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Inhibition of Escherichia coli O157:H7 and Salmonella enterica virulence factors by benzyl isothiocyanate
Food Microbiology 86 (2020) 103303
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Inhibition of Escherichia coli O157:H7 and Salmonella enterica virulence factors by benzyl isothiocyanate
T
⁎
Jitendra Patela, , Hsin-Bai Yina, Gary Bauchanb, Joseph Moweryb a b
U.S. Department of Agriculture, Agricultural Research Service, Environmental and Microbial Food Safety Laboratory, Beltsville, MD 20705, USA U.S. Department of Agriculture, Agricultural Research Service, SGIL Electron and Confocal Microscopy Unit, Beltsville, MD 20705, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Foodborne pathogens Virulence factors Shiga toxins Microscopic analysis
Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P < 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.
1. Introduction
followed by the release of Shiga toxin subunit A into the cytosol. The Nglycosidase activity of Shiga toxin subunit A removes an adenine group from the 28S rRNA, ultimately leading to the inhibition of protein synthesis (O'Brien et al., 1992) and apoptosis within eukaryotic cells (Monnens et al., 1998). Antibiotics are generally not recommended for treating E. coli O157:H7 infections because antibiotics increase the expression of Shiga toxin producing genes through the activation of phage lytic cycle, thereby resulting in increased Shiga toxin production (Neely and Friedman, 1998). Targeting bacterial virulence such as bacterial motility and toxin production is an alternative approach to inhibit specific mechanisms that promote bacterial infections to the hosts (Cegelski et al., 2008; Shakhnovich et al., 2007). The development of anti-virulence strategies allows the host immune system to prevent bacterial colonization or remove any established infections without killing bacteria or preventing bacterial growth, which ultimately place less selective pressure on the target microorganisms (Cegelski et al., 2008; Escaich, 2008; Rasko and Sperandio, 2010). Bacterial virulence factors contribute to the initiation and onset of a bacterial infection in the human host; therefore, an effective antimicrobial against virulence could prevent the
Pathogenic bacteria utilize several virulence factors once they are in the host and cause infections in human hosts. These virulence factors help bacteria to invade the host, cause disease, and evade host defenses (Wilson et al., 2002). Escherichia coli O157:H7 and Salmonella enterica are important foodborne pathogens in the United States, causing an estimated 2100 and 19,000 cases of hospitalizations annually, respectively (Centers for Disease Control and Prevention (CDC), 2012). Bacterial motility in E. coli O157:H7 and Salmonella is an essential virulence factor for bacterial infections in the host, because it allows these pathogens to traverse through the intestine to reach a favorable niche (Tobe et al., 2011), where flagella serves as adhesive appendages in the initial phases of colonization (Josenhans and Suerbaum, 2002). The pathogenicity of E. coli O157:H7 is also associated with the production of Shiga toxins (stx1 and stx2), which may be characterized by life threatening conditions, such as hemorrhagic colitis and hemolytic uremic syndrome in humans. Once E. coli O157:H7 colonizes human gut mucosa, Shiga toxins produced by the pathogen binds to the cell surface receptor, glycosphingolipid globotriaosylceramide,
⁎ Corresponding author. Environmental Microbial & Food Safety Laboratory, USDA-ARS, 10300 Baltimore Avenue, Building 201 BARC-East, Beltsville, MD 20705 USA. E-mail address: [email protected] (J. Patel).
https://doi.org/10.1016/j.fm.2019.103303 Received 11 April 2019; Received in revised form 3 August 2019; Accepted 10 August 2019 Available online 16 August 2019 0740-0020/ Published by Elsevier Ltd.
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compound was selected for the subsequent analyses. The sub-inhibitory concentration (SIC) and minimum inhibitory concentration (MIC) values of BIT were determined for the representative Salmonella and E. coli O157:H7 strains (S. Braenderup USDA 4559, S. Tennessee USDA 4556, E. coli O157:H7 strain ATCC 43895, and E. coli O157:H7 strain EDL 933). The susceptibility tests were carried out to determine SIC and MIC values by the standard broth microdilution method as previously described (Chen et al., 2006; Johny et al., 2010; Kim et al., 1995; Kollanoor-Johny et al., 2012). The highest concentration of the BIT that permitted bacterial growth similar to that of the DMSO control culture after incubation at 37 °C for 24 h was considered as SIC. The lowest concentration of BIT that inhibited the visible growth of bacteria after 24 h incubation at 37 °C was reported as the MIC values. At 0, 6, 12, 18, and 24 h, bacterial populations were determined by serially dilutions of samples followed by spiral plating on TSA and selective agars, where sorbitol MacConkey Agar (Neogen) and XLT4 Agar (Neogen) were used as selective agars for enumeration of E. coli O157:H7 and Salmonella, respectively.
disease progression in hosts (Lee et al., 2003; Marra, 2004; Rasko and Sperandio, 2010). In the past decade, the use of plant-derived compounds has gained significant attention due to the increasing concerns over the safety of synthetic antimicrobial agents and the emergence of antibiotic-resistant strains of microorganisms. Glucosinolates are natural compounds present in cruciferous vegetables such as cauliflower, broccoli, cabbage, turnip, and radishes (Fenwick et al., 1983; Keck and Finley, 2004). When plant tissues are disrupted, glucosinolate hydrolysis compounds including substituted isothiocyanates, nitriles, thiocyanates, and epithionitriles are produced by the enzymatic reaction of myrosinase (AlGendy et al., 2009; Delaquis and Mazza, 1995; Mari et al., 2008). Aires et al. (2009) examined the in vitro antimicrobial properties of intact glucosinolates and their hydrolysis compounds against human pathogenic and gastrointestinal tract bacteria, where isothiocyanates were found to be the most effective compounds. Jang et al. (2010) reported the antimicrobial activities of four isothiocyanates (butenyl-, pentenyl-, phenyl-ethyl-, and benzyl-) against pathogenic bacteria including Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enterica. However, the anti-virulence activities of the isothiocyanates against foodborne bacterial pathogens still need further investigation. In this study, we investigated the antimicrobial activity of seven glucosinolate hydrolysis isothiocyanate compounds (benzyl-, butyl-, ethyl-, isopropyl-, methyl-, phenethyl-, and allyl-) against five Salmonella enterica serovars (S. Braenderup, S. Negev, S. Newport, S. Thompson, and S. Tennessee) and Escherichia coli O157:H7 strains. A compound with the highest antimicrobial activity, namely the benzyl isothiocyanate (BIT), was chosen to determine its efficacy for controlling E. coli O157:H7 and Salmonella virulence in vitro. Additionally, effect of BIT on Salmonella cell viability and cell morphology was determined using confocal microscopy and transmission electron microscopy.
