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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03497</article-id><article-id pub-id-type="doi">10.7554/eLife.03497</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Short report</subject></subj-group><subj-group subj-group-type="heading"><subject>Microbiology and infectious disease</subject></subj-group></article-categories><title-group><article-title>Evolutionary consequences of intra-patient phage predation on microbial populations</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-14685"><name><surname>Seed</surname><given-names>Kimberley D</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14686"><name><surname>Yen</surname><given-names>Minmin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14687"><name><surname>Shapiro</surname><given-names>B Jesse</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14688"><name><surname>Hilaire</surname><given-names>Isabelle J</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14689"><name><surname>Charles</surname><given-names>Richelle C</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff5"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14690"><name><surname>Teng</surname><given-names>Jessica E</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff6"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14691"><name><surname>Ivers</surname><given-names>Louise C</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff6"/><xref ref-type="aff" rid="aff8"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14692"><name><surname>Boncy</surname><given-names>Jacques</given-names></name><xref ref-type="aff" rid="aff7"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" id="author-14693"><name><surname>Harris</surname><given-names>Jason B</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff5"/><xref ref-type="aff" rid="aff9"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><contrib contrib-type="author" corresp="yes" id="author-7578"><name><surname>Camilli</surname><given-names>Andrew</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/></contrib><aff id="aff1"><institution content-type="dept">Department of Molecular Biology and Microbiology</institution>, <institution>Howard Hughes Medical Institute, Tufts University School of Medicine</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Département de sciences biologiques</institution>, <institution>Université de Montréal</institution>, <addr-line><named-content content-type="city">Montreal</named-content></addr-line>, <country>Canada</country></aff><aff id="aff3"><institution>Partners In Health</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Division of Infectious Diseases</institution>, <institution>Massachusetts General Hospital</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Department of Medicine</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff6"><institution content-type="dept">Division of Global Health Equity</institution>, <institution>Brigham and Women's Hospital</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff7"><institution>National Public Health Laboratory</institution>, <addr-line><named-content content-type="city">Port-au-Prince</named-content></addr-line>, <country>Haiti</country></aff><aff id="aff8"><institution content-type="dept">Department of Global Health and Social Medicine</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff9"><institution content-type="dept">Department of Pediatrics</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Cossart</surname><given-names>Pascale</given-names></name><role>Reviewing editor</role><aff><institution>Institut Pasteur</institution>, <country>France</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>andrew.camilli@tufts.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>26</day><month>08</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03497</elocation-id><history><date date-type="received"><day>04</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>24</day><month>07</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Seed et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Seed et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03497.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03497.001</object-id><p>The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, <italic>Vibrio cholerae</italic>, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient <italic>V. cholerae</italic> populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant <italic>V. cholerae</italic> populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of <italic>V. cholerae</italic> shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.001">http://dx.doi.org/10.7554/eLife.03497.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03497.002</object-id><title>eLife digest</title><p>Cholera epidemics occur seasonally in areas such as Bangladesh, and outbreaks can also strike in vulnerable regions, as has occurred recently in Haiti. The disease is caused by <italic>Vibrio cholerae</italic>, a water-borne bacterium that colonizes the small intestine, and its symptoms include severe diarrhea and vomiting which can lead to death if the patient is not treated promptly.</p><p>Lytic phages are viruses that specifically attack and kill bacteria. After replicating many times inside the bacterial cell, the phages break open and destroy the cell. Over time a bacterial population can evolve to resist this phage ‘predation’; however, it is not known if bacterial pathogens need to defend themselves against phage attack when they infect humans. It had been suggested that phages might affect the progress of cholera infections in people, but molecular evidence that supports this hypothesis was lacking.</p><p>When testing stool samples from Haitian cholera patients, Seed et al. found one sample contained a lot of lytic phage relative to the amount of <italic>V. cholerae</italic> present. This phage was very similar to—but distinct from—a phage found in Bangladeshi patients.