# A novel green synthesized ZnO-based antimicrobial nanocomposite: synergistic action, in vitro cytotoxicity, and molecular docking studies of ceftazidime, metformin, and chitosan against multidrug-resistant Salmonella enterica

## TL;DR

A new green-made nanocomposite with zinc oxide, chitosan, ceftazidime, and metformin shows strong antibacterial effects against drug-resistant Salmonella and is safe for human cells.

## Contribution

A novel green-synthesized ZnO-based nanocomposite with synergistic antimicrobial activity against MDR Salmonella is developed and tested.

## Key findings

- The nanocomposite showed a 42 mm inhibition zone and 8 µg/ml MIC against MDR Salmonella.
- Molecular docking revealed strong interactions between the nanocomposite and Salmonella proteins.
- The nanocomposite had an IC₅₀ of 84.26 µg/ml in human lung fibroblast cells, indicating good biocompatibility.

## Abstract

The alarming rise of multidrug-resistant (MDR) bacteria, particularly Salmonella spp., has prompted an urgent search for alternative and synergistic antimicrobial strategies. In this study, a novel, green, and multicomponent nanocomposite was synthesized by integrating zinc oxide nanoparticles (ZnO NPs), chitosan (CS), the β-lactam antibiotic ceftazidime (CAZ), and the antidiabetic agent metformin (MTF) straightforward and economical manner.

Bacillus subtilis strain ATCC 6633 was used to biosynthesize ZnO NPs, acting as a reliable bio-nanofactory. Various characterization techniques such as FTIR, XRD, TEM, and zeta potential analysis verified the successful integration and structural integrity of the ZnO NPs within the CS nanocomposite containing CAZ and MTF (ZnO/CS/CAZ/MTF). The FTIR spectra confirmed the presence of proteins that act as binding and supportive agents during the biosynthesis process. The produced nanomaterials have a significant positive surface charge of +28.61 mV, which enhances their stability. The particle sizes of the NPs ranged from 9.93 to 17.44 nm. The nanocomposite exhibited strong antibacterial activity against MDR Salmonella enterica subsp., enterica serovar Typhi ATCC 19214, showing a significantly increased inhibition zone of 42 mm and a greatly reduced minimum inhibitory concentration (MIC) value of 8 µg/ml, compared to the separate components. The minimum bactericidal concentration (MBC) value was found to be consistent with the MIC result, emphasizing the potent bactericidal action of the prepared nanocomposite. In silico molecular docking further supported these findings by revealing favorable interactions between the nanocomposite constituents and the outer membrane proteins (OMPs) of Salmonella enterica serovar Typhimurium (PDB ID: 4W4M) and S. typhi (PDB ID: 3UU2). Key interactions included hydrogen bonding, ionic forces, and metal coordination with critical residues. Cytotoxicity assessment using WI-38 lung fibroblast cells revealed an IC₅₀ of 84.26 µg/ml, indicating acceptable preliminary biocompatibility.

The present study demonstrates the novelty of a ZnO-based multicomponent nanocomposite that uniquely integrates CAZ, MTF, and CS. This novel formulation exhibited synergistic antibacterial effects against multidrug-resistant Salmonella enterica alongside acceptable in vitro safety. The findings underscore the potential of microbially synthesized nanocomposites as promising candidates for combating antibiotic-resistant bacterial infections and support further preclinical investigations.