{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:28:15Z","timestamp":1785403695822,"version":"3.56.0"},"reference-count":85,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI182745"],"award-info":[{"award-number":["R21AI182745"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,10,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Heteroresistance describes the phenomenon where seemingly isogenic bacterial populations contain subpopulations with elevated resistance compared to the susceptible majority that are often missed in routine susceptibility testing. The enhanced resistance of those subpopulations can either be maintained (stable) or quickly lost (unstable\/transient) after treatment, where transient cases return susceptibility results identical to those of original cultures. Recent work has implicated increased gene dosage of resistance determinants as a major cause of unstable heteroresistance in clinical isolates. Inspired by that work, we sought to systematically evaluate how gene dosage of an antibiotic\u2019s target network components impacted heteroresistance and its stability. To accomplish that, we used\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      MG1655 as a model organism, and D-cycloserine (DCS), a cell-wall synthesis inhibitor that enters through a transporter (CycA) and inhibits multiple enzymes (DdlA, DdlB, DadX, and Alr), as a model antibiotic. To measure heteroresistance, we used population analysis profiling, and to quantify stability, we used a population-level model to define a heteroresistance stability index (\n                      <jats:inline-formula>\n                        <jats:alternatives>\n                          <jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"mbio.01490-25.im001.tif\"\/>\n                          <mml:math xmlns:mml=\"http:\/\/www.w3.org\/1998\/Math\/MathML\" display=\"inline\" overflow=\"scroll\">\n                            <mml:mstyle displaystyle=\"true\" scriptlevel=\"0\">\n                              <mml:mrow>\n                                <mml:mrow>\n                                  <mml:msub>\n                                    <mml:mrow>\n                                      <mml:mi mathvariant=\"normal\">H<\/mml:mi>\n                                      <mml:mi mathvariant=\"normal\">S<\/mml:mi>\n                                      <mml:mi mathvariant=\"normal\">I<\/mml:mi>\n                                    <\/mml:mrow>\n                                    <mml:mi mathvariant=\"normal\">j<\/mml:mi>\n                                  <\/mml:msub>\n                                <\/mml:mrow>\n                                <mml:msub>\n                                  <mml:mo stretchy=\"false\">\u2223<\/mml:mo>\n                                  <mml:mi>t<\/mml:mi>\n                                <\/mml:msub>\n                              <\/mml:mrow>\n                            <\/mml:mstyle>\n                          <\/mml:math>\n                        <\/jats:alternatives>\n                      <\/jats:inline-formula>\n                      ), which quantifies the proportion of heteroresistant subpopulation\n                      <jats:italic toggle=\"yes\">j<\/jats:italic>\n                      that maintained elevated resistance over\n                      <jats:italic toggle=\"yes\">t<\/jats:italic>\n                      generations. We found that increased sensitivity to DCS through gene dosage variation of\n                      <jats:italic toggle=\"yes\">cycA<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">ddlB<\/jats:italic>\n                      divergently impacted heteroresistance stability, with\n                      <jats:italic toggle=\"yes\">ddlB<\/jats:italic>\n                      enhancing stability and\n                      <jats:italic toggle=\"yes\">cycA<\/jats:italic>\n                      fostering transience. These findings translated to uropathogenic\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      (UTI89) and suggested that increasing the number of antibiotic targets and\/or points of antibiotic entry could decrease the propensity of heteroresistance to yield stable resistance. This knowledge could impact the development of new antibiotics and improve understanding of antibiotic treatment failure.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Heteroresistance is a concern because heteroresistant strains escape clinical detection and facilitate treatment failure. Heteroresistant cells can produce stably resistant or transiently resistant populations, and enhanced understanding of genetic factors that influence the level of heteroresistance and its stability has the potential to improve treatment strategies. Here, we introduce the heteroresistance stability index, which is a quantitative metric of heteroresistance stability, and use it to analyze heteroresistance of\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      to D-cycloserine. We investigated how gene dosage of antibiotic target network components (transporter, enzymatic targets) influences heteroresistance and its stability and found diverging outcomes on stability for comparable declines in heteroresistance. Specifically, these results suggest that designing antibiotics to enter through multiple transporters or target multiple enzymes would reduce the emergence of stable resistance.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01490-25","type":"journal-article","created":{"date-parts":[[2025,8,22]],"date-time":"2025-08-22T13:00:13Z","timestamp":1755867613000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["An investigation of gene dosage reveals that increased sensitivity to D-cycloserine divergently impacts the transience of heteroresistance in\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-9072-8680","authenticated-orcid":false,"given":"Katherine J.","family":"Sniezek","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00hx57361","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemical and Biological Engineering, Princeton University","place":["Princeton, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5707-8894","authenticated-orcid":true,"given":"Mark P.","family":"Brynildsen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00hx57361","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemical and Biological Engineering, Princeton University","place":["Princeton, USA"]},{"id":[{"id":"https:\/\/ror.org\/00hx57361","id-type":"ROR","asserted-by":"publisher"}],"name":"Omenn-Darling Bioengineering Institute, Princeton University","place":["Princeton, USA."]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0196-3"},{"key":"e_1_3_3_3_2","unstructured":"World Health Organization. 2022. Global Antimicrobial Resistance and Use Surveillance System (GLASS) report 2022"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(21)02724-0"},{"key":"e_1_3_3_5_2","unstructured":"Review on Antimicrobial Resistance. 2016. Tackling drug-resistant infections globally: final report and recommendations"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.9b00076"},{"key":"e_1_3_3_7_2","unstructured":"The Pew Charitable Trusts. 