2.3. Confocal microscopy A Live/Dead BacLight bacterial viability kit (Thermo Fisher, Waltham, MA) was used to evaluate the viability of bacterial cells treated with BIT using confocal microscopy. S. Braenderup was selected as a representative bacterium for the confocal and transmission electron microscopic (TEM) analyses. TSB inoculated with Salmonella was treated with 200 μg/ml BIT (MIC) for 24 h or 2000 μg/ml BIT for 1 h at 37 °C. A TSB tube containing 0.2% DMSO served as the control. After incubation, bacterial culture was treated with fluorescent dyes as per manufacturer's recommendations. Viability of bacterial cells were visualized and photographed by utilizing a Zeiss 710 laser scanning confocal microscopy (Zeiss, Richmond, VA). 2.4. Transmission electron microscopy
2. Materials and methods Transmission electron microscopy (TEM) was used to visualize ultrastructural changes in Salmonella at the cellular level. A 10-ml TSB tube was inoculated with overnight culture of S. Braenderup to obtain ~5 log CFU/ml, BIT was added at 200 μg/ml or 2000 μg/ml concentrations and incubated at 37 °C for 24 h or 1 h, respectively. A TSB containing Salmonella and supplemented with DMSO for 24 h incubation at 37 °C was used as the control. For negative staining, few drops of bacterial suspension were absorbed onto carbon-formvar coated grids for 30 s and then stained with 1% (w/v) phosphotungstic acid for 5 s. For ultrastructural examination, bacterial suspensions were centrifuged at 5000 rpm for 3 min to form a pellet. Bacteria pellets were fixed for 2 h at room temperature (22 °C) in 2.5% glutaraldehyde, 0.05M NaCacodylate, 0.005M CaCl2 (pH 7.0), then refrigerated at 4 °C overnight. Pellets were rinsed 6 times with 0.05M NaCacodylate, 0.005M CaCl2 buffer and post-fixed in 1% buffered osmium tetroxide for 2 h at room temperature (22 °C). Samples were then rinsed again and dehydrated in a graded series of ethanol followed by 3 exchanges of propylene oxide (Electron Microscopy Sciences, Hatfield, PA). Pellets were then infiltrated in a graded series of LX-112 resin/propylene oxide, and polymerized in LX-112 resin (LADD Research Industries, Williston, VT) at 65 °C for 24 h. Silver-gold ultrathin (60–90 nm) sections were cut on a Reichert/AO Ultracut ultramicrotome with a Diatome diamond knife and mounted onto 100 mesh carbon-formvar coated grids. Grids were stained with 4% uranyl acetate for 10 min followed by 5 min of 3% lead citrate and imaged at 80 kV with HT-7700 transmission electron microscope (TEM, Hitachi, Schaumburg, IL).
2.1. Antibacterial activity of glucosinolate compounds Antimicrobial activities of seven glucosinolate compounds: benzyl isothiocyanate (BIT), butyl isothiocyanate (BIC), ethyl isothiocyanate (EIT), isopropyl isothiocyanate (IIT), methyl isothiocyanate (MIT), phenethyl isothiocyanate (PIT), and allyl isothiocyanate (AIT) (SigmaAldrich, Allentown, PA) against bacterial pathogens were determined by the disc diffusion assay (Kim et al., 1995). Five Salmonella enterica serovars (S. Braenderup, S. Negev, S. Newport, S. Thompson, and S. Tennessee) and five E. coli O157:H7 strains (1918, 4406, 4407, 4688, and 5279) were used in this study. Briefly, an overnight culture of each bacterial isolate in trypticase soy broth (TSB; Fisher, Waltham, MA) was adjusted to the concentration of ~6 log CFU/ml with absorbance value at 600nm, followed by spread plating 100 μl of each adjusted culture on trypticase soy agar (TSA; Fisher). A sterile filter paper disc (6 mm diameter; Fisher) was impregnated with 10 μl of each glucosinolate compound at the concentration of 10 mg/ml (100 μg/disc). The discs were placed on bacterial pathogen-seeded TSA plates and incubated at 37 °C overnight. Gentamicin antibiotic (10 μg/disc; Sigma) was used as a positive control. A sterile filter disc loaded with 10 μl dimethyl sulfoxide (DMSO; Fisher) was included as the diluent control since it was used to dissolve these glucosinolate compounds. Following overnight incubation, the zone of inhibition (mm) surrounding each disc was measured. 2.2. Determination of sub-inhibitory and minimum inhibitory concentrations
2.5. Bacterial motility assay Among 7 glucosinolate hydrolysis compounds, BIT exerted the highest antimicrobial activity against bacterial pathogens; this
The effect of BIT on motility of bacterial pathogens was determined 2
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as previously described (Wang et al., 2006). The BIT at SIC levels (125 μg/ml for Salmonella and 150 μg/ml for E. coli O157:H7) or 25 μg/ ml (below SIC) was added to each petri plate containing 20 ml of semisolid TSB (0.3% agar w/v) cooled at 45 °C. The plates with BIT were swirled for 15–20 s, and then 10 μl of overnight culture of individual E. coli O157:H7 (ATCC 43895 and EDL 933) or Salmonella strain (S. Braenderup USDA 4559 and S. Tennessee USDA 4556) containing ~6 log CFU was placed at the center of the plate. The plates were kept at room temperature for 1 h, followed by overnight incubation at 37 °C. After incubation, the diameter of the motility zone (mm) was measured.