</p><p>The <italic>V. cholerae</italic> bacteria isolated from the stool sample were resistant to attack by the phage. Sequencing the genome of individual bacteria from this sample revealed that each had a mutation that made them resistant to the phage; and while many types of these mutations were found, these were the only differences between all the <italic>V. cholerae</italic> bacteria in this patient sample. This suggests that this resistance developed independently many different times within the patient due to strong selective pressure from phage predation.</p><p>When Seed et al. looked at a phage-positive stool sample from a Bangladeshi patient, more mutations that made the bacteria resistant to this phage were found; however, these mutations were different again from the ones in the Haitian bacteria. Because of the nature of these mutations the bacteria from this patient were rendered unable to cause disease and non-transmissible.</p><p>This work shows that phages can indeed have access to pathogenic bacteria during human infection. It also indicates that the pressure imposed by phage predation can, in some cases, be so strong that the bacteria lose their virulence and ability to spread to other humans in order to become resistant to the phage.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.002">http://dx.doi.org/10.7554/eLife.03497.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd><italic>Vibrio cholerae</italic></kwd><kwd>cholera</kwd><kwd>bacteriophage</kwd><kwd>phage</kwd><kwd>OmpU</kwd><kwd>ToxR</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>viruses</kwd><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Camilli</surname><given-names>Andrew</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AI055058</award-id><principal-award-recipient><name><surname>Camilli</surname><given-names>Andrew</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100005294</institution-id><institution>Massachusetts General Hospital</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Charles</surname><given-names>Richelle C</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000038</institution-id><institution>Natural Sciences and Engineering Research Council of Canada</institution></institution-wrap></funding-source><award-id>950-228996</award-id><principal-award-recipient><name><surname>Shapiro</surname><given-names>B Jesse</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001804</institution-id><institution>Canada Research Chairs</institution></institution-wrap></funding-source><award-id>950-228996</award-id><principal-award-recipient><name><surname>Shapiro</surname><given-names>B Jesse</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>Charles A. King Trust</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Seed</surname><given-names>Kimberley D</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AI099243</award-id><principal-award-recipient><name><surname>Ivers</surname><given-names>Louise C</given-names></name><name><surname>Harris</surname><given-names>Jason B</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AI089721</award-id><principal-award-recipient><name><surname>Charles</surname><given-names>Richelle C</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Bacterial viruses are an unexpected ‘third party’ that imposes a strong predatory pressure on a bacterial pathogen during the natural course of infection in humans.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1"><title>Main Text</title><p>Traditional views of host–pathogen interactions are of a battle between two opposing organisms. This perspective is being challenged with greater appreciation of the influence of the host's microbial ecosystem on these interactions (<xref ref-type="bibr" rid="bib17">Lozupone et al., 2012</xref>). Bacteriophages influence bacterial populations in many ecosystems and specifically temperate phages play a role in many diseases through lysogenic conversion (<xref ref-type="bibr" rid="bib2">Brüssow et al., 2004</xref>). In contrast, the impact of lytic phage predation on bacterial pathogens in the context of human disease is under-appreciated. <italic>Vibrio cholerae</italic> is a water-borne pathogen responsible for the diarrheal disease cholera. In order for <italic>V. cholerae</italic> to colonize the human small intestine and elicit diarrhea, it produces a set of virulence determinants including cholera toxin (<xref ref-type="bibr" rid="bib12">Herrington et al., 1988</xref>). The ToxR signaling cascade activates expression of these virulence factors in response to host environmental stimuli (<xref ref-type="bibr" rid="bib21">Miller et al., 1987</xref>; <xref ref-type="bibr" rid="bib30">Taylor et al., 1987</xref>; <xref ref-type="bibr" rid="bib7">DiRita et al., 1991</xref>; <xref ref-type="bibr" rid="bib3">Childers and Klose, 2007</xref>). Cholera epidemics appear with regular seasonality in regions like Bangladesh (<xref ref-type="bibr" rid="bib9">Faruque et al., 2005</xref>) and can arise unpredictably in vulnerable regions as exemplified by the epidemic that recently began in Haiti following the single-source introduction of a pandemic <italic>V. cholerae</italic> O1 strain from another continent (<xref ref-type="bibr" rid="bib4">Chin et al., 2011</xref>; <xref ref-type="bibr" rid="bib5">Cravioto et al., 2011</xref>; <xref ref-type="bibr" rid="bib10">Frerichs et al., 2012</xref>; <xref ref-type="bibr" rid="bib14">Katz et al., 2013</xref>). Lytic phages are hypothesized to impact cholera disease burden in Bangladesh (<xref ref-type="bibr" rid="bib8">Faruque et al., 2005</xref>). The predation of <italic>V. cholerae</italic> by phages following their co-ingestion from the environment is central to this hypothesis; however, molecular evidence in support of this hypothesis is lacking.