Abstract
We analysed whether the hydroethanolic extracts from leaves of Haplopappus baylahuen Remy (bailahuen) and Aloysia citriodora Palau (cedron) inhibit the growth and ability of Salmonella Enteritidis to form biofilms and to adhere to human intestinal epithelial cells. Herein, we first determined the total phenolic content and antioxidant and antibacterial activities of the extracts. Then, Salmonella Enteritidis was treated with the extracts to analyse biofilm formation by scanning electronic microscopy and the violet crystal test. We also measured the efflux pump activity of Salmonella Enteritidis since biofilm formation is associated with this phenomenon. Furthermore, the human intestinal cell line Caco-2 was infected with Salmonella Enteritidis pretreated with the extracts, and 30 min later, the number of bacteria that adhered to the cell surface was quantified. Finally, we determined by qPCR the expression of genes associated with biofilm formation, namely, the diguanilate cyclase AdrA protein gene (adrA) and the BapA protein gene (bapA), and genes associated with adhesion, namely, the transcriptional regulator HilA (hilA). The phenolic content and antioxidant and bactericide activities were higher in bailahuen than in the cedron extract. Biofilm formation was inhibited by the extracts in a dose-dependent manner, while the activity of efflux pumps was decreased only with the cedron extract. Adhesion to Caco-2 cells was also inhibited without differences between doses and extracts. The extracts decreased the expression of adrA; with the cedron extract being the most efficient. The expression of hilA is affected only with the cedron extract. We concluded that hydroethanolic extracts of bailahuen and cedron differentially inhibit the growth of Salmonella Enteritidis and affect its the ability to form biofilms and to adhere to human intestinal epithelial cells. These results highlight the presence of molecules in bailahuen and cedron with a high potential for the control of the Salmonella Enteritidis pathogenesis.
Highlights
Salmonella enterica subspecies enterica is a worldwide common pathogen that causes foodborne diseases [1]
The antioxidant activity of the extract expressed as inhibition of DPPH percent showed that the bailahuen extract inhibits by 74:5 ± 4:8% and the cedron extract inhibits by 40 ± 7:8%, indicating that bailahuen is a better antioxidant than cedron (p < 0:05)
The cedron extract inhibits the functioning of the RND pumps between 5 and 20 minutes, while the bailahuen extract stimulated efflux pump activity at 25 and 50 minutes (Figure 3), with significant differences observed between them and the control condition (p < 0:05); these results indicate that only cedron utilizes this mechanism to decrease biofilm formation in Salmonella Enteritidis
Summary
Salmonella enterica subspecies enterica is a worldwide common pathogen that causes foodborne diseases [1]. From 2013 to 2014, the number of salmonellosis cases in the European Union increased by 19%, being more prevalent in children between 0 and 4 years old than in adults. In Africa, a high prevalence of invasive salmonellosis has been observed in people with HIV and malaria, and in children under 5 years old with an estimated fatality rate of 20.6% [3]. In Chile, the Institute of Public Health (ISPCH) reported that 74% of outbreaks of BioMed Research International foodborne diseases in 2015 were caused by Salmonella infections [6]. From the two serotypes that cause nontyphoid disease, Salmonella Enteritidis is the predominant strain in most countries, and the most common in clinical isolates [1, 7]
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