However, the effect of BIT was significantly higher than other isothiocyanates and gentamicin against Salmonella. Additionally, Salmonella were more sensitive to BIT than E. coli O157:H7; the inhibition zones for Salmonella ranged from 35 mm for S. Newport to 64 mm for S. Negev, whereas the inhibition zones for E. coli O157:H7 strains were in 9–14 mm range. Overall, the BIT exerted the highest antibacterial activity against E. coli O157:H7 and Salmonella compared to other six compounds.
2.6. Shiga toxin concentrations
The SIC values of BIT were determined as 125 μg/ml and 150 μg/ml (0.0125% and 0.015% v/v for Salmonella and E. coli O157:H7, respectively. The average initial Salmonella and E. coli O157:H7 population in the untreated control, DMSO control, and BIT-treated samples was approximately 5 log CFU/ml in TSB, bacterial populations in the controls and the treatment with SICs of BIT reached approximately 8.5 log CFU/ml after 24 h incubation at 37 °C, thereby confirming that the SIC values were not inhibitory for these bacteria. The MIC values were 200 μg/ml (0.02% v/v) for both Salmonella and E. coli O157:H7. At the MIC level, bacterial populations maintained at 5 log CFU/ml after 24 h of incubation at 37 °C.
3.2. Sub-inhibitory and minimum inhibitory concentrations
The effect of BIT at 150 μg/ml (SIC) and 125 μg/ml (below SIC) on Shiga toxin production by E. coli O157:H7 (ATCC 43895 and EDL 933) was investigated as described by Baskaran et al. (2016). Tubes containing TSB inoculated with each strain of E. coli O157:H7 (~5 log CFU/ml) were incubated with or without BIT at 37 °C for 24 h. Shiga toxin concentrations were determined in the control (0.2% DMSO) and BIT treated E. coli O157:H7 cultures by an enzyme-linked immunosorbent assay (ELISA) using the Premier EHEC test kit (Meridian Bioscience, Cincinnati, OH). Absorbance at 450 nm were measured using a microplate reader (BioTek Instruments, Winnoski, VT) to determine Shiga toxin production and results were expressed in percentage (%) in comparison to the DMSO control.
3.3. Confocal and transmission electron microscopy The antimicrobial effect of BIT against E. coli O157:H7 and Salmonella was also confirmed by visualizing the bacterial cell viability using confocal microscopy using S. Braenderup as a representative bacterium (Fig. 1). There was no antimicrobial effect of DMSO control as evidenced by green-colored live cells (Fig. 1A). When S. Braenderup was treated with BIT at 200 μg/ml (MIC) for 24 h at 37 °C, the presence of bacteria with disrupted membranes (red-colored dead cells) by Live/ Dead staining was observed, where propidium iodide (red) only stains bacterial cells with damaged membranes (Fig. 1B). To study the rapid antimicrobial effect of BIT at higher concentration on Salmonella, a BIT treatment at 2000 μg/ml for 1 h contact time with bacteria was included. The presence of the membrane-damaged dead cells was also observed by confocal microscopy images when Salmonella was treated with 2000 μg/ml BIT for 1 h (Fig. 1C). The TEM micrographs of S. Braenderup treated with DMSO and BIT are shown in Fig. 2. Following DMSO treatment, the bacterial cell membranes slightly wrinkled, but the cytoplasm remained uniform and evenly distributed (Fig. 2D). The treatment with BIT at 200 μg/ml for 24 h or 2000 μg/ml for 1 h caused disruption of the outer membrane, large membrane protrusions on the cell surface, uneven cytoplasmic content with dark deposits around the perimeter of the cell, and cell lysis leading to release of cytoplasmic content (Fig. 2E and F). Overall, TEM examination revealed that Salmonella treated with 200 μg/ml BIT
2.7. Statistical analysis All the experiments had duplicate samples for each treatment and control, and the experiments were repeated three times. The data were analyzed using PROC-GENMOD procedure of SAS version 9.4 (SAS Institute, Cary, NC). Antimicrobial activity tests (zone of inhibition) were presented as mean ± SD (standard deviation). Motility and Shiga toxin production were presented as mean ± SEM (standard error of the mean). Differences among the means were analyzed at P < 0.05 using Fisher's least significance difference test. 3. Results 3.1. Antibacterial activity The inhibition zones by seven glucosinolate hydrolysis compounds against E. coli O157:H7 and Salmonella are shown in Table 1. No inhibition zone was observed in the discs with DMSO (negative control). The inhibition pattern varied with isothiocyanates and bacterial strains. The inhibitory effect of six isothiocyanates (butyl-, ethyl-, isopropyl-, methyl-, phenethyl-, and allyl-) were marginal against bacterial pathogens and lower than the positive control (Gentamicin at 10 μg/disc).