</p><p>We recently described the ICP2 species of <italic>V. cholerae</italic>-specific, virulent podoviruses that are found sporadically in cholera patient stool samples collected since 2001 in Bangladesh (<xref ref-type="bibr" rid="bib27">Seed et al., 2011</xref> and present study). To begin to address the geographic diversity of cholera phages in patient stools, we tested by plaque assay for the presence of phages within nine Haitian cholera patient stool samples collected in 2013. We identified one sample that had a high titer of phage (10<sup>8</sup> PFU/ml) relative to <italic>V. cholerae</italic> (10<sup>5</sup> CFU/ml). Whole genome sequencing and comparative analysis of this phage, named ICP2_2013_A_Haiti, revealed 84% identity over 93% of its genome to ICP2_2011_A, a phage isolated from Bangladesh in 2011. An alignment of the annotated ICP2 genome (<xref ref-type="bibr" rid="bib27">Seed et al., 2011</xref>) with the three available ICP2-related isolates shows that synteny is conserved between these geographically distinct phages, with no significant genome rearrangements observed (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). The Haitian ICP2 isolate is strikingly similar, although clearly distinct, from Bangladeshi ICP2 isolates, and is the first lytic phage reported to be associated with epidemic <italic>V. cholerae</italic> in Haiti.</p><p>Remarkably, we observed that most <italic>V. cholerae</italic> isolates recovered from the same stool sample as ICP2_2013_A_Haiti were resistant to infection by this phage. We investigated phenotypic heterogeneity within this stool sample by testing 269 single colony isolates for sensitivity to ICP2_2013_A_Haiti and found that 267 (>99%) were phage-resistant. Eight phage-resistant isolates and the two phage-sensitive isolates from this sample were subjected to whole genome sequencing. All 10 isolates were isogenic except for mutations within <italic>ompU</italic> encoding the major outer membrane porin (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). Furthermore, the <italic>ompU</italic> mutations in the eight phage-resistant isolates were heterogeneous. We sequenced <italic>ompU</italic> from an additional 11 phage-resistant isolates from this stool sample and found a total of six unique <italic>ompU</italic> alleles (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). When each of these alleles was used to replace <italic>ompU</italic> in a clean genetic background, all conferred resistance to ICP2, yet produced normal amounts of OmpU (<xref ref-type="fig" rid="fig2">Figure 2</xref> and data not shown), suggesting that ICP2 uses OmpU as a receptor to initiate infection and that the mutations disrupt this interaction. These data showing that intra-host <italic>V. cholerae</italic> isolates are isogenic, only differing in ICP2 resistance mutations, indicates that they diverged within the patient. Furthermore, the presence of multiple different ICP2 resistance mutations within this single host suggests that selection of phage resistance occurred multiple, independent times during the course of infection. We also found evidence for ICP2-mediated selection of OmpU mutants in isolates from Bangladesh. Analysis of the <italic>ompU</italic> sequence from 54 clinical isolates collected between 2001 and 2011 showed that 15% had non-synonomous mutations (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>) and these alleles were sufficient for ICP2 resistance (<xref ref-type="fig" rid="fig2">Figure 2</xref>). One of the OmpU mutants, G325D, was observed in clinical isolates from Haiti and Bangladesh (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Interestingly, seven of the eight mutant OmpU proteins detected in the Haitian and Bangladeshi clinical isolates had alterations in two predicted extracellular loops, while the eighth had an altered transmembrane segment adjacent to the first of these extracellular loops (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). This suggests that ICP2 interacts specifically with these exposed loops to initiate infection.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03497.003</object-id><label>Figure 1.</label><caption><title>The presence of <italic>V. cholerae</italic> OmpU and ToxR mutants present within and between cholera patients.</title><p>(<bold>A</bold>) Graphical depiction and frequency of OmpU mutants found within a stool sample containing ICP2_2013_A_Haiti phage (10<sup>8</sup> PFU/ml) from a single Haitian patient (top) and from different patients in Bangladesh (n = 54) (bottom). (<bold>B</bold>) Predicted membrane topology of mature OmpU generated using Pred-TMBB (<xref ref-type="bibr" rid="bib1">Bagos et al., 2004</xref>). Locations of amino acid substitutions or insertions carried by <italic>V. cholerae</italic> clinical isolates are indicated. (<bold>C</bold>) Graphical depiction and frequency of ToxR mutants found within a stool sample containing ICP2_2011_A (10<sup>9</sup> PFU/ml) from a single Bangladeshi patient. Amino acid substitutions or nonsense mutations (asterisks) are in orange and duplications are in blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.003">http://dx.doi.org/10.7554/eLife.03497.003</ext-link></p></caption><graphic xlink:href="elife03497f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03497.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>ICP2_2013_A_Haiti is closely related to ICP2 bacteriophages from Bangladesh.</title><p>Comparison of ICP2 genomes collected from cholera patients in Dhaka, Bangladesh in 2004 (ICP2), 2006 and 2011, and in Haiti in 2013 using progressiveMauve software. The degree of nucleotide similarity between aligned regions is indicated by the height of the similarity profile (colored blocks), where mauve represents the highly conserved backbone genome and other colors represent segments whose presence varies between isolates. Annotated genes in the ICP2 genome are shown as white boxes, with genes transcribed from the negative strand displaced downward. The numbers above the ICP2 genome show distance in kilobases.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.004">http://dx.doi.org/10.7554/eLife.03497.004</ext-link></p></caption><graphic xlink:href="elife03497fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03497.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Identification of OmpU mutants in samples collected at the International Centre for Diarrheal Disease Research, Bangladesh between 2001–2011.