanding of the gut ecosystem. How can several close bacterial strains with very similar metabolic functions inhabit the same host without occupying the same ecological niches? Why does a bacterial strain carry a bacteriocin that limits its own growth? Can we restore the P. copri diversity in Westernized populations and is this desirable? Only by improving our understanding of the gut microbiome will we be able to answer these questions and many more. Above all, these studies underscore that understanding precisely the functionality of every single strain that should be used in the future to restore an impoverished microbiome is pristine to ensure efficient and safe microbiota-based therapies.
578 Cell Host & Microbe 26, November 13, 2019
REFERENCES Costea, P.I., Hildebrand, F., Arumugam, M., Backhed, F., Blaser, M.J., Bushman, F.D., de Vos, W.M., Ehrlich, S.D., Fraser, C.M., Hattori, M., et al. (2018). Enterotypes in the landscape of gut microbial community composition. Nature Microbiology 3, 1–9. De Filippis, F., Pasolli, E., Tett, A., Tarallo, S., Naccarati, A., De Angelis, M., Neviani, E., Cocolin, L., Gobbetti, M., Segata, N., and Ercolini, D. (2019). Distinct genetic and functional traits of human intestinal Prevotella copri strains are associated with different habitual diets. Cell Host Microbe 25, 444–453.e3. De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., and Lionetti, P. (2010). Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. USA 107, 14691–14696. Fehlner-Peach, H., Magnabosco, C., Raghavan, V., Scher, J.U., Tett, A., Cox, L.M., Gottsegen, C., Watters, A., Wiltshire-Gordon, J.D., Segata, N., et al. (2019). Distinct polysaccaride growth profiles of human intestinal Prevotella copri isolates. Cell Host Microbe 26, this issue, 680–690. Hayashi, H., Shibata, K., Sakamoto, M., Tomita, S., and Benno, Y. (2007). Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 57, 941–946. Kovatcheva-Datchary, P., Nilsson, A., Akrami, R., Lee, Y.S., De Vadder, F., Arora, T., Hallen, A., €ckhed, F. (2015). Martens, E., Bjo¨rck, I., and Ba Dietary Fiber-Induced Improvement in Glucose Metabolism Is Associated with Increased Abundance of Prevotella. Cell Metab. 22, 971–982. Li, J., Zhao, F., Wang, Y., Chen, J., Tao, J., Tian, G., Wu, S., Liu, W., Cui, Q., Geng, B., et al. (2017). Gut microbiota dysbiosis contributes to the development of hypertension. Microbiome 5, 14. Pasolli, E., Asnicar, F., Manara, S., Zolfo, M., Karcher, N., Armanini, F., Beghini, F., Manghi, P., Tett, A., Ghensi, P., et al. (2019). Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle. Cell 176, 649–662.e20. Rolhion, N., Chassaing, B., Littman, D.R., Van de Wiele, T., and Cossart, P. (2019). Targeting of intestinal Prevotella copri by a Listeria monocytogenes bacteriocin. Cell Host Microbe 26, this issue, 691–701. Scher, J.U., Sczesnak, A., Longman, R.S., Segata, N., Ubeda, C., Bielski, C., Rostron, T., Cerundolo, V., Pamer, E.G., Abramson, S.B., et al. (2013). Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife 2, e01202. Tett, A., Huang, K.D., Asnicar, F., Fehlner-Peach, H., Pasolli, E., Karcher, N., Armanini, F., Manghi, P., Bonham, K., Zolfo, M., et al. (2019). The Prevotella copri complex comprises four distinct clades that are underrepresented in Westernised populations. Cell Host Microbe 26, this issue, 666–679. Truong, D.T., Tett, A., Pasolli, E., Huttenhower, C., and Segata, N. (2017). Microbial strain-level population structure and genetic diversity from metagenomes. Genome Res. 27, 626–638.
