Elsevier

LWT

Volume 118, January 2020, 108806
LWT

Comparison of the inhibitory potential of benzyl isothiocyanate and phenethyl isothiocyanate on Shiga toxin-producing and enterotoxigenic Escherichia coli

https://doi.org/10.1016/j.lwt.2019.108806Get rights and content

Highlights

  • Two isothiocyanates had antibacterial effect against two strains of E. coli.

  • Benzyl isothiocyanate (BITC) was stronger than phenethyl isothiocyanate (PEITC).

  • BITC disrupted the surface morphology and reduced the total ATP levels in E. coli.

  • Virulence genes were downregulated by BITC and PEITC in dose-dependent fashion.

  • Virulence reduction of two ITCs was effective in Shiga toxin-producing E. coli.

Abstract

The aim of this study was to investigate the inhibitory effects of benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) against Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC). Compared with that of PEITC, the antibacterial effect of BITC was more obvious. The minimal inhibitory concentration (MIC) of BITC was 60 and 160 μmol/L for STEC and ETEC, while that of PEITC was 400 μmol/L for both strains. Scanning electron microscopy (SEM) images indicated that treatment with BITC severely affected the cell integrity and led to partly sunken. Incubation of STEC and ETEC with BITC and PEITC for 1 and 4 h decreased total ATP level, while increased extracellular ATP level. Virulence genes were downregulated in dose-dependent fashion upon treatment with subinhibitory concentration of BITC and PEITC, as determined by real-time quantitative polymerase chain reaction (RT-qPCR). The expressions of stx2, eaeA and ehxA, was significantly lower in STEC treated with 1/4 MIC of PEITC than that of BITC. The inhibitory effects of BITC and PEITC on virulence genes were more obvious in STEC than ETEC. These results suggest that BITC and PEITC affect the strains growth, biological structure and expression of virulence genes of both strains.

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–Ndouble bondCdouble bondS, are especially abundant in cruciferous plants and are produced by glucosinolate hydrolysis. ITCs are formed by substituting the oxygen in the isocyanate group (R–Ndouble bondCdouble bondO) with a sulfur. The isocyanate group is arranged differently from the cyanate functional group (R–O–Ctriple bondN). Thiocyanate is analogous to the cyanate ion, wherein oxygen is replaced by sulfur (R–S–Ctriple bondN). 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).

References (46)

  • E. Dean-nystrom et al.

    Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves

    Infection and Immunity

    (1998)
  • S.U. Din et al.

    Level of pathogenic Escherichia coli on animal's body coat and in meat under slaughter house environment

    Journal of Biology, Agriculture and Healthcare

    (2015)
  • V. Dufour et al.

    Insights into the mode of action of benzyl isothiocyanate on Campylobacter jejuni

    Applied and Environmental Microbiology

    (2013)
  • G. Frankel

    Enteropathogenic and enterohaemorrhagic Escherichia coli: More subversive elements

    Molecular Microbiology

    (1998)
  • P. Gupta et al.

    Molecular targets of isothiocyanates in cancer: Recent advances

    Molecular Nutrition & Food Research

    (2014)
  • I.M. Helander

    Characterization of the action of selected essential oil components on gram-negative bacteria

    Journal of Agricultural and Food Chemistry

    (1998)
  • C. Herranz

    Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136

    Applied and Environmental Microbiology

    (2001)
  • L. Johannes et al.

    Shiga toxins from cell biology to biomedical applications

    Nature Reviews Microbiology

    (2010)
  • D. Kalemba et al.

    Antibacterial and antifungal properties of essential oils

    Current Medicinal Chemistry

    (2003)
  • J.B. Kaper

    The locus of enterocyte effacement pathogenicity island of Shiga toxin-producing Escherichia coli O157:H7 and other attaching and effacing E. coli

    Medical Science & Biology

    (1998)
  • F. Kassie et al.

    Genotoxic effects of benzyl isothiocyanate, a natural chemopreventive agent

    Mutagenesis

    (1999)
  • M.G. Kim et al.

    Growth-inhibiting activities of phenethyl isothiocyanate and its derivatives against intestinal Bacteria

    Journal of Food Science

    (2009)
  • J.H. Lewerenzh et al.

    Subacute oral toxicity study of benzyl isothiocyanate in rats

    Nahrung

    (1992)
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