</title><p>(a) Single <italic>V. cholerae</italic> O1 El Tor isolates from different stool samples collected within a given year are indicated as closed circles. (b) The number of isolates with the noted mutation is indicated in a given year. If left blank, OmpU was wild-type.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.005">http://dx.doi.org/10.7554/eLife.03497.005</ext-link></p></caption><graphic xlink:href="elife03497fs002"/></fig></fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03497.006</object-id><label>Figure 2.</label><caption><title>OmpU expression of OmpU and ToxR mutants and their sensitivity to ICP2.</title><p>Outer membrane fractions were prepared from samples matched by equivalent OD<sub>600</sub> units. Samples were separated by SDS-PAGE and subjected to Western blot analysis using rabbit polyclonal antisera against OmpU. The sensitivity of each strain to ICP2_2013_A_Haiti is represented as a histogram of the efficiency of plaquing, which is the plaque count ratio of a mutant <italic>V. cholerae</italic> strain to that of the wild-type strain. The limit of detection for plaque assays was 10<sup>−7</sup>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.006">http://dx.doi.org/10.7554/eLife.03497.006</ext-link></p></caption><graphic xlink:href="elife03497f002"/></fig></p><p>We also tested for <italic>V. cholerae</italic> population heterogeneity within an ICP2-positive stool sample isolated in Bangladesh in 2011. Of 46 single colony isolates tested for sensitivity to ICP2_2011_A, 22% were resistant to the phage. Whole genome sequencing of two phage-sensitive and four phage-resistant isolates from this sample revealed that the strains were isogenic, except that phage-resistant isolates had nonsense mutations in <italic>toxR</italic>, the direct transcriptional activator of <italic>ompU</italic> (<xref ref-type="bibr" rid="bib6">Crawford et al., 1998</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref>). We sequenced <italic>toxR</italic> from an additional eight phage-resistant isolates from this stool sample and found that, analogous to what was observed in the Haitian patient sample, there were multiple unique <italic>toxR</italic> alleles, which included two non-synonomous and two nonsense mutations (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Each of the four mutant <italic>toxR</italic> alleles conferred ICP2 resistance and abrogated expression of OmpU when used to replace wild-type <italic>toxR</italic> in a clean genetic background (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Both ICP2-sensitivity and OmpU expression were restored to the clinical <italic>toxR</italic> mutants by expressing <italic>ompU in trans</italic> or by reverting the <italic>toxR</italic> allele to wild-type (data not shown), indicating that these mutant <italic>toxR</italic> alleles are necessary and sufficient for ICP2 resistance and that this resistance is mediated through loss of OmpU expression.</p><p>To address the potential consequences of the phage-resistance mutations on <italic>V. cholerae</italic> fitness<italic>,</italic> we tested the four most frequently isolated <italic>ompU</italic> alleles and all four <italic>toxR</italic> alleles in survival and growth assays. Recent Tn-seq analyses of <italic>V. cholerae</italic> showed that OmpU is critical for fitness in an infant rabbit infection model (<xref ref-type="bibr" rid="bib11">Fu et al., 2013</xref>; <xref ref-type="bibr" rid="bib13">Kamp et al., 2013</xref>) and for dissemination from the host into pond water (<xref ref-type="bibr" rid="bib13">Kamp et al., 2013</xref>). OmpU has also been shown to be important for protection against the bactericidal effect of bile salts (<xref ref-type="bibr" rid="bib25">Provenzano et al., 2001</xref>), cationic peptides (<xref ref-type="bibr" rid="bib18">Mathur and Waldor, 2004</xref>), and intestinal organic acids (<xref ref-type="bibr" rid="bib19">Merrell et al., 2001</xref>). When we tested for survival in bile and for competitive fitness in pond water we found that the four OmpU mutants are fully fit (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>), consistent with their wild-type levels of OmpU expression (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Moreover, the OmpU(G325D) mutant was fully virulent in a single round, competitive infection in infant rabbits (<xref ref-type="fig" rid="fig4">Figure 4</xref>, first column). However, the OmpU mutants, particularly the A195T and G325D mutants, showed a mild competitive defect after multiple passaging in growth medium (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). The mild growth defect of the OmpU mutants in conjunction with the low prevalence of ICP2 (<xref ref-type="bibr" rid="bib27">Seed et al., 2011</xref>) may explain why the ICP2-resistant <italic>ompU</italic> variants do not become fixed in the population (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). In sharp contrast, in the presence of ICP2 we observed a 10,000-fold enrichment of the OmpU(G325D) mutant over the wild-type after infection of infant rabbits (<xref ref-type="fig" rid="fig4">Figure 4</xref>), indicating that strong selective pressure is imposed by phage predation during <italic>V. cholerae</italic> infection. This emulates what we hypothesize happens in human infections in the presence of ICP2 and is the first demonstration of phage predation of <italic>V. cholerae</italic> in the context of a diarrheal disease model.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03497.007</object-id><label>Figure 3.</label><caption><title>The fitness cost of clinically relevant OmpU and ToxR mutations.</title><p>(<bold>A</bold>) Clinically relevant OmpU mutants retain fitness in the presence of bile. *p < 0.05 significantly different means for the compared data sets (Mann–Whitney U Test). (<bold>B</bold>) OmpU mutants retain competitive fitness in pond water. *p < 0.05 significantly different from wild-type control (Kruskal–Wallis and <italic>post hoc</italic> Dunn's multiple comparison tests). (<bold>C</bold>) OmpU mutants have slight competitive fitness defects when serially passaged in Luria–Bertani broth (for ca. 58 generations). *p < 0.05 or ****p < 0.0001 significantly different from wild-type control (Kruskal–Wallis and <italic>post hoc</italic> Dunn's multiple comparison tests). (<bold>D</bold>) ToxR mutants are attenuated in vivo using the infant mouse colonization model. **p < 0.01 or ***p < 0.001 significantly different from the in vitro median (Mann–Whitney U Tests). The horizontal bars indicate the median of each data set. Open symbols represent data below the limit of detection.