In Vitro Antimicrobial and Antibiofilm Activity of S-(-)-Limonene and R-(+)-Limonene against Fish Bacteria

Abstract

Brazilian fish farming goes together with the emergence of numerous bacterial diseases, with Aeromonas hydrophila being the main bacterial pathogen. As a consequence, antimicrobials are excessively used. Considering that antimicrobials are relatively stable and nonbiodegradable, medicinal plants and their phytochemicals have been used as alternative treatments of bacteriosis in fish farming. Limonene is a monoterpene available in two enantiomers: R-(+)-limonene and S-(-)-limonene. This study analyzed the antibacterial activity of the phytochemicals S-(-)-limonene and R-(+)-limonene against some bacteria isolated from silver catfish (Rhamdia quelen). Furthermore, by means of spectrophotometry and atomic force microscopy, we also investigated the combination therapy of phytochemicals with antimicrobials and their activity in terms of inhibiting biofilm formation. Six clinical isolates and a standard strain were selected for antimicrobial activity testing. Biofilm formation was tested in 96-well plates and nylon cubes. The most sensitive of the strains tested was the A. hydrophila strain (MF 372510). S-(-)-limonene and R-(+)-limonene had high minimum inhibitory concentrations; however, they strongly inhibited A. hydrophila biofilm formation. R-(+)-limonene and S-(-)-limonene had an additive effect when combined with florfenicol and an antagonistic effect with oxytetracycline. In general, the phytochemicals tested showed strong antibiofilm activity against A. hydrophila, and when in combination therapy with florfenicol, they showed an additive effect against the treatment of A. hydrophila.

Antimicrobial and Antivirulence Activities of Carvacrol against Pathogenic Aeromonas hydrophila

“…Besides the antibacterial capability, Silva et al (2021) found that tambaqui (C. macropomum) fed with carvacrol had higher monocyte and neutrophil counts, phagocytic activity, and a higher survival rate when exposed to A. hydrophila infection [56]. Thus, we inferred that carvacrol could enhance the non-specific cellular immune function of grass carp and improve their ability to resist bacterial invasion Essential oils are commonly considered to have multi-target inhibitory effects on pathogenic bacteria and, in combination with conventional antibiotics, may enhance the activity, avoid the emergence of antibiotic resistance, and reduce drug use [59,60]. Pirog et al (2019) found that essential oils combined with other antimicrobials could destroy yeast and bacterial biofilm, thereby significantly decreasing their MIC [61].…”

The action of phytochemicals in biofilm control

“…The combination of phytochemicals and antibiotics can cause antagonistic antimicrobial effects. For instance, Silva et al 326 combined two isoforms of limonene (Fig. 2 AZ ), ( S -(−)-limonene and R -(+)-limonene), and with oxytetracycline to apply against biofilms of A. hydrophila.…”

D-Limonene: Promising and Sustainable Natural Bioactive Compound

“…It is classified as "generally recognized as safe" (GRAS) by the U.S. Food and Drug Administration and is widely used in the food and cosmetic industries [13]. Additionally, D-limonene also has analgesic, anti-inflammatory [17,18], antioxidant [19], neuroprotective [20], and antimicrobial [21] effects and has hence received extensive attention from researchers.…”