Previews The Strange Case of Prevotella copri: Dr. Jekyll or Mr. Hyde? Sandrine P. Claus1,* 1LNC Therapeutics, 17 place de la Bourse, 33076 Bordeaux, France *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.10.020
This issue of Cell Host & Microbe highlights the opposite faces of Prevotella copri (P. copri). Tett et al. (2019) and Fehlner-Peach et al. (2019) unravel the complexity of gut dwelling P. copri, whereas Rolhion et al. (2019) explore the P. copri dark side with its ability to potentiate Listeria monocytogenes pathogenicity. The human gut microbiota of Westernized populations is dominated by two bacterial phyla, the Firmicutes and the Bacteroidetes, the latter being mostly dominated by either Bacteroides or Prevotella species. Both are highly saccharolytic bacteria, but they tend to mutually exclude one another, and therefore have been proposed to form two distinct gut enteroytpes (Costea et al., 2018). Prevotelladominated microbiomes have been shown to occur in populations fed a fiber-rich diet, such as rural African children (De Filippo et al., 2010), where these bacteria were also associated with a reduction of Enterobacteriaceae, a bacterial family comprising a number of opportunistic pathogens. Prevotella increase has also been associated with improvement in glucose metabolism and has been suggested as a potentially beneficial group of bacteria (Kovatcheva-Datchary et al., 2015). Yet, a number of studies have also reported increased Prevotella spp. levels in patients suffering from hypertension (Li et al., 2017) and of new onset rheumatoid arthritis (NORA) (Scher et al., 2013), which have led to suspicions that Prevotella spp. potentiate a pro-inflammatory status. These seemingly contradictory visions of Prevotella’s role on human physiology is likely due to a poor understanding of their diversity. Indeed, despite their widespread prevalence, little is known about the role of Prevotella spp. in human health because most of our current knowledge has been derived from the study of the two type strains isolated from intestinal samples: P. copri and P. stercorea (Hayashi et al., 2007). In 2017, Truong et al. observed that P. copri species are highly diverse and are characterized by an important inter-
individual strain specificity. More recently, De Filippis et al. (2019) analyzed the pangenome of P. copri recovered from nearly 50 Italian subjects and demonstrated species-specific diet associations. In this issue of Cell Host & Microbe, Tett et al. (2019) investigated the diversity of human-gut-associated P. copri by using a large-scale cross-continent metaanalysis of reconstructed P. copri genomes. From an initial dataset comprising > 6,800 metagenomes of Westernized and non-Westernized humans, they recovered 1,023 high quality P. copri genomes for population analysis. This genome catalog was then used to identify population structure and revealed that P. copri genomes clustered within 4 distinct clades (A, B, C, and D) that were defined on the basis of average nucleotide identity (ANI) scores. Intra-clade variation was low (ANI < 5%) but inter-clade distances was very high (ANI range from 13% to 21.4%), which according to previous large-scale metagenome studies would be considered as different species (Pasolli et al., 2019). Yet, the authors argue that the clade distinction is supported by core genome single-nucleotide distance and gene content distance and propose the naming ‘‘Prevotella copri complex’’ to encompass these 4 clades. In a parallel study, Fehlner-Peach et al. (2019) newly isolated 83 clones of P. copri from 11 donors. All fell within one of the four clades identified by Tett et al. (2019), although clades B and D had only one representing clone each. Most clones clustered within clade A. The authors further characterized all isolates by biochemical and genomic methods to confirm membership to the P. copri complex despite some noticeable variations in genomes and enzyme activities. Further-
more, they confirmed a high genomic diversity within the P. copri complex that was illustrated by a relatively modest core genome of 1,750 genes, whereas the accessory genome ranged from 1,250 to up to 2,250 genes in some strains. Because Prevotella spp. are well known for their highly saccharolytic activity, the authors hypothesized that genes involved in carbohydrate metabolism would likely reflect a broad range of saccharolytic functions and thus focused on studying the genomic diversity of well characterized genes encoding polysaccharide catabolism enzymes that are commonly grouped in polysaccharide utilization loci (PULs). They then used this information to predict growth on specific polysaccharides, which was further validated in vitro. This revealed that some polysaccharide utilizing abilities were clade specific. For example, all isolates except the only isolate in clade B, were able to grow on arabinoxylan and arabinan. On the contrary, only the clade B isolate was able to degrade heparin. These observations prompt the conclusion that P. copri strain selection must be driven by diet, especially plant-based food. Unfortunately, the authors did not include any diet record along these investigations, which would have been a nice addition to demonstrate P. copri strain selection by the type of fiber intake. Interestingly, Tett et al. (2019) also questioned the ability of P. copri to degrade plant-derived carbohydrates but focused on carbohydrate active enzymes (CAZymes). They discovered that even if all clades had the potential to degrade plant-derived carbohydrates, some CAZy families were only found in specific clades. This observation might explain how several close P. copri strains
