# Overcoming Polymyxin Resistance in Klebsiella pneumoniae with Ocotea Essential Oils: Insights from In Vitro and In Vivo Analyses

## TL;DR

This study explores how Ocotea essential oils can help overcome antibiotic resistance in a dangerous type of bacteria by working with existing drugs.

## Contribution

The study introduces Ocotea essential oils as potential adjuvants to polymyxin B against resistant Klebsiella pneumoniae.

## Key findings

- Ocotea essential oils combined with polymyxin B reduced the required drug concentration by 32-fold against resistant Klebsiella pneumoniae.
- The essential oil combinations inhibited biofilm formation and increased bacterial death.
- Both treatments improved survival rates in a C. elegans infection model by over 50%.

## Abstract

Antimicrobial resistance,
particularly in carbapenem-polymyxin-resistant Klebsiella
pneumoniae (CPR-Kp),
is a major challenge associated with severe infections. In this study,
we assessed the chemical composition and antimicrobial properties
of essential oils (EOs) from Ocotea diospyrifolia (OdEO) and Ocotea velloziana (OvEO)
against CPR-Kp. The EOs were extracted from the leaves
via hydrodistillation and analyzed using gas chromatography–mass
spectrometry. The antimicrobial activities of the EOs, alone and along
with polymyxin B (OdEO-PMB and OvEO-PMB), were assessed through checkerboard
assays, survival curves, and biofilm inhibition. Cell membrane permeability,
reactive oxygen species levels, and scanning electron microscopy (SEM)
were used to investigate antimicrobial mechanisms. Safety was evaluated
by conducting hemolysis and toxicity tests in Caenorhabditis
elegans. An in vivo infection model was constructed
using C. elegans larvae to assess survival
rates. OdEO and OvEO exhibited distinct chemical compositions, with
α-bisabolol and viridiflorene as the major components, respectively.
In silico analyses revealed that OdEO components can modulate the
porin OmpK36, suggesting their ability to serve as antibiotic adjuvants.
OdEO-PMB and OvEO-PMB reduced the PMB concentration required to inhibit
CPR-Kp growth by 32-fold. Both combinations inhibited
biofilm formation and caused bacterial death. The OdEO-PMB combination
induced oxidative stress and increased protein leakage. Both treatments
were nontoxic and nonhemolytic in the assay performed. In the infection
models, OdEO-PMB and OvEO-PMB improved C. elegans survival rates to 72.4% and 50.9%, respectively. These results indicated
that the Ocotea essential oils investigated are effective adjuvants
for PMB, suggesting that they can be used to develop novel therapeutic
strategies against CPR-Kp strains.