Table 1 Antimicrobial activity of seven glucosinolate hydrolysis compounds against E. coli O157:H7 and Salmonella enterica serovars by disc diffusion assaya. Bacteria E. coli O157:H7
Salmonella enterica
Strain
BIT
1918 4406 4407 4688 5279 S. Braenderup S. Negev S. Newport S. Thompson S. Tennessee
B
BIC ab
13 ± 6 9 ± 3 ab B 12 ± 1 ab B 14 ± 3 ab B 10 ± 3 a A 43 ± 11 a A 64 ± 12 a A 35 ± 0 a A 45 ± 1 a A 39 ± 5 a B
A
EIT b
10 ± 1 11 ± 1 ab A 10 ± 1 c A 11 ± 1 ab A 10 ± 0 a A 10 ± 0 c A 10 ± 1 c A 10 ± 0 c B 6 ± 1 ab B 8±1c A
A A A A A A A A A A
9 8 7 9 6 6 7 9 5 9
IIT ± ± ± ± ± ± ± ± ± ±
b
2 1 ab 1c 2b 6a 5c 2c 2c 1b 2c
A A A A A A A A A A
MIT b
9±2 9 ± 2 ab 4±4c 9 ± 2b 9±2a 7±6c 9±2c 9±2c 8 ± 0b 4±3c
A
PIT b
8±2 4 ± 4b A 7±1c A 9 ± 2b A 8±2a AB 7±6c AB 7 ± 6 bc AB 6±5c B 0±0c AB 5±4c A
A A A A A A A A A A
AIT b
9±1 9 ± 3 ab 8 ± 3 bc 10 ± 2 ab 9±3a 10 ± 1 c 10 ± 1 c 9±2c 9 ± 0b 9±1c
A A A A A A A A A A
Gentamicin b
10 ± 0 9 ± 1 ab 9 ± 1 bc 10 ± 1 ab 9±1a 10 ± 1 c 9±1c 8±2c 11 ± 0 b 10 ± 0 c
A A A A A A A A A A
18 18 19 18 17 15 16 19 17 16
± ± ± ± ± ± ± ± ± ±
2a 6a 5a 4a 5a 2b 3b 2b 0a 1b
a The tested hydrolysis glucosinolate compounds included benzyl isothiocyanate (BIT), butyl isothiocyanate (BIC), ethyl isothiocyanate (EIT), isopropyl isothiocyanate (IIT), methyl isothiocyanate (MIT), phenethyl isothiocyanate (PIT), and allyl isothiocyanate (AIT). A DMSO (diluent) and antibiotic (Gentamicin at 10 μg/disc) control were also included. Dara are presented as mean ± SD (mm) (n = 3). Means with different superscripts a-c in a row and A−B in a column differ significantly (P < 0.05).
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Fig. 1. Confocal microscopic images of Salmonella Braenderup treated with benzyl isothiocyanate (BIT). Salmonella Braenderup was treated with (A) DMSO for 24 h, (B) BIT at 200 μg/ml for 24 h, or (C) 2000 μg/ml for 1 h at 37 °C, stained by the Live/Dead assay, and imaged using a confocal microscope. Panels show the merge of the green and red channel. Scale bar is valid for all panels.
4. Discussion
for 24 h showed more pronounced ultrastructural cell damages as compared to the treatment with 2000 μg/ml BIT for 1 h.
This study was conducted to evaluate the in vitro antimicrobial activities of seven isothiocyanates against Salmonella and E. coli O157:H7 and the potential inhibitory effect of BIT on the virulence factors of these bacteria. The preliminary screening for antibacterial activity of seven isothiocyanates against Salmonella and E. coli O157:H7 revealed that BIT was the most potent antibacterial among these compounds. The results of the current study are in the agreement with previous studies (Aires et al., 2009; Jang et al., 2010), where authors reported that BIT exerted the highest antimicrobial activity among the isothiocyanates against pathogenic bacteria in vitro. The antimicrobial activities of isothiocyanates can be attributed to their ability to nonenzymatically react with thiol groups such as glutathione and amino acid in proteins forming dithiocarbamates and thioureas, respectively (Holst and Williamson, 2004; Juge et al., 2007). These reactions can lead to bacterial death because of the increased oxidation and the inhibition of essential proteins and enzymes. Role of glucosinolate hydrolysis compounds against plant pathogens and plant diseases have been previously reported but still not yet clear in foodborne pathogens (Becker and Juvik, 2016). In this study, the antimicrobial effect of BIT at the cellular level on Salmonella was visualized by Live/Dead staining and TEM technique. Confocal microscopic images of Salmonella treated with BIT and stained with Live/
3.4. Bacterial motility assay The effect of BIT on motility of Salmonella and E. coli O157:H7 is shown in Fig. 3. Motility zones of DMSO-treated E. coli O157:H7 were ~75 mm, whereas exposure to BIT at 25 μg/ml (below SIC) reduced the motility zones by 42% to less than 40 mm and BIT completely inhibited the bacterial motility at the SIC levels (P < 0.05). Similarly, BIT concentrations at 25 μg/ml (below SIC) and 125 μg/ml (SIC) significantly reduced motility zone of Salmonella by 60% and 96% to less than 30 mm and 3 mm, respectively (P < 0.05). 3.5. Shiga toxin production The efficacy of BIT in reducing Shiga toxin production by E. coli O157:H7 is shown in Fig. 4. The Shiga toxin production by E. coli O157:H7 in 0.2% DMSO control measured at absorbance 450nm was considered as 100%. The BIT at 150 μg/ml (SIC) significantly inhibited Shiga toxin production by 32% and 36% in E. coli O157:H7 strains ATCC43895 and EDL933, respectively. Similarly, a lower concentration of BIT at 125 μg/ml significantly reduced (≤18%) Shiga toxin production by E. coli O157:H7.