Cell Host & Microbe 26, November 13, 2019 ª 2019 Elsevier Inc. 577
Cell Host & Microbe
Previews of different clades might co-inhabit the same host, given that they likely use complementary enzymatic activities to extract usable carbons from complex plantderived polysaccharides. Tett et al. (2019) further investigated how these four clades are geographically distributed but little geographical stratification could be identified among the 22 countries investigated, indicating that the P. copri complex is widely distributed. An interesting feature of the distribution of these four clades is their distribution among non-Westernized populations. Indeed, the authors were able to identify P. copri in nearly all investigated samples (95.4% prevalence compared with only 29.6% prevalence in Westernized populations). This high prevalence of Prevotella bacteria, and of P. copri in particular, had already been reported by previous studies (De Filippo et al., 2010) whose datasets were included in the present analysis. Still, the increased diversity of the P. copri complex among non-Westernized populations was striking. Most Westernized microbiomes were colonized with a single clade, whereas most of the non-Westernized microbiomes hosted at least two or three clades and 61.6% of them contained genomes from the four clades. On the contrary, only 4.6% of Westernized individuals contained the four P. copri complex clades, indicating an impoverishment of Westernized P. copri diversity. Consistent with Fehlner-Peach et al. (2019), this translated into a much larger P. copri functional potential in non-Westernized humans. The authors further questioned whether the high P. copri prevalence and co-presence of the four clades in non-Westernized populations was a pattern similarly observed in ancient human civilizations. For this purpose, they obtained metagenomic data from the O¨tzi Iceman who lived 5,300 years ago in the Southern Alps, and from 3 Mexican coprolites extracted from a Mexican cave that was inhabited by pre-Columbian humans about 1,300 B.P. Examining the metagenomes from these samples revealed that the P. copri complex distribution was very similar to this of non-Westernized individuals, both in terms of high prevalence and co-presence of more than one clade within the same individual. On the basis of these observations, one could expect that some clades might therefore be associated with specific dis-
eases or clinical markers. On the contrary, there was no evidence in this work that any of the four clades were associated with a disease among the five investigated conditions (i.e., colorectal cancer, type 2 diabetes, hypertension, liver cirrhosis, and inflammatory bowel diseases), nor did they associate with body mass index (BMI) or age. The authors went deeper into the analysis of sub-clades associations with these disorders but could not identify any significant association either. Yet, such association with a disease was suggested by findings of Rolhion et al. (2019). In this fascinating work, they discovered that P. copri bacteria potentiate growth of the pandemic pathogen Listeria monocytogenes. Using the L. monocytogenes strain EGD-e, a Lineage II strain, they demonstrated that Listeria produce a bacteriocin, Lmo2776, which impedes L. monocytogenes infection potential by targeting Prevotella spp. both in vivo and in vitro. Remarkably, the Lmo2776 seemed specific to P. copri strains, given that seven other Prevotella spp. were tested but remained unaffected. Finally, P. copri was found to degrade the mucus layer in vivo, which facilitated access of L. monocytogenes to the epithelium and was associated with an increased in lipocalin-2, a marker of intestinal inflammation. Although these latter findings support a pro-inflammatory role of P. copri in the context of gastrointestinal disorders, and in light of the newly discovered functional heterogeneity in the P. copri complex, it remains difficult to conclude about the potentially beneficial or detrimental influence of P. copri on human health. Altogether, these studies challenge our current understanding of the gut ecosystem. How can several close bacterial strains with very similar metabolic functions inhabit the same host without occupying the same ecological niches? Why does a bacterial strain carry a bacteriocin that limits its own growth? Can we restore the P. copri diversity in Westernized populations and is this desirable? Only by improving our understanding of the gut microbiome will we be able to answer these questions and many more. Above all, these studies underscore that understanding precisely the functionality of every single strain that should be used in the future to restore an impoverished microbiome is pristine to ensure efficient and safe microbiota-based therapies.
