Comparison of the inhibitory potential of benzyl isothiocyanate and phenethyl isothiocyanate on Shiga toxin-producing and enterotoxigenic Escherichia coli
Introduction
Shiga toxin-producing Escherichia coli (STEC), a significant type of pathogenic E. coli that secrete intestinal toxins, cause serious public health incidents due to foodborne outbreaks, and epidemics in developed countries around the world; for example, there was a STEC outbreak in Germany in 2011 and an outbreak of enterohemorrhagic E, coli, in Japan in the same year (Asadishad, Hidalgo, & Tufenkji, 2012; Yahata et al., 2015). The characteristic symptoms of STEC infection include diarrhea, hemorrhagic colitis and even hemolytic-uremic syndrome (Spinale, Ruebner, Copelovitch, & Kaplan, 2013). Some previous studies have reported the prevalence of multi-antimicrobial-resistant E. coli isolates positive for virulence determinants for enterotoxigenic E. coli (ETEC) in surface waters being used as raw water to supply drinking water (Begum et al., 2005; Ram, Vajpayee, & Shanker, 2007; Singh, Vajpayee, Ram, & Shanker, 2010). ETEC is a common cause of diarrhea in children and in travelers in developing countries and may result in death in severe cases.
The pathogenesis of STEC is induced by some normal mechanisms, and the Shiga toxins are among the most significant virulence factors (Din, Rahman, Uddin, & Khan, 2015). The Shiga toxins intensify the severity and persistence of enterohemorrhagic E. coli induced diarrhea and affect the host inflammatory response to EHEC (Ritchie, Thorpe, Rogers, & Waldor, 2003). STEC adhere to the surface of intestinal epithelial cells and produce a characteristic histopathology (Kalemba & Kunicka, 2003; Kaper, 1998). Other genes, such as the eae gene, influence attaching and effacing lesions formation, and a role in intestinal colonization has also been reported (Ritchie et al., 2003; Dean-nystrom, Bosworth, Moon, & O'brien, 1998). One or more colonization factors allow ETEC to adhere to the small intestine and secrete heat-labile enterotoxin (lt) and/or heat-stable enterotoxin (st), inducing aqueous diarrhea (Sofrata et al., 2011). The lt toxin is highly similar to cholera toxin in structure and function, and the lt1 gene is associated with human illness (Sjöling et al., 2006). St is a small, poorly immunogenic molecule that is highly similar in function and structure to the endogenic peptide hormones guanylin and uroguanylin (Nakazato, 2001). It has been reported that natural chemical products showed significant antibacterial, antiviral and antifungal activities (Yiannakopoulou, 2012). In particular, foodborne exogenous substances that affect virulence genes have received much attention as germicide and natural agents to combat bacterial and fungal contamination of food (Liu et al., 2016).
Isothiocyanates (ITCs), containing the chemical group R–NCS, are especially abundant in cruciferous plants and are produced by glucosinolate hydrolysis. ITCs are formed by substituting the oxygen in the isocyanate group (R–NCO) with a sulfur. The isocyanate group is arranged differently from the cyanate functional group (R–O–CN). Thiocyanate is analogous to the cyanate ion, wherein oxygen is replaced by sulfur (R–S–CN). Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) are aromatic compounds containing benzene ring structure. Some ITCs have been reported to cause toxic effects in different rat models (Akagi, J., et al., 2018; Suzuki, I., et al., 2017). Both BITC and PEITC can induce toxicity by oral administration in urinary bladder of rats (Masutomi et al., 2001; Akagi et al., 2003). BITC shows subacute toxicity in male rats (Lewerenzh, Bleyl, & Plass, 1992), and the doses of BITC required to cause measurable DNA damage in laboratory rodents exceeded by far the dietary exposure levels of humans (Kassie, Pool-Zobel, Parzefall, & Knasmüller, 1999). Studies have suggested that ITCs act as anticancer agents and exert antibacterial, chemopreventive and antiparasitic activities (Gupta, Kim, Kim, & Srivastava, 2014; Wagner, Terschluesen, & Rimbach, 2014). Other research has reported that PITC inhibits the growth and Shiga toxin production of EHEC (Nowicki et al., 2014). It has been shown that BITC and PEITC are the most potent isothiocyanates against enterohemorrhagic E. coli strains (Nowicki, Rodzik, Herman-Antosiewicz, & Szalewska-Pałasz, 2016). However, little evidence has been reported regarding the effects of BITC and PEITC on relevant molecular mechanisms of action, intracellular compounds and bacterial morphology.
The objective of this study was to investigate the effects of sub-inhibitory concentrations of BITC and PEITC on relevant STEC and ETEC virulence genes expression, morphological features and ATP levels. We observed that BITC and PEITC could decrease the expression of relevant virulence genes and impair bacterial membrane integrity, resulting in major cell leakage.
Section snippets
Bacterial strains and reagents
The STEC strain CICC 10670 and ETEC strain CICC 10667 were purchased from the China Center of Industrial Culture Collection. The strains were cultured in bouillon culture-medium at 37 °C with shaking at 150 rpm. BITC and PEITC were purchased from the Sigma (U.S.A).
Minimal inhibitory concentration (MIC) determination and growth curve assay
The MICs of BITC and PEITC against STEC and ETEC were determined using the microbroth dilution method in Mueller-Hinton broth (MHB) as described by Burt (2004). For the growth curve assay, bacteria were cultivated overnight at 37 °C
MIC and growth curves
Our preliminary studies have shown that BITC and PEITC have strong inhibitory effect on some gram-negative bacteria by comparing with other isothiocyanates, including isopropyl isothiocyanate, butyl isothiocyanate, 2-butyl isothiocyanate, isobutyl isothiocyanate, isoamyl isothiocyanate, 4-pentenyl isothiocyanate, 3-methylthiopropyl isothiocyanate (data not shown). The MICs of BITC and PEITC for complete inhibition of STEC cell growth were 60 and 400 μmol/L, respectively. The MICs of BITC and
Discussion
The incidence of pathogenic E. coli infection is increasing dramatically, and conventional treatments have resulted in severe medical complications and increased mortality in recent years. Thus, the search for new antibacterial agents has strengthened. A novel strategy to prevent the production of drug resistance is to prevent virulence genes activity rather than to kill pathogen cells (Vila et al., 2017). ITCs have aroused great attention for their potent antimicrobial properties against a
Conclusion
In the present study, it was shown that BITC and PEITC could inhibit the growth of SETC and ETEC. The surface morphology of cells treated with BITC was more severely disrupted than PEITC for both STEC and ETEC. Total ATP levels were reduced and extracellular ATP levels were increased by the addition of BITC and PEITC. BITC and PEITC at subinhibitory concentrations decreased the stx2, eaeA and ehxA in SETC and eltA and estA in ETEC virulence genes expression. Moreover, the effect of virulence
Declaration of competing interest
No conflict of interest exists. No financial arrangement between an author and a company whose product figures prominently in the submitted manuscript exists. The materials have not been published, and this paper has not been submitted to any other journal and is not under consideration for publication elsewhere. The corresponding author and all of the authors have read and approved the final submitted manuscript. Your kind review of the manuscript for publication will be greatly appreciated.
Acknowledgement
This work was supported financially by the National Key R&D Program of China (2017YFC1600403), the National Natural Science Foundation of China (31571888) and the Liaoning Provincial Natural Science Foundation of China (No. 2019-MS-021).
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