Antibacterial and antibiofilm activities of star anise-cinnamon essential oil against multidrug-resistant Salmonella Thompson

Abstract

Introduction: The emergence of foodborne multidrug-resistant (MDR) Salmonella has attracted considerable global attention. Given that food is the primary transmission route, our study focuses on Bellamya quadrata, a freshwater snail that is commonly consumed as a specialty food in Guangxi, China.

Methods: Eight MDR Salmonella strains were isolated from Bellamya quadrata samples collected across various markets. Previous animal experiments have confirmed their lethality in mice. We determined the minimum inhibitory concentrations (MICs) and fractional inhibitory concentration (FIC) indices of cinnamon essential oil (CEO) and star anise essential oil (SAEO) using the microdilution plate and checkerboard methods. The time-kill curve method was employed to assess the antibacterial activity of the cinnamon-star anise essential oil (SCEO) against planktonic MDR Salmonella. The alkaline phosphatase assay and fluorescence microscopy demonstrated that SCEO causes damage to bacterial cell walls and membranes. Crystal violet staining and scanning electron microscopy (SEM) were used to observe changes in biofilms after SCEO treatment. Quantitative real-time PCR was utilized to analyze the expression of genes related to biofilm formation following SCEO treatment.

Results: The MIC of SAEO was determined to be 25 mg/mL, whereas that of CEO was significantly lower at 0.62 mg/mL. The FIC index calculated was 0.375, which suggests a synergistic interaction between the two. When SCEO was used in combination at specific ratios, it demonstrated enhanced antibacterial and anti-biofilm capabilities compared to the individual effects of CEO or SAEO, potentially through the disruption of bacterial cell membranes and cell walls. However, in Salmonella treated with SCEO, an upregulation in the expression of biofilm-associated genes was observed, including csgA, adrA, bcsA, and csgD. This increase may be attributed to stress-induced transcriptional responses within the bacteria.

Discussion: SCEO significantly impacts cell wall integrity, suggesting its crucial role in reducing biofilm formation. These findings indicate that SCEO holds potential as an alternative to traditional antibiotics and merits further scientific investigation and development.

Keywords: Bellamya quadrata; Salmonella Thompson; anti-biofilm; antibacterial; star anise-cinnamon essential oil.

Figure 1

SCEO time-kill curve test. (A) Bacterial colony counts at different time points after treatment with various essential oils. (B) Bar chart representing the bacterial colony counts at different time points. Compared to the blank group, * indicates p<0.05 (significant difference), ** indicates p<0.01 (highly significant difference), and ns indicates no significant difference.

Figure 2

SEM showed changes in cell morphology. (A) blank control group 10000×; (B) SAEO group 10000×; (C) CEO group 10000×; (D) SCEO group 10000×.

Figure 3

The absorbance value of SCEO on the inhibitory effect of Salmonella BF was detected by crystal violet method. (A) Inhibition of biofilm formation by different essential oils at various MICs (n=6). The biofilm inhibition rate was calculated using the formula: (Absorbance of control group - Absorbance of drug-treated group)/Absorbance of control group × 100%. The results demonstrate the inhibitory effects of different essential oils on biofilm formation at their respective MICs. *: ** (p < 0.01), and **** (p < 0.0001). (B) This figure presents the raw absorbance data at 570 nm for various drug treatment groups (n=6). *: **** (p < 0.0001).

Figure 4

The morphology of adherent-state MDR Salmonella organisms treated with the SCEO was detected by the SEM in vitro. (A1) blank control group 1000×; (B1) medium control containing Tween-80 1000×; (C1) CEO group 1000×; (D1): SAEO group 1000×; (E1) SCEO group 1000×; (A2) blank control group 5000×; (B2) medium containing Tween-80 control 5000×; (C2) CEO group 5000×; (D2) SAEO group 5000×; (E2) SCEO group 5000×.

Figure 5

Quantification of bacterial adhesion after drug treatment using SEM. This figure displays the results of a quantitative analysis of bacterial adhesion inhibition following drug treatment, as observed by SEM at 1000× magnification(n = 6). The y-axis of the bar graph is logarithmically scaled to represent the area of bacterial clusters, expressed in pixels. Higher values on the y-axis indicate larger bacterial cluster areas, which correspond to greater bacterial adhesion. The x-axis lists the different drug treatment groups. *: **** (p < 0.0001).

Figure 6

The related genes of BF were detected by the method of qPCR. Figures (A–D) represent the gene expression levels of adrA (A), bcsA (B), csgA (C), and csgD (D) as measured by qPCR (n=3). *: * (p < 0.05), *** (p < 0.001).

Figure 7

The influence of SCEO on the release of ALP. (A) Overview of alkaline phosphatase absorbance values in different treatment groups at three concentration levels: MIC, 1/2MIC, and 1/4MIC. Higher absorbance indicates greater disruption of bacterial cell walls (n=6). (B–D) Detailed alkaline phosphatase absorbance values for different treatment groups at specific concentration levels: (B) MIC: Alkaline phosphatase absorbance in different treatment groups at 1/4MIC concentration. (C) 1/2MIC: Alkaline phosphatase absorbance in different treatment groups at 1/2MIC concentration. (D) 1/4MIC: Alkaline phosphatase absorbance in different treatment groups at MIC concentration. *: * (p < 0.05), ** (p < 0.01), *** (p < 0.001)and **** (p < 0.0001).

Figure 8

CLSM of SCEO-treated Salmonella with live/dead staining. Figure (A–C) correspond to the control group, 1/4 MIC, and 1/2 MIC, respectively. Red represents PI staining, and green represents Syto9 staining. Figure (A1–C1) show the merged images of PI and Syto9. Figure (A2–C2) display the images of PI staining alone, where an increase in the number of bacteria with membrane damage (indicated by PI staining) is observed with increasing SCEO concentrations. Figure (A3–C3) show the images of Syto9 staining alone, where a decrease in the number of bacteria with intact cell membranes (indicated by Syto9 staining) is observed as the SCEO concentration increases.