{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T03:08:51Z","timestamp":1792638531724,"version":"4.5.0"},"reference-count":36,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T00:00:00Z","timestamp":1759708800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Many bacterial species in the viable but non-culturable (VBNC) state pose a considerable risk to public health, primarily because of their ability to persist and resuscitate in the environment and their enhanced ability to escape detection. Our research revealed that high alcohol-producing <jats:italic>Klebsiella pneumoniae<\/jats:italic> (HiAlc <jats:italic>Kpn<\/jats:italic>) could cause non-alcoholic fatty liver disease. Notably, HiAlc <jats:italic>Kpn<\/jats:italic> is prevalent in the gut microbiome, and tracing pathogens in intestinal samples requires identifying and quantifying viable cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>For precise quantification, we optimized PMA concentration between 5 \u03bcM and 200 \u03bcM and adjusted the incubation time from 5 to 30 minutes. Subsequently, we established real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) methods to count HiAlc <jats:italic>Kpn<\/jats:italic> viable cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>These methods produced methodological and technically comparable counts using KP, <jats:italic>rpoB<\/jats:italic>, and <jats:italic>adhE<\/jats:italic>. In mice fecal samples, we observed activity reductions ranging from 1.13 to 0.64 log<jats:sub>10<\/jats:sub> DNA copies\/mL. Interestingly, ciprofloxacin inhibited the recovery of VBNC-state cells while maintaining resuscitation and ethanol production after the removal of antibiotics in HiAlc <jats:italic>Kpn<\/jats:italic>, and we quantified the number of viable cells directly by PMA-ddPCR. This is a method for direct quantification that does not require an external standard curve reference.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>This is a method for direct quantification that does not require an external standard curve reference. This method could be an innovative tool for quantifying viable cells of <jats:italic>K. pneumoniae<\/jats:italic>, with potential applications in quantifying bacterial cells in the VBNC state, resuscitation, and assessing the persistence of pathogens in clinical samples.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2025.1627943","type":"journal-article","created":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T05:21:57Z","timestamp":1759728117000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Absolute quantification of VBNC state formation and resuscitation in alcohol-producing Klebsiella pneumoniae"],"prefix":"10.3389","volume":"15","author":[{"given":"Shuo","family":"Zhao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenpu","family":"Dou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiazheng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2020.116499","article-title":"Resistance and induction of viable but non culturable states (VBNC) during inactivation of E. coli and Klebsiella pneumoniae by addition of H2O2 to natural well water under simulated solar irradiation","volume":"188","author":"Alvear-Daza","year":"2021","journal-title":"Water. 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