Fig. 2. Transmission Electron Microscopy (TEM) micrographs of Salmonella Braenderup exposed to benzyl isothiocyanate (BIT). (A–C) Salmonella negatively stained with 1% PTA. (D–F) Cross sections of resin embedded Salmonella. (A, D) DMSO Control, (B, E) Salmonella treated with 2000 μg/ml BIT for 1 h. (C, F) Salmonella treated with 200 μg/ml BIT for 24 h. Scale bars = 500 nm. 4
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cellular contents, and cell lysis when cells were treated with BIT. In the current study, the BIT at SIC values was used to determine the anti-virulence activity on Salmonella and E. coli O157:H7. Determination of SIC is essential to confirm that the attenuation of bacterial virulence is not due to the bacterial growth inhibition (Bhattaram et al., 2017). We observed that BIT concentrations at SIC levels or even below the SICs significantly inhibited bacterial motility and Shiga toxin production, suggesting that the lower concentration could be used to control bacterial infections by inhibiting bacterial virulence factors. Recently, anti-virulence strategies developed for infectious diseases included preventing bacterial adhesion to host tissue, interfering with bacterial toxins and modulating virulence gene expression and bacterial chemical communication (Rasko and Sperandio, 2010). These anti-virulence therapeutic strategies target bacterial virulence factors, thereby preventing bacterial pathogenesis without threatening their existence because most virulence factors are not essential for bacterial survival (Cegelski et al., 2008; Rasko and Sperandio, 2010). In addition, using antimicrobials at SIC levels as an anti-virulence approach may decrease the selection pressure for the development of bacterial drug resistance since SIC levels are neither bacteriostatic nor bactericidal (Hung et al., 2005; Mellbye and Schuster, 2011). Similar studies have been conducted on the efficacy of other plant compounds for inhibiting Salmonella and E. coli O157:H7 virulence factors. Inamuco et al. (2012) observed that carvacrol at lower concentrations completely inhibited the motility of Salmonella Typhimurium. Burt et al. (2007) stated that carvacrol significantly reduced E. coli O157:H7 motility by preventing the synthesis of bacterial flagellin due to the over-production of a heat shock protein. Other studies have also reported role of antimicrobials at their SIC levels in controlling the transcription of specific genes associated with virulence of microorganisms (Bhattaram et al., 2017; Yin et al., 2015). Results of this study confirmed that BIT significantly reduced motility of Salmonella and E. coli O157:H7 as well as inhibited Shiga toxin production by E. coli O157:H7. Bacterial pathogens with reduced motility are less likely to adhere to epithelial cells for the initiation of bacterial infections (Burt et al., 2007; Girón et al., 2002). As Shiga toxins are the key virulence factors that cause the E. coli O157:H7 infection associated diseases such as hemolytic-uremic syndrome (Goldwater and Bettelheim, 2012), therapeutic interventions that can impede toxin production are potential approaches for the treatment of E. coli O157:H7 infections. In conclusion, our results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by inhibiting bacterial virulence factors including bacterial motility and Shiga toxin production. The toxicity and metabolic activities of BIT in human subjects should be evaluated for potential use of BIT as an alternative therapeutic treatment.
Fig. 3. Effect of benzyl isothiocyanate (BIT) on Salmonella spp. and Escherichia coli O157:H7 motility. Two strains of Salmonella (S. Braenderup and S. Tennessee) and E. coli O157:H7 (ATCC 43895 and EDL933) were included. BIT concentrations tested included sub-inhibitory concentrations (SICs) of BIT (125 μg/ml for Salmonella and 150 μg/ml E. coli O157:H7) and an additional lower concentration at 25 μg/ml. Treatments: Control-0.2% DMSO (■); BIT at 25 μg/ml (□); BIT at SIC levels (▨). Zones of motility are presented in cm and data are presented as mean ± SEM (n = 6/group). * Means of the sample are significantly different from the Control (P < 0.05).
Conflicts of interest None. Acknowledgments Fig. 4. Effect of sub-inhibitory concentration of benzyl isothiocyanate (BIT) on E. coli O157:H7 Shiga toxin production. Two strains of E. coli O157:H7 (ATCC 43895 and EDL 933) were included. BIT concentrations tested included subinhibitory concentration (SIC) at 150 μg/ml E. coli O157:H7 and an additional lower concentration at 125 μg/ml. Treatments: Control-0.2% DMSO (■); BIT at 125 μg/ml ( ); BIT at SIC level (□). Data are presented as mean ± SEM (n = 6/group), where Control (DMSO control) was considered as 100%. * Means of the sample are significantly different from the Control (P < 0.05).
This study was funded in part by the Center for Product Safety grant (SCB-11068). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fm.2019.103303. References
Dead staining showed disruption of cell membrane and uptake of propidium iodide dye by dead cells. The TEM images also revealed rough surface morphology, shrinkage of cell in the bacterial cells, leakage of
Al-Gendy, A.A., El-gindi, O.D., Hafez, A.S., Ateya, A.M., 2009. Glucosinolates, volatile constituents and biological activities of Erysimum corinthium Boiss. (Brassicaceae).
