# Synergistic Anti-quorum Sensing and Anti-biofilm Activities of Crude Terminalia catappa Leaf Extract Combined With Ciprofloxacin Against Pseudomonas aeruginosa

## TL;DR

This study shows that combining Terminalia catappa leaf extract with ciprofloxacin can enhance anti-biofilm and anti-quorum sensing effects against Pseudomonas aeruginosa.

## Contribution

The novel finding is the synergistic interaction between Terminalia catappa extract and low-dose ciprofloxacin in inhibiting Pseudomonas aeruginosa biofilms.

## Key findings

- Terminalia catappa extract alone inhibited biofilm formation by up to 67.23% at 64 µg/mL.
- Combining low-dose ciprofloxacin with Terminalia catappa extract achieved 72.94% biofilm inhibition.
- Checkerboard analysis confirmed synergistic interactions at specific concentration ranges.

## Abstract

The increasing prevalence of biofilm-associated infections and antimicrobial resistance highlights the need for alternative and complementary therapeutic strategies. This study investigated the anti-quorum-sensing, anti-biofilm, and antibacterial activities of Terminalia catappa ethanolic crude leaf extract combined with ciprofloxacin against Pseudomonas aeruginosa BIOTECH 1335 using an in vitro quantitative experimental design. The crude extract was obtained from fresh leaves, yielding 2.46% (2.46 g/100 g), and phytochemical screening revealed the presence of flavonoids, tannins, cardiac glycosides, reducing sugars, and fixed oils, while alkaloids and anthraquinone glycosides were absent. Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) analysis was performed using an IRPrestige-21 FTIR spectrophotometer (Shimadzu Corporation, Kyoto, Japan) equipped with a MIRacle ATR accessory (PIKE Technologies, Madison, WI, USA), confirming functional groups associated with phenolic and flavonoid compounds. Biofilm inhibition assays showed that the extract alone achieved up to 67.23% inhibition at 64 µg/mL. Ciprofloxacin demonstrated an inverse dose-response pattern in biofilm inhibition, wherein higher concentrations exhibited lower inhibitory effects, an atypical finding of potential mechanistic relevance. The combined treatment produced enhanced inhibition at defined concentration ranges, with the highest biofilm inhibition (72.94%) observed at 64 µg/mL extract and 0.0625 µg/mL ciprofloxacin. Quorum-sensing assays showed dose-dependent reductions in swarming and swimming motility across treatments. Checkerboard analysis revealed concentration-dependent synergistic interactions (∑FIC ≤ 0.5) specifically at low-to-moderate extract concentrations (16-32 µg/mL) combined with sub-inhibitory ciprofloxacin doses (0.015625-0.03125 µg/mL), while higher-dose combinations demonstrated predominantly additive effects. Two-way ANOVA confirmed significant main effects of extracts and ciprofloxacin concentrations. Overall, the findings demonstrate that Terminalia catappa crude extract can potentiate the antibacterial and anti-biofilm activity of ciprofloxacin at specific concentration combinations, supporting the potential of plant-antibiotic combination strategies as a multi-targeted approach against biofilm-related infections and antimicrobial resistance.