{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T02:42:43Z","timestamp":1783996963545,"version":"3.55.0"},"reference-count":33,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T00:00:00Z","timestamp":1755648000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T00:00:00Z","timestamp":1755648000000},"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":["R01AI165582"],"award-info":[{"award-number":["R01AI165582"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2025,8,20]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Measuring bacterial growth rates\n in vitro<\/jats:italic>\n is routine; however, determining growth rates during infection within a host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin (\n ori<\/jats:italic>\n ) copies to those of the terminus (\n ter<\/jats:italic>\n ), as originally defined by whole-genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of\n ori<\/jats:italic>\n - and\n ter<\/jats:italic>\n -adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic\n Escherichia coli<\/jats:italic>\n (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent\n ori:ter<\/jats:italic>\n (O:T\n PCR<\/jats:sup>\n ). We found that PTR and O:T\n PCR<\/jats:sup>\n were highly correlated and that O:T\n PCR<\/jats:sup>\n reliably predicted growth rates calculated by conventional methods. O:T\n PCR<\/jats:sup>\n was then used to calculate the\n in situ E. coli<\/jats:italic>\n growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that the growth rate of UPEC strains gradually increased during the early stages of infection (0\u20136 h), followed by a slow decrease in growth rates during later time points (1\u20137 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Accurately measuring bacterial growth rates in the host, which plays a crucial role in determining the success of pathogens in establishing infections, poses significant challenges. To address this, bacterial replication rate has been measured as a proxy for the growth rate estimation. While whole-genome sequencing (WGS) has been used for this purpose, it comes with drawbacks such as high costs and difficulties in analyzing bacterial sequences due to the overwhelming presence of host DNA. In this study, we validate a more accessible PCR-based approach compared to the established WGS method and confirmed the reliability of our PCR-based technique. We then applied it to measure the growth rate of\n Escherichia coli<\/jats:italic>\n during experimental urinary tract infection in a mouse model. This study provides a cost-effective and efficient alternative to WGS for studying bacterial replication dynamics during infection, potentially offering new insights into pathogen behavior and host-microbe interactions.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/aem.02382-24","type":"journal-article","created":{"date-parts":[[2025,7,31]],"date-time":"2025-07-31T13:00:13Z","timestamp":1753966813000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Multiplexed PCR to measure\n in situ<\/i>\n growth rates of uropathogenic\n E. coli<\/i>\n during experimental urinary tract infection"],"prefix":"10.1128","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5266-391X","authenticated-orcid":true,"given":"Santosh","family":"Paudel","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2441-5916","authenticated-orcid":true,"given":"Geoffrey B.","family":"Severin","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Pirani","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4553-3276","authenticated-orcid":true,"given":"Melanie M.","family":"Pearson","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5097-3597","authenticated-orcid":true,"given":"Mark T.","family":"Anderson","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8409-278X","authenticated-orcid":true,"given":"Evan S.","family":"Snitkin","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Michigan Medical School","place":["Ann Arbor, USA"]},{"name":"Department of Internal Medicine, University of Michigan Medical School","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9195-7665","authenticated-orcid":true,"given":"Harry L. 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