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.007">http://dx.doi.org/10.7554/eLife.03497.007</ext-link></p></caption><graphic xlink:href="elife03497f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03497.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Clinical isolates harboring ToxR mutations are severely attenuated for infection.</title><p>Competitions indices (CI) in infant mice after 24 hr of infection were determined between each clinical ToxR mutant strain and its isogenic ToxR wild-type revertant strain carrying a <italic>lacZ</italic> deletion. The <italic>lacZ</italic> deletion allowed for differentiation of the competing strains upon plating on LB agar plates supplemented with X-gal. Each symbol represents the CI for an individual animal. The horizontal bars indicate the median of each data set. The open symbols represent data below the limit of detection for the ToxR mutant strain.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.008">http://dx.doi.org/10.7554/eLife.03497.008</ext-link></p></caption><graphic xlink:href="elife03497fs003"/></fig></fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03497.009</object-id><label>Figure 4.</label><caption><title>Phage predation leads to enrichment of OmpU mutant over wild-type in vivo.</title><p>Competitive indices (CI) were determined between wild-type Δ<italic>lacZ</italic> and OmpU G325D in the absence or presence of ICP2_2013_A_Haiti at the multiplicity of infection (MOI) indicated in infant rabbits 12 hr post-infection. Each symbol represents the CI for an individual rabbit and the horizontal lines indicate the median for each condition. The open symbols represent data below the limit of detection for the wild-type strain. **p < 0.01, significantly different from no phage control (Kruskal–Wallis and <italic>post hoc</italic> Dunn's multiple comparison tests).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.009">http://dx.doi.org/10.7554/eLife.03497.009</ext-link></p></caption><graphic xlink:href="elife03497f004"/></fig></p><p><italic>V. cholerae</italic> strains harboring any of the four ToxR mutations were avirulent in the infant mouse model of infection and were indistinguishable from a Δ<italic>toxR</italic> mutant (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). The clinical isolates containing these mutations were also at least 100-fold attenuated (<xref ref-type="fig" rid="fig3s1">Figure 3D—figure supplement 1</xref>), indicating that these isolates do not harbor compensatory mutations. The cytoplasmic amino terminus (D17-E112) of ToxR where the two non-synonomous mutations mapped has homology to the winged helix-turn-helix family of transcription activators and is involved in DNA binding and transcriptional activation (<xref ref-type="bibr" rid="bib21">Miller et al., 1987</xref>; <xref ref-type="bibr" rid="bib24">Ottemann et al., 1992</xref>). A number of point mutations in this domain were previously shown to abrogate activation of <italic>ompU</italic> and virulence genes (<xref ref-type="bibr" rid="bib24">Ottemann et al., 1992</xref>; <xref ref-type="bibr" rid="bib22">Morgan et al., 2011</xref>). The severe virulence defects of the two non-synonomous <italic>toxR</italic> mutants from the Bangladeshi patient sample (<xref ref-type="fig" rid="fig3">Figure 3D</xref>) are consistent with an inability of the encoded mutant ToxR proteins to activate these genes. These <italic>toxR</italic> mutants would not colonize the human small intestine and be recovered in the secretory diarrhea if ingested (<xref ref-type="bibr" rid="bib12">Herrington et al., 1988</xref>), which further supports our conclusion that these mutants arose due to ICP2 predation during cholera infection in this individual. This also indicates that, in this patient, predation and selection of phage-resistant mutants likely occurred late in infection when colonization and virulence gene expression were no longer required for the development of acute symptoms (<xref ref-type="bibr" rid="bib20">Merrell et al., 2002</xref>).</p><p>We have shown that lytic phages are an unexpected ‘third party’ that impose significant bactericidal pressure on an environmentally transmitted pathogen during the natural course of infection in humans. We observed that adaptations to phage predation involve tradeoffs in evolutionary fitness and provide a molecular mechanism for phage predation impacting <italic>V. cholerae</italic> transmission and seeding of environmental reservoirs. Our results highlight that host–pathogen interactions are embedded within and strongly affected by a complex microbial ecosystem. Predator-prey dynamics are notably absent between microbiotal phage and their bacterial hosts in the intestinal ecosystem in healthy humans (<xref ref-type="bibr" rid="bib26">Reyes et al., 2010</xref>). In contrast, our findings indicate that diseased states, which are often accompanied by significant bacterial proliferation, likely facilitate these predatory interactions.