Red Seaweeds Sarcodiotheca gaudichaudii and Chondrus crispus down Regulate Virulence Factors of Salmonella Enteritidis and Induce Immune Responses in Caenorhabditis elegans

Abstract

Red seaweeds are a rich source of unique bioactive compounds and secondary metabolites that are known to improve human and animal health. S. Enteritidis is a broad range host pathogen, which contaminates chicken and poultry products that end into the human food chain. Worldwide, Salmonella outbreaks have become an important economic and public health concern. Moreover, the development of resistance in Salmonella serovars toward multiple drugs highlights the need for alternative control strategies. This study evaluated the antimicrobial property of red seaweeds extracts against Salmonella Enteritidis using the Caenorhabditis elegans infection model. Six red seaweed species were tested for their antimicrobial activity against S. Enteritidis and two, Sarcodiotheca gaudichaudii (SG) and Chondrus crispus (CC), were found to exhibit such properties. Spread plate assay revealed that SG and CC (1%, w/v) significantly reduced the growth of S. Enteritidis. Seaweed water extracts (SWE) of SG and CC, at concentrations from 0.4 to 2 mg/ml, significantly reduced the growth of S. Enteritidis (log CFU 4.5-5.3 and log 5.7-6.0, respectively). However, methanolic extracts of CC and SG did not affect the growth of S. Enteritidis. Addition of SWE (0.2 mg/ml, CC and SG) significantly decreased biofilm formation and reduced the motility of S. Enteritidis. Quantitative real-time PCR analyses showed that SWE (CC and SG) suppressed the expression of quorum sensing gene sdiA and of Salmonella Pathogenesis Island-1 (SPI-1) associated genes sipA and invF, indicating that SWE might reduce the invasion of S. Enteritidis in the host by attenuating virulence factors. Furthermore, CC and SG water extracts significantly improved the survival of infected C. elegans by impairing the ability of S. Enteritidis to colonize the digestive tract of the nematode and by enhancing the expression of C. elegans immune responsive genes. As the innate immune response pathways of C. elegans and mammals show a high degree of conservation, these results suggest that these SWE may also impart beneficial effects on animal and human health.

Keywords: Caenorhabditis elegans; Chondrus crispus; Salmonella enteritidis; Sarcodiotheca gaudichaudii; immune response; virulence factors.

Figure 1

The procedure for the extraction of red seaweed water extract.

Figure 2

Effect of red seaweeds on the growth of Salmonella Enteritidis growth. Six red seaweed species Chondrus crispus (CC), Gymnogongrus devoniensis (GD), Palmaria palmate (PPMS), Sarcodiotheca gaudichaudii (SG), Solieria chordalis (SC), and Sarcodiotheca spp (SUK) were tested against S. Enteritidis. A hundred μl of fresh overnight culture was spread plated on the TSA plates containing ground seaweed (1% w/v). Log CFU/mL was calculated after incubating the plates at 37°C for 24 h. Values with different superscript letters (Tukey multiple mean comparison) are significantly different (one-way Anova; p < 0.05). Values represent mean ± standard deviation from three independent experiments (n = 9).

Figure 3

Antimicrobial activities of red seaweeds. Chondrus crispus (CC) and Sarcodiotheca gaudichaudii (SG) were tested at different concentration against S. Enteritidis by agar well diffusion method and liquid culture broth inoculation method. Solid agar showing the zone of growth inhibition at different concentration of (A) SG (B) CC. Liquid culture showing the bacterial colony count at different concentrations of (C) CC extract and (D) SG extract. Values with different superscript letters (Tukey multiple mean comparison) are significantly different (one-way Anova; p < 0.05). Values represent mean ± standard deviation from three independent experiments (n = 9).

Figure 4

Effect of SWE on the motility of bacteria. (A) SWE effect on S. Enteritidis swimming motility. Swimming motility of S. Enteritidis was determined by adding 200 μl/ml SWE into the agar plates. Single purified colony was inoculated with a toothpick from an overnight TSA plate onto a swim plate (tryptone broth plus 0.3% agar) to observe for effect on motility after overnight incubation at 30°C. (B) SWE effect on S. Enteritidis swarming motility. S. Enteritidis showed deficient movement when inoculated onto swarm plates (Difco bacto-agar, 0.5% glucose) after 24 h incubation at 30°C. (C) Effect of SWE on the relative gene expression of fliD gene required for polymerization of flagellin on the tip of growing flagella. Values represent Mean ± Standard deviation from three independent experiments; each experiment had three biological replicates.

Figure 5

Effect of SWE treatment on biofilm formation of S. Enteritidis. S. Enteritidis culture was statically grown for 24 h at 37°C in polyvinyl chloride microtitre plates in the presence of 200 μl/ml SWE (SG or CC). (A) Biofilm formation was quantified by staining with crystal violet and determining the optical density at 600 nm. (B) Effect of SWE on the relative gene expression of sdiA gene (homolog of quorum-sensing regulators LuxR). Values with different superscript letters (Tukey multiple mean comparison) are significantly different (one-way Anova; p < 0.05). Values represent Mean ± Standard deviation from three independent experiments; each experiment had three biological replicates. Picture insert: C, positive control; B, negative control; SG200, 200 μl/ml SG extract; CC200, 200 μl/ml CC extract.

Figure 6

Effect of SWE on the relative expression of virulent genes. SPI-1 encodes genes (sdiA, hilA, fliD, sipA, and invF) required for the invasion of S. Enteritidis into the host epithelium (Type 3 secretion system). Values with different superscript letters are significantly different (Tukey multiple mean comparison, p < 0.05). Values represent Mean ± Standard deviation from three independent experiments; each experiment had three biological replicates.

Figure 7

The effect of SWE on the survival of nematodes infected with S. Enteritidis. Three concentrations of SWE (200, 400, and 800 μg/ml) were used with. Worms were grown with seaweed water extract as food supplements and were exposed to S. Enteritidis. (A) SG treatment and (B) CC treatment. Values represent Mean ± Standard deviation from two experiments with six biological replicates.

Figure 8

S. Enteritidis colony counts in the gut of C. elegans. (A) Effect of SWE on bacterial CFU count in the C. elegans gut. (B) Microscopic images showing the gut of C. elegans infected with S. enteritidis. In control the gut is swelled while much less swelling was observed when infected nematodes were treated with SWE. Values with different superscript letters are significantly different (Tukey multiple mean comparison, p < 0.05). Values represent Mean ± Standard deviation from two experiments with six biological replicates.

Figure 9

Effect of SWE on the relative expression of immune responsive genes. Expression of the immune response genes spp-1 (saponin like protein), abf-1 (antibacterial protein), f49f1.6 (ShK domain-like, PMK-1) and f38a1.5 (lectin family protein) after 5 days of exposure to (A) SG or (B) CC was analyzed by quantitative reverse transcription PCR. SG-inf, C. elegans fed on SG and infected with S. Enteritidis; SG, C. elegans fed on SG; E. coli, C. elegans fed on heat killed E. coli; SE, C. elegans fed on S. Enteritidis. Values with different superscript letters are significantly different (Tukey multiple mean comparison, p < 0.05). Values represent Mean ± Standard deviation from three independent experiments; each experiment had three biological replicates.