# Intrinsic Acidity of N-Acetylcysteine Mediates Enhanced Inhibition of Klebsiella pneumoniae and Its Biofilms by Polymyxin B

## TL;DR

Combining polymyxin B with acidic N-acetylcysteine improves antibacterial effects and biofilm inhibition against Klebsiella pneumoniae, especially for resistant strains.

## Contribution

The intrinsic acidity of N-acetylcysteine is shown to be critical for its enhanced antibacterial and anti-biofilm activity when combined with polymyxin B.

## Key findings

- The PB–NAC combination showed additive effects against PB-resistant and intermediate K. pneumoniae strains.
- Sub-inhibitory concentrations of PB–NAC significantly inhibited biofilm formation compared to individual agents.
- Neutralizing NAC reduced its antibacterial and anti-biofilm effects unless used at high concentrations.

## Abstract

This study investigated the combined antibacterial and anti-biofilm activity of polymyxin B (PB) with intrinsically acidic N-acetylcysteine (NAC) against Klebsiella pneumoniae. The minimum inhibitory concentrations (MICs) of PB, acidic NAC, and neutralized NAC against 34 K. pneumoniae strains were determined using the broth microdilution. Drug interactions were assessed by checkerboard assays and the fractional inhibitory concentration index (FICI), while biofilm inhibition was quantified using crystal violet staining. Polymyxin B resistance was identified in the reference multidrug-resistant strain K. pneumoniae ATCC BAA-1705. The PB–NAC combination showed an additive effect (FICI 0.53–0.63) against PB-resistant and PB-intermediately susceptible strains, whereas indifferent interactions were observed in PB-susceptible strains. Furthermore, sub-inhibitory concentrations of the combination produced significantly stronger biofilm inhibition than either agent alone. Neutralization of NAC markedly reduced its antibacterial and anti-biofilm activities, with substantial inhibition observed only at concentrations ≥ 32 mg/mL. These findings demonstrate that the combination of PB and acidic NAC exerts additive antibacterial effects, particularly against resistant K. pneumoniae strains, and enhances biofilm inhibition. Notably, the intrinsic acidity of NAC is essential for its antimicrobial and anti-biofilm activity.