</p></sec><sec id="s2" sec-type="materials|methods"><title>Materials and methods</title><sec id="s2-1"><title>Growth conditions, strains and genomic analysis</title><p>Strains utilized in this study are listed in <xref ref-type="supplementary-material" rid="SD3-data">Supplementary file 3</xref>. Stool specimens and <italic>V. cholerae</italic> isolates from the International Centre for Diarrheal Disease Research, Bangladesh were collected and stored from previous studies (<xref ref-type="bibr" rid="bib23">Nelson et al., 2008</xref>; <xref ref-type="bibr" rid="bib27">Seed et al., 2011</xref>, <xref ref-type="bibr" rid="bib28">2012</xref>, <xref ref-type="bibr" rid="bib29">2013</xref>). Phage (ICP2_2011_A and ICP2_2013_A_Haiti) were isolated from cholera rice-water stool samples as described (<xref ref-type="bibr" rid="bib27">Seed et al., 2011</xref>). <italic>V. cholerae</italic> isolates were isolated from stool samples on Luria–Bertani agar with sulfamethoxazole (24 µg/ml), trimethoprim (32 μg/ml), nalidixic acid (20 µg/ml) and streptomycin (Sm) (100 µg/ml). Mutations were introduced using pCVD442-lac as previously described (<xref ref-type="bibr" rid="bib28">Seed et al., 2012</xref>). Strains containing the pMMB67EH vector were grown in the presence of 50 μg/ml ampicillin and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG).</p><p><italic>V. cholerae</italic> and phage genomic libraries were generated (<xref ref-type="bibr" rid="bib16">Lazinski and Camilli, 2013</xref>) and sequenced using an Illumina HiSeq2000 (Tufts University Core Facility). Sequence reads from each <italic>V. cholerae</italic> isolate were mapped to the reference genome <italic>V. cholerae</italic> O1 2010EL-1786 (for Haitian isolates, accession numbers NC_016445.1 and NC_016446.1) or to <italic>V. cholerae</italic> O1 N16961 (for Bangladeshi isolates, accession numbers NC_002505.1 and NC_002506.1) and mutations were identified using Varscan v2.3.6 (<xref ref-type="bibr" rid="bib15">Koboldt et al., 2012</xref>) and CLC Genomic Workbench software (Version 6.8; CLC Bio, Denmark). Variants were called as being present (different allele than reference), absent (the same allele as reference) or low-coverage (<15×; in which case no call was made).</p></sec><sec id="s2-2"><title>Preparation of outer membrane fractions and Western blotting</title><p>Cultures were grown to mid-exponential growth phase in Luria–Bertani (LB) broth at 37°C with aeration. Bacteria were re-suspended in 200 mM Tris–HCl, pH 8.0 and 2 mM EDTA. Sucrose was added to a final concentration of 20% followed by lysozyme treatment for 10 min at 37°C. Using a dry ice/ethanol bath, samples were freeze-thawed twice followed by DNAse I treament for 20 min at room temperature. Samples were spun for 5 min at 16,100×<italic>g</italic> to pellet the membrane fraction. The pellets were re-suspended in 1% Triton X-100, 10 mM MgCl<sub>2</sub>, and 50 mM Tris–HCl, pH 8.0 and incubated at 37°C for 20 min. Samples were spun again for 5 min at 16,100×<italic>g</italic> to separate the inner membrane from the outer membrane. The pellets, which contain the outer membrane proteins, were re-suspended in 200 mM Tris–HCl, pH 8.0.</p><p>Outer membrane fractions were boiled for 10 min in sample buffer containing sodium dodecyl sulfate and β-mercaptoethanol and separated on a NuPAGE 4–12% Bis-Tris polyacrylamide gel (Life Technologies, Carlsbad, CA). Protein gels were transferred to a nitrocellulose membrane for Western blotting. Membranes were probed with rabbit polyclonal antisera against <italic>V. cholerae</italic> OmpU (gift of James Kaper). A Cy5 goat anti-rabbit antibody was used to develop the blot.</p></sec><sec id="s2-3"><title>Evaluation of in vitro fitness</title><p>Strains (grown to OD<sub>600</sub> = 0.5) were assessed for their ability to survive 0.2% porcine bile (Sigma, St. Louis, MO) in 0.85% NaCl for 1 hr at room temperature. Pond water survival assays were done as described previously (<xref ref-type="bibr" rid="bib13">Kamp et al., 2013</xref>) at 30°C for 48 hr. Competition experiments were performed between the strain of interest (<italic>lacZ</italic><sup>+</sup>) and the appropriate control strain (Δ<italic>lacZ</italic>), and outputs were plated on LB agar plates containing 100 µg/ml Sm and 40 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal).</p></sec><sec id="s2-4"><title>Animals</title><p>All animal experiments were in accordance with the rules of the Department of Laboratory Animal Medicine at Tufts Medical Center. Infant mouse colonization assays and in vitro controls were done as described previously (<xref ref-type="bibr" rid="bib28">Seed et al., 2012</xref>). 3-day old infant rabbits were pre-treated with Cimetidine-HCL (Morton Grove Pharmaceuticals, Morton Grove, IL) 3 hr prior to infection (<xref ref-type="bibr" rid="bib13">Kamp et al., 2013</xref>) and infected with ∼5 × 10<sup>8</sup> CFU in 2.5% sodium bicarbonate buffer (pH 9). A 1:1 mixture of wild-type to mutant (for experiments without phage) or 10:1 wild-type to mutant (for experiments including phage) was used. For experiments involving the addition of phage, phage were added immediately before intragastric inoculation to each rabbit inoculum to limit phage adsorption ex vivo (phage were in contact with the bacterial inoculum for ∼30–60 s prior to gavage of each animal). Rabbits were euthanized ∼12 hr post-inoculation and cecal and/or small intestinal fluid was collected by puncture. As with the in vitro assays, the competing strains were enumerated on LB agar plates containing 100 µg/ml Sm and 40 µg/ml X-gal.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors thank the Tufts University Core Facility for sequencing and computational support, James Kaper for providing OmpU antisera, and David Lazinski and Linc Sonenshein for commenting on the manuscript. This work was supported by US National Institutes of Health grants AI055058 (AC), AI099243 (JH/LI), AI089721 (RCC), a Massachusetts General Hospital Physician Scientist Development Award (RCC), the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs program (BJS), and the Charles A King Trust Postdoctoral Fellowship Program (KDS). AC is a Howard Hughes Medical Institute Investigator.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>KDS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>MY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>AC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>BJS, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>IJH, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>RCC, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>JET, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>LCI, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con9"><p>JB, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con10"><p>JBH, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Human subjects: The studies were approved by the institutional review boards of Partners HealthCare (Brigham and Women's Hospital and Massachusetts General Hospital) and Tufts University School of Medicine, and the Haitian National Ethics Committee. Written informed consent was obtained from adult participants and from guardians of children.