578 Cell Host & Microbe 26, November 13, 2019
REFERENCES Costea, P.I., Hildebrand, F., Arumugam, M., Backhed, F., Blaser, M.J., Bushman, F.D., de Vos, W.M., Ehrlich, S.D., Fraser, C.M., Hattori, M., et al. (2018). Enterotypes in the landscape of gut microbial community composition. Nature Microbiology 3, 1–9. De Filippis, F., Pasolli, E., Tett, A., Tarallo, S., Naccarati, A., De Angelis, M., Neviani, E., Cocolin, L., Gobbetti, M., Segata, N., and Ercolini, D. (2019). Distinct genetic and functional traits of human intestinal Prevotella copri strains are associated with different habitual diets. Cell Host Microbe 25, 444–453.e3. De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., and Lionetti, P. (2010). Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. USA 107, 14691–14696. Fehlner-Peach, H., Magnabosco, C., Raghavan, V., Scher, J.U., Tett, A., Cox, L.M., Gottsegen, C., Watters, A., Wiltshire-Gordon, J.D., Segata, N., et al. (2019). Distinct polysaccaride growth profiles of human intestinal Prevotella copri isolates. Cell Host Microbe 26, this issue, 680–690. Hayashi, H., Shibata, K., Sakamoto, M., Tomita, S., and Benno, Y. (2007). Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 57, 941–946. Kovatcheva-Datchary, P., Nilsson, A., Akrami, R., Lee, Y.S., De Vadder, F., Arora, T., Hallen, A., €ckhed, F. (2015). Martens, E., Bjo¨rck, I., and Ba Dietary Fiber-Induced Improvement in Glucose Metabolism Is Associated with Increased Abundance of Prevotella. Cell Metab. 22, 971–982. Li, J., Zhao, F., Wang, Y., Chen, J., Tao, J., Tian, G., Wu, S., Liu, W., Cui, Q., Geng, B., et al. (2017). Gut microbiota dysbiosis contributes to the development of hypertension. Microbiome 5, 14. Pasolli, E., Asnicar, F., Manara, S., Zolfo, M., Karcher, N., Armanini, F., Beghini, F., Manghi, P., Tett, A., Ghensi, P., et al. (2019). Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle. Cell 176, 649–662.e20. Rolhion, N., Chassaing, B., Littman, D.R., Van de Wiele, T., and Cossart, P. (2019). Targeting of intestinal Prevotella copri by a Listeria monocytogenes bacteriocin. Cell Host Microbe 26, this issue, 691–701. Scher, J.U., Sczesnak, A., Longman, R.S., Segata, N., Ubeda, C., Bielski, C., Rostron, T., Cerundolo, V., Pamer, E.G., Abramson, S.B., et al. (2013). Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife 2, e01202. Tett, A., Huang, K.D., Asnicar, F., Fehlner-Peach, H., Pasolli, E., Karcher, N., Armanini, F., Manghi, P., Bonham, K., Zolfo, M., et al. (2019). The Prevotella copri complex comprises four distinct clades that are underrepresented in Westernised populations. Cell Host Microbe 26, this issue, 666–679. Truong, D.T., Tett, A., Pasolli, E., Huttenhower, C., and Segata, N. (2017). Microbial strain-level population structure and genetic diversity from metagenomes. Genome Res. 27, 626–638.