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Food Chem. 118, 519–524. Aires, A., Mota, V.R., Saavedra, M.J., Rosa, E.A.S., Bennett, R.N., 2009. The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract. J. Appl. Microbiol. 106, 2086–2095. Baskaran, S.A., Kollanoor-Johny, A., Nair, M.S., Venkitanarayanan, K., 2016. Efficacy of plant-derived antimicrobials in controlling enterohemorrhagic Escherichia coli virulence in vitro. J. Food Prot. 79, 1965–1970. Becker, T.M., Juvik, J.A., 2016. The role of glucosinolate hydrolysis products from Brassica vegetable consumption in inducing antioxidant activity and reducing cancer incidence. Diseases 4, 22. https://doi.org/10.3390/diseases-4020022. Bhattaram, V., Upadhyay, A., Yin, H.B., Mooyottu, S., Venkitanarayanan, K., 2017. Effect of dietary minerals on virulence attributes of Vibrio cholerae. Front. Microbiol. 8, 911. https://doi.org/10.3389/fmicb.2017.00911. Burt, S.A., van der Zee, R., Koets, A.P., de Graaff, A.M., van Knapen, F., Gaastra, W., Haagsman, H.P., Veldhuizen, E.J., 2007. Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157: H7. Appl. Environ. Microbiol. 73, 4484–4490. Cegelski, L., Marshall, G.R., Eldridge, G.R., Hultgren, S.J., 2008. The biology and future prospects of antivirulence therapies. Nat. Rev. Microbiol. 6, 17–27. Centers for Disease Control and Prevention (CDC), 2012. Pathogen causing US foodborne illness, hospitalizations, and deaths, 2000-2008. https://www.cdc.gov/ foodborneburden/PDFs/pathogens-complete-list-01-12.pdf, Accessed date: 9 September 2018. Chen, J.C., Ho, T.Y., Chang, Y.S., Wu, S.L., Hsiang, C.Y., 2006. Anti-diarrheal effect of Galla Chinensis on the Escherichia coli heat-labile enterotoxin and gangloside interaction. J. Ethnopharmacol. 103, 385–391. Delaquis, P.J., Mazza, G., 1995. Antimicrobial properties of isothiocyanates if food preservation. Food Technol. 49, 73–84. Escaich, S., 2008. Antivirulence as a new antibacterial approach for chemotherapy. Curr. Opin. Chem. Biol. 12, 400–408. Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates and their breakdown products in food and food plants. Crit. Rev. Food Sci. Nutr. 18, 123–201. Girón, J.A., Torres, A.G., Freer, E., Kaper, J.B., 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44, 361–379. Goldwater, P.N., Bettelheim, K.A., 2012. Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS). BMC Med. 10, 12. https:// doi.org/10.1186/1741-7015-10-12. Holst, B., Williamson, G., 2004. A critical review of the bioavailability of glucosinolates and related compounds. Nat. Prod. Rep. 21, 425–447. Hung, D.T., Shakhnovich, E.A., Pierson, E., Mekalanos, J.J., 2005. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310, 670–674. Inamuco, J., Veenendaal, A.K., Burt, S.A., Post, J.A., Tjeerdsma-van Bokhoven, J.L., Haagsman, H.P., Veldhuizen, E.J., 2012. Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells. Vet. Microbiol. 157, 200–207. Jang, M., Hong, E., Kim, G.H., 2010. Evaluation of antibacterial activity of 3‐butenyl, 4‐pentenyl, 2‐phenylethyl, and benzyl isothiocyanate in Brassica vegetables. J. Food Sci. 75, 412–416. Johny, A.K., Hoagland, T., Venkitanarayanan, K., 2010. Effect of subinhibitory concentrations of plant-derived molecules in increasing the sensitivity of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 to antibiotics. Foodb.
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Inhibition of Escherichia coli O157:H7 and Salmonella enterica virulence factors by benzyl isothiocyanate
T
⁎
Jitendra Patela, , Hsin-Bai Yina, Gary Bauchanb, Joseph Moweryb a b
U.S. Department of Agriculture, Agricultural Research Service, Environmental and Microbial Food Safety Laboratory, Beltsville, MD 20705, USA U.S. Department of Agriculture, Agricultural Research Service, SGIL Electron and Confocal Microscopy Unit, Beltsville, MD 20705, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Foodborne pathogens Virulence factors Shiga toxins Microscopic analysis
Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P < 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.
1. Introduction
followed by the release of Shiga toxin subunit A into the cytosol. The Nglycosidase activity of Shiga toxin subunit A removes an adenine group from the 28S rRNA, ultimately leading to the inhibition of protein synthesis (O'Brien et al., 1992) and apoptosis within eukaryotic cells (Monnens et al., 1998). Antibiotics are generally not recommended for treating E. coli O157:H7 infections because antibiotics increase the expression of Shiga toxin producing genes through the activation of phage lytic cycle, thereby resulting in increased Shiga toxin production (Neely and Friedman, 1998). Targeting bacterial virulence such as bacterial motility and toxin production is an alternative approach to inhibit specific mechanisms that promote bacterial infections to the hosts (Cegelski et al., 2008; Shakhnovich et al., 2007). The development of anti-virulence strategies allows the host immune system to prevent bacterial colonization or remove any established infections without killing bacteria or preventing bacterial growth, which ultimately place less selective pressure on the target microorganisms (Cegelski et al., 2008; Escaich, 2008; Rasko and Sperandio, 2010). Bacterial virulence factors contribute to the initiation and onset of a bacterial infection in the human host; therefore, an effective antimicrobial against virulence could prevent the
Pathogenic bacteria utilize several virulence factors once they are in the host and cause infections in human hosts. These virulence factors help bacteria to invade the host, cause disease, and evade host defenses (Wilson et al., 2002). Escherichia coli O157:H7 and Salmonella enterica are important foodborne pathogens in the United States, causing an estimated 2100 and 19,000 cases of hospitalizations annually, respectively (Centers for Disease Control and Prevention (CDC), 2012). Bacterial motility in E. coli O157:H7 and Salmonella is an essential virulence factor for bacterial infections in the host, because it allows these pathogens to traverse through the intestine to reach a favorable niche (Tobe et al., 2011), where flagella serves as adhesive appendages in the initial phases of colonization (Josenhans and Suerbaum, 2002). The pathogenicity of E. coli O157:H7 is also associated with the production of Shiga toxins (stx1 and stx2), which may be characterized by life threatening conditions, such as hemorrhagic colitis and hemolytic uremic syndrome in humans. Once E. coli O157:H7 colonizes human gut mucosa, Shiga toxins produced by the pathogen binds to the cell surface receptor, glycosphingolipid globotriaosylceramide,
⁎ Corresponding author. Environmental Microbial & Food Safety Laboratory, USDA-ARS, 10300 Baltimore Avenue, Building 201 BARC-East, Beltsville, MD 20705 USA. E-mail address: [email protected] (J. Patel).
https://doi.org/10.1016/j.fm.2019.103303 Received 11 April 2019; Received in revised form 3 August 2019; Accepted 10 August 2019 Available online 16 August 2019 0740-0020/ Published by Elsevier Ltd.