</p><p>Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol (#B2013-44) of Tufts University School of Medicine.</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03497.010</object-id><label>Supplementary file 1.</label><caption><p><italic>V. cholerae</italic> isolates recovered from the ICP2-positive Haitian patient sample are isogenic except for mutations in <italic>ompU</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.010">http://dx.doi.org/10.7554/eLife.03497.010</ext-link></p></caption><media mime-subtype="xls" mimetype="application" xlink:href="elife03497s001.xls"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03497.011</object-id><label>Supplementary file 2.</label><caption><p><italic>V. cholerae</italic> isolates recovered from the ICP2-positive Bangladeshi patient sample are isogenic except for mutations in <italic>toxR</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.011">http://dx.doi.org/10.7554/eLife.03497.011</ext-link></p></caption><media mime-subtype="xls" mimetype="application" xlink:href="elife03497s002.xls"/></supplementary-material><supplementary-material id="SD3-data"><object-id pub-id-type="doi">10.7554/eLife.03497.012</object-id><label>Supplementary file 3.</label><caption><p>Strains used in this study.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03497.012">http://dx.doi.org/10.7554/eLife.03497.012</ext-link></p></caption><media mime-subtype="xls" mimetype="application" xlink:href="elife03497s003.xls"/></supplementary-material><sec sec-type="datasets"><title>Major datasets</title><p>The following datasets were generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Seed</surname><given-names>KD</given-names></name>, <name><surname>Yen</surname><given-names>M</given-names></name>, <name><surname>Shapiro</surname><given-names>BJ</given-names></name>, <name><surname>Hilaire</surname><given-names>IJ</given-names></name>, <name><surname>Charles</surname><given-names>RC</given-names></name>, <name><surname>Teng</surname><given-names>JE</given-names></name>, <name><surname>Ivers</surname><given-names>LC</given-names></name>, <name><surname>Boncy</surname><given-names>J</given-names></name>, <name><surname>Harris</surname><given-names>JB</given-names></name>, <name><surname>Camilli</surname><given-names>A</given-names></name>, <year>2014</year><x>, </x><source>Sequence for ICP2_2011_A</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=KM224878">http://www.ncbi.nlm.nih.gov/nuccore/?term=KM224878</ext-link><x>, </x><comment>Publicly available at NCBI GenBank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Seed</surname><given-names>KD</given-names></name>, <name><surname>Yen</surname><given-names>M</given-names></name>, <name><surname>Shapiro</surname><given-names>BJ</given-names></name>, <name><surname>Hilaire</surname><given-names>IJ</given-names></name>, <name><surname>Charles</surname><given-names>RC</given-names></name>, <name><surname>Teng</surname><given-names>JE</given-names></name>, <name><surname>Ivers</surname><given-names>LC</given-names></name>, <name><surname>Boncy</surname><given-names>J</given-names></name>, <name><surname>Harris</surname><given-names>JB</given-names></name>, <name><surname>Camilli</surname><given-names>A</given-names></name>, <year>2014</year><x>, </x><source>Sequence for ICP2_2013_A_Haiti</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=KM224879">http://www.ncbi.nlm.nih.gov/nuccore/?term=KM224879</ext-link><x>, </x><comment>Publicly available at NCBI GenBank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro3"><name><surname>Seed</surname><given-names>KD</given-names></name>, <name><surname>Yen</surname><given-names>M</given-names></name>, <name><surname>Shapiro</surname><given-names>BJ</given-names></name>, <name><surname>Hilaire</surname><given-names>IJ</given-names></name>, <name><surname>Charles</surname><given-names>RC</given-names></name>, <name><surname>Teng</surname><given-names>JE</given-names></name>, 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person-group-type="author"><name><surname>Seed</surname><given-names>KD</given-names></name><name><surname>Lazinski</surname><given-names>DW</given-names></name><name><surname>Calderwood</surname><given-names>SB</given-names></name><name><surname>Camilli</surname><given-names>A</given-names></name></person-group><year>2013</year><article-title>A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity</article-title><source>Nature</source><volume>494</volume><fpage>489</fpage><lpage>491</lpage><pub-id pub-id-type="doi">10.1038/nature11927</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>RK</given-names></name><name><surname>Miller</surname><given-names>VL</given-names></name><name><surname>Furlong</surname><given-names>DB</given-names></name><name><surname>Mekalanos</surname><given-names>JJ</given-names></name></person-group><year>1987</year><article-title>Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>84</volume><fpage>2833</fpage><lpage>2837</lpage><pub-id pub-id-type="doi">10.1073/pnas.84.9.2833</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03497.013</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Cossart</surname><given-names>Pascale</given-names></name><role>Reviewing editor</role><aff><institution>Institut Pasteur</institution>, <country>France</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Evolutionary Consequences of Intra-Patient Phage Predation on Microbial Populations” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Richard Losick (Senior editor), a Reviewing editor and 2 reviewers, one of whom, Marie-Agnès Petit, has agreed to reveal her identity.