# Absolute quantification of VBNC state formation and resuscitation in alcohol-producing Klebsiella pneumoniae

## TL;DR

This study introduces a new method to directly quantify viable but non-culturable Klebsiella pneumoniae cells, which could improve detection and understanding of these hard-to-detect pathogens.

## Contribution

A novel direct quantification method for VBNC Klebsiella pneumoniae using PMA-ddPCR without needing an external standard curve.

## Key findings

- Optimized PMA concentration and incubation time for accurate quantification of HiAlc Kpn.
- Ciprofloxacin inhibits VBNC-state recovery but allows resuscitation and ethanol production after antibiotic removal.
- PMA-ddPCR enables direct quantification of viable cells without an external standard curve.

## Abstract

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 Klebsiella pneumoniae (HiAlc Kpn) could cause non-alcoholic fatty liver disease. Notably, HiAlc Kpn is prevalent in the gut microbiome, and tracing pathogens in intestinal samples requires identifying and quantifying viable cells.

For precise quantification, we optimized PMA concentration between 5 μM and 200 μM 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 Kpn viable cells.

These methods produced methodological and technically comparable counts using KP, rpoB, and adhE. In mice fecal samples, we observed activity reductions ranging from 1.13 to 0.64 log10 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 Kpn, 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.

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 K. pneumoniae, with potential applications in quantifying bacterial cells in the VBNC state, resuscitation, and assessing the persistence of pathogens in clinical samples.