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compound was selected for the subsequent analyses. The sub-inhibitory concentration (SIC) and minimum inhibitory concentration (MIC) values of BIT were determined for the representative Salmonella and E. coli O157:H7 strains (S. Braenderup USDA 4559, S. Tennessee USDA 4556, E. coli O157:H7 strain ATCC 43895, and E. coli O157:H7 strain EDL 933). The susceptibility tests were carried out to determine SIC and MIC values by the standard broth microdilution method as previously described (Chen et al., 2006; Johny et al., 2010; Kim et al., 1995; Kollanoor-Johny et al., 2012). The highest concentration of the BIT that permitted bacterial growth similar to that of the DMSO control culture after incubation at 37 °C for 24 h was considered as SIC. The lowest concentration of BIT that inhibited the visible growth of bacteria after 24 h incubation at 37 °C was reported as the MIC values. At 0, 6, 12, 18, and 24 h, bacterial populations were determined by serially dilutions of samples followed by spiral plating on TSA and selective agars, where sorbitol MacConkey Agar (Neogen) and XLT4 Agar (Neogen) were used as selective agars for enumeration of E. coli O157:H7 and Salmonella, respectively.
disease progression in hosts (Lee et al., 2003; Marra, 2004; Rasko and Sperandio, 2010). In the past decade, the use of plant-derived compounds has gained significant attention due to the increasing concerns over the safety of synthetic antimicrobial agents and the emergence of antibiotic-resistant strains of microorganisms. Glucosinolates are natural compounds present in cruciferous vegetables such as cauliflower, broccoli, cabbage, turnip, and radishes (Fenwick et al., 1983; Keck and Finley, 2004). When plant tissues are disrupted, glucosinolate hydrolysis compounds including substituted isothiocyanates, nitriles, thiocyanates, and epithionitriles are produced by the enzymatic reaction of myrosinase (AlGendy et al., 2009; Delaquis and Mazza, 1995; Mari et al., 2008). Aires et al. (2009) examined the in vitro antimicrobial properties of intact glucosinolates and their hydrolysis compounds against human pathogenic and gastrointestinal tract bacteria, where isothiocyanates were found to be the most effective compounds. Jang et al. (2010) reported the antimicrobial activities of four isothiocyanates (butenyl-, pentenyl-, phenyl-ethyl-, and benzyl-) against pathogenic bacteria including Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enterica. However, the anti-virulence activities of the isothiocyanates against foodborne bacterial pathogens still need further investigation. In this study, we investigated the antimicrobial activity of seven glucosinolate hydrolysis isothiocyanate compounds (benzyl-, butyl-, ethyl-, isopropyl-, methyl-, phenethyl-, and allyl-) against five Salmonella enterica serovars (S. Braenderup, S. Negev, S. Newport, S. Thompson, and S. Tennessee) and Escherichia coli O157:H7 strains. A compound with the highest antimicrobial activity, namely the benzyl isothiocyanate (BIT), was chosen to determine its efficacy for controlling E. coli O157:H7 and Salmonella virulence in vitro. Additionally, effect of BIT on Salmonella cell viability and cell morphology was determined using confocal microscopy and transmission electron microscopy.
2.3. Confocal microscopy A Live/Dead BacLight bacterial viability kit (Thermo Fisher, Waltham, MA) was used to evaluate the viability of bacterial cells treated with BIT using confocal microscopy. S. Braenderup was selected as a representative bacterium for the confocal and transmission electron microscopic (TEM) analyses. TSB inoculated with Salmonella was treated with 200 μg/ml BIT (MIC) for 24 h or 2000 μg/ml BIT for 1 h at 37 °C. A TSB tube containing 0.2% DMSO served as the control. After incubation, bacterial culture was treated with fluorescent dyes as per manufacturer's recommendations. Viability of bacterial cells were visualized and photographed by utilizing a Zeiss 710 laser scanning confocal microscopy (Zeiss, Richmond, VA). 2.4. Transmission electron microscopy
2. Materials and methods Transmission electron microscopy (TEM) was used to visualize ultrastructural changes in Salmonella at the cellular level. A 10-ml TSB tube was inoculated with overnight culture of S. Braenderup to obtain ~5 log CFU/ml, BIT was added at 200 μg/ml or 2000 μg/ml concentrations and incubated at 37 °C for 24 h or 1 h, respectively. A TSB containing Salmonella and supplemented with DMSO for 24 h incubation at 37 °C was used as the control. For negative staining, few drops of bacterial suspension were absorbed onto carbon-formvar coated grids for 30 s and then stained with 1% (w/v) phosphotungstic acid for 5 s. For ultrastructural examination, bacterial suspensions were centrifuged at 5000 rpm for 3 min to form a pellet. Bacteria pellets were fixed for 2 h at room temperature (22 °C) in 2.5% glutaraldehyde, 0.05M NaCacodylate, 0.005M CaCl2 (pH 7.0), then refrigerated at 4 °C overnight. Pellets were rinsed 6 times with 0.05M NaCacodylate, 0.005M CaCl2 buffer and post-fixed in 1% buffered osmium tetroxide for 2 h at room temperature (22 °C). Samples were then rinsed again and dehydrated in a graded series of ethanol followed by 3 exchanges of propylene oxide (Electron Microscopy Sciences, Hatfield, PA). Pellets were then infiltrated in a graded series of LX-112 resin/propylene oxide, and polymerized in LX-112 resin (LADD Research Industries, Williston, VT) at 65 °C for 24 h. Silver-gold ultrathin (60–90 nm) sections were cut on a Reichert/AO Ultracut ultramicrotome with a Diatome diamond knife and mounted onto 100 mesh carbon-formvar coated grids. Grids were stained with 4% uranyl acetate for 10 min followed by 5 min of 3% lead citrate and imaged at 80 kV with HT-7700 transmission electron microscope (TEM, Hitachi, Schaumburg, IL).