</p><p>The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>Seed et al. report a very clear cut and elegant set of data providing evidence of a natural, <italic>in vivo</italic> predator prey relationship between <italic>Vibrio cholerae</italic> and ICP2-like virulent phages of the podoviridae family, upon human infection. In two patients infected with <italic>V. cholerae</italic>, and having high loads of ICP2 phage, they discovered two different panels of Vibrio mutations, in ompU and toxR, resulting in the mutation or repression of the OmpU receptor, respectively. They also establish that in a collection of 54 isolates of <italic>V. cholerae</italic>, 15% had an ompU mutation.</p><p>To our knowledge, this is the first report of a predator-prey evolution taking place in the human intestine. In addition, the toxR mutants have attenuated virulence, suggesting that the presence of the virulent phage may have contributed to the elimination of the infection by natural evolution. This is not true however for the ompU mutants that appear almost not attenuated in virulence/ survival phenotypes. The contrast between the two sets of mutants isolated is striking. As suggested by the authors, such an evolution, not reported until now for bacterial strains in healthy microbiota, may have taken place due to the disequilibrium of the resident microbiota in patients. This is a new concept, and the study certainly deserves publication.</p><p>This study also represents an extremely interesting and timely topic in light of the recent phage therapy re-birth. One of the reviewers wrote: “I was amazed by the results on ompU mutant selection by phage co-infection in these conditions and was surprised that this selective effect had never been observed in the cholerae OmpU. I went to Genbank and retrieved the various cholerae OmpU (VC0633) sequences and compared its variation to its neighboring D-alanyl-D-alanine carboxypeptidase (VC0632). I did not perform a statistical analysis, but it is obvious that OmpU is more variable than VC0632. However, this can have many different causes, but strikingly, most of the OmpU variants carry mutation(s) in the very same positions as those identified in this study (A182, VG 324/5...). A simple but thorough analysis of the OmpU mutations could add more weight to the conclusions presented in this manuscript on the selective force exerted by phage over the time.”</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03497.014</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>[…] This study also represents an extremely interesting and timely topic in light of the recent phage therapy re-birth. One of the reviewers wrote: “I was amazed by the results on ompU mutant selection by phage co-infection in these conditions and was surprised that this selective effect had never been observed in the cholerae OmpU. I went to Genbank and retrieved the various cholerae OmpU (VC0633) sequences and compared its variation to its neighboring D-alanyl-D-alanine carboxypeptidase (VC0632). I did not perform a statistical analysis, but it is obvious that OmpU is more variable than VC0632. However, this can have many different causes, but strikingly, most of the OmpU variants carry mutation(s) in the very same positions as those identified in this study (A182, VG 324/5...). A simple but thorough analysis of the OmpU mutations could add more weight to the conclusions presented in this manuscript on the selective force exerted by phage over the time.”</italic></p><p>We thank the reviewers for their comments and idea to include more OmpU sequences. We used BLASTP to identify OmpU in other <italic>Vibrio cholerae</italic> isolates in GenBank. Homologues were aligned and WebLogo (Crooks <italic>et al.</italic>, 2004) was used to generate a sequence logo in order to gain insight into variable or conserved regions. We have included this analysis in a figure below (<xref ref-type="fig" rid="fig5">Author response image 1</xref>).<fig id="fig5" position="float"><label>Author response image 1.</label><graphic xlink:href="elife03497f005"/></fig></p><p>We have also included a reference image equal to <xref ref-type="fig" rid="fig1">Figure 1b</xref> but with numbering of the extracellular loops to orient the reader. The mutations observed in <italic>V. cholerae</italic> isolates in this study, and experimentally validated to confer ICP2 resistance, are highlighted in yellow. The results of this analysis indicate that there is significant variability in OmpU, specifically in the predicted extracellular loops. As the reviewers point out, there are other <italic>V. cholerae</italic> isolates in GenBank that share the mutations identified in this study. There are also highly variable loops (i.e., loops 2 and 6) that we did not observe mutations in our data set, and it is not known if variability in these loops can confer phage resistance.</p><p>The strains included in the analysis below represent a broad collection of both environmental and clinical <italic>V. cholerae</italic> isolates from geographically disparate locations. Trying to identify the source of these deposited sequences is not always possible. Since OmpU represents an abundant outer membrane protein it is not surprising to observe this variability, which as the reviewers point out, is likely due to many different causes. One could imagine that the variability may be due to immune pressure, or phage pressure from ICP2 and/or other uncharacterized phages. To-date ICP2 like phages have only been isolated from clinical samples in Haiti and Bangladesh and we do not know if they co-exist with environmental non-toxigenic <italic>V. cholerae</italic> in these locations, or for that matter anywhere else in the world. We feel that the analysis provided in this manuscript which is the analysis of over 50 clinical strains in Bangladesh collected over a 10 year period, which share a niche with ICP2, is particularly relevant to making conclusions about the selective force exerted by ICP2 over time. In addition, we were able to experimentally confirm our hypothesis that these mutations confer ICP2 resistance.</p></body></sub-article></article> |