2016. A scientific roadmap for antibiotic discovery"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00050-19"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms24065777"},{"key":"e_1_3_3_10_2","unstructured":"Federal Task Force on Combating Antibiotic-Resistant B. 2020. United States: second national action plan for combating antibiotic-resistant bacteria 2020-2025"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1007726"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00058-14"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0218-1"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.85.6.607"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3001346"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmi.2014.12.013"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-019-0480-z"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijantimicag.2017.10.014"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-018-0342-0"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00284-020-02064-6"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.14240"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01509-20"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkw260"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.2147\/IDR.S273784"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2016.53"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02448-17"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.05055-14"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkaa131"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jgar.2020.04.026"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.05793-11"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkaa288"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02066-13"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1086\/596630"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00437-10"},{"key":"e_1_3_3_35_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2017.00037"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02881-20"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.682239"},{"key":"e_1_3_3_38_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-48233-0"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00892a001"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00220a033"},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00383013"},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2009.09.006"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.1017\/s0031182000085966"},{"key":"e_1_3_3_44_2","volume-title":"D-cycloserine package label","author":"Parsolex Gmp Center I","year":"2023","unstructured":"Parsolex Gmp Center I. 2023. D-cycloserine package label"},{"key":"e_1_3_3_45_2","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-021-02052-x"},{"key":"e_1_3_3_46_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-46571-7"},{"key":"e_1_3_3_47_2","doi-asserted-by":"publisher","DOI":"10.1128\/ecosalplus.esp-0002-2023"},{"key":"e_1_3_3_48_2","doi-asserted-by":"publisher","DOI":"10.1038\/msb4100050"},{"key":"e_1_3_3_49_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.120163297"},{"key":"e_1_3_3_50_2","doi-asserted-by":"publisher","DOI":"10.1038\/srep19452"},{"key":"e_1_3_3_51_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-52166-z"},{"key":"e_1_3_3_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijantimicag.2018.09.013"},{"key":"e_1_3_3_53_2","doi-asserted-by":"publisher","DOI":"10.1021\/cb3005116"},{"key":"e_1_3_3_54_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a025320"},{"key":"e_1_3_3_55_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciz826"},{"key":"e_1_3_3_56_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01454-17"},{"key":"e_1_3_3_57_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41429-023-00687-y"},{"key":"e_1_3_3_58_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.72.8.2999"},{"key":"e_1_3_3_59_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01598-12"},{"key":"e_1_3_3_60_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2008.00104.x"},{"key":"e_1_3_3_61_2","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.054064-0"},{"key":"e_1_3_3_62_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuz016"},{"key":"e_1_3_3_63_2","doi-asserted-by":"publisher","DOI":"10.1038\/ijos.2016.34"},{"key":"e_1_3_3_64_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1990.tb08192.x"},{"key":"e_1_3_3_65_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a016592"},{"key":"e_1_3_3_66_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-102108-134805"},{"key":"e_1_3_3_67_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2318600121"},{"key":"e_1_3_3_68_2","doi-asserted-by":"publisher","DOI":"10.1128\/Spectrum.00289-21"},{"key":"e_1_3_3_69_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.110.3.978-987.1972"},{"key":"e_1_3_3_70_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.185.19.5673-5684.2003"},{"key":"e_1_3_3_71_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00296-11"},{"key":"e_1_3_3_72_2","doi-asserted-by":"publisher","DOI":"10.1016\/S2666-5247(21)00271-8"},{"key":"e_1_3_3_73_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(21)00194-8"},{"key":"e_1_3_3_74_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics11060723"},{"key":"e_1_3_3_75_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(20)30796-9"},{"key":"e_1_3_3_76_2","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.001080"},{"key":"e_1_3_3_77_2","doi-asserted-by":"publisher","DOI":"10.1128\/aac.00234-23"},{"key":"e_1_3_3_78_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.05.005"},{"key":"e_1_3_3_79_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-6-39"},{"key":"e_1_3_3_80_2","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.052043-0"},{"key":"e_1_3_3_81_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0093110"},{"key":"e_1_3_3_82_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2023.05.016"},{"key":"e_1_3_3_83_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2021.10.005"},{"key":"e_1_3_3_84_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms10020358"},{"key":"e_1_3_3_85_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2015.06.005"},{"key":"e_1_3_3_86_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2018.10.435"}],"container-title":["mBio"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mbio.01490-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mbio.01490-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T13:04:44Z","timestamp":1759928684000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/mbio.01490-25"}},"subtitle":[],"editor":[{"given":"Vanessa","family":"Sperandio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]},{"given":"David S.","family":"Weiss","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,10,8]]},"references-count":85,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2025,10,8]]}},"alternative-id":["10.1128\/mbio.01490-25"],"URL":"https:\/\/doi.org\/10.1128\/mbio.01490-25","relation":{},"ISSN":["2150-7511"],"issn-type":[{"value":"2150-7511","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,8]]},"assertion":[{"value":"2025-05-12","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-07-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-08-22","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e01490-25"}}