2.1. Antibacterial activity of glucosinolate compounds Antimicrobial activities of seven glucosinolate compounds: benzyl isothiocyanate (BIT), butyl isothiocyanate (BIC), ethyl isothiocyanate (EIT), isopropyl isothiocyanate (IIT), methyl isothiocyanate (MIT), phenethyl isothiocyanate (PIT), and allyl isothiocyanate (AIT) (SigmaAldrich, Allentown, PA) against bacterial pathogens were determined by the disc diffusion assay (Kim et al., 1995). Five Salmonella enterica serovars (S. Braenderup, S. Negev, S. Newport, S. Thompson, and S. Tennessee) and five E. coli O157:H7 strains (1918, 4406, 4407, 4688, and 5279) were used in this study. Briefly, an overnight culture of each bacterial isolate in trypticase soy broth (TSB; Fisher, Waltham, MA) was adjusted to the concentration of ~6 log CFU/ml with absorbance value at 600nm, followed by spread plating 100 μl of each adjusted culture on trypticase soy agar (TSA; Fisher). A sterile filter paper disc (6 mm diameter; Fisher) was impregnated with 10 μl of each glucosinolate compound at the concentration of 10 mg/ml (100 μg/disc). The discs were placed on bacterial pathogen-seeded TSA plates and incubated at 37 °C overnight. Gentamicin antibiotic (10 μg/disc; Sigma) was used as a positive control. A sterile filter disc loaded with 10 μl dimethyl sulfoxide (DMSO; Fisher) was included as the diluent control since it was used to dissolve these glucosinolate compounds. Following overnight incubation, the zone of inhibition (mm) surrounding each disc was measured. 2.2. Determination of sub-inhibitory and minimum inhibitory concentrations
2.5. Bacterial motility assay Among 7 glucosinolate hydrolysis compounds, BIT exerted the highest antimicrobial activity against bacterial pathogens; this
The effect of BIT on motility of bacterial pathogens was determined 2
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as previously described (Wang et al., 2006). The BIT at SIC levels (125 μg/ml for Salmonella and 150 μg/ml for E. coli O157:H7) or 25 μg/ ml (below SIC) was added to each petri plate containing 20 ml of semisolid TSB (0.3% agar w/v) cooled at 45 °C. The plates with BIT were swirled for 15–20 s, and then 10 μl of overnight culture of individual E. coli O157:H7 (ATCC 43895 and EDL 933) or Salmonella strain (S. Braenderup USDA 4559 and S. Tennessee USDA 4556) containing ~6 log CFU was placed at the center of the plate. The plates were kept at room temperature for 1 h, followed by overnight incubation at 37 °C. After incubation, the diameter of the motility zone (mm) was measured.
However, the effect of BIT was significantly higher than other isothiocyanates and gentamicin against Salmonella. Additionally, Salmonella were more sensitive to BIT than E. coli O157:H7; the inhibition zones for Salmonella ranged from 35 mm for S. Newport to 64 mm for S. Negev, whereas the inhibition zones for E. coli O157:H7 strains were in 9–14 mm range. Overall, the BIT exerted the highest antibacterial activity against E. coli O157:H7 and Salmonella compared to other six compounds.
2.6. Shiga toxin concentrations
The SIC values of BIT were determined as 125 μg/ml and 150 μg/ml (0.0125% and 0.015% v/v for Salmonella and E. coli O157:H7, respectively. The average initial Salmonella and E. coli O157:H7 population in the untreated control, DMSO control, and BIT-treated samples was approximately 5 log CFU/ml in TSB, bacterial populations in the controls and the treatment with SICs of BIT reached approximately 8.5 log CFU/ml after 24 h incubation at 37 °C, thereby confirming that the SIC values were not inhibitory for these bacteria. The MIC values were 200 μg/ml (0.02% v/v) for both Salmonella and E. coli O157:H7. At the MIC level, bacterial populations maintained at 5 log CFU/ml after 24 h of incubation at 37 °C.
3.2. Sub-inhibitory and minimum inhibitory concentrations
The effect of BIT at 150 μg/ml (SIC) and 125 μg/ml (below SIC) on Shiga toxin production by E. coli O157:H7 (ATCC 43895 and EDL 933) was investigated as described by Baskaran et al. (2016). Tubes containing TSB inoculated with each strain of E. coli O157:H7 (~5 log CFU/ml) were incubated with or without BIT at 37 °C for 24 h. Shiga toxin concentrations were determined in the control (0.2% DMSO) and BIT treated E. coli O157:H7 cultures by an enzyme-linked immunosorbent assay (ELISA) using the Premier EHEC test kit (Meridian Bioscience, Cincinnati, OH). Absorbance at 450 nm were measured using a microplate reader (BioTek Instruments, Winnoski, VT) to determine Shiga toxin production and results were expressed in percentage (%) in comparison to the DMSO control.
3.3. Confocal and transmission electron microscopy The antimicrobial effect of BIT against E. coli O157:H7 and Salmonella was also confirmed by visualizing the bacterial cell viability using confocal microscopy using S. Braenderup as a representative bacterium (Fig. 1). There was no antimicrobial effect of DMSO control as evidenced by green-colored live cells (Fig. 1A). When S. Braenderup was treated with BIT at 200 μg/ml (MIC) for 24 h at 37 °C, the presence of bacteria with disrupted membranes (red-colored dead cells) by Live/ Dead staining was observed, where propidium iodide (red) only stains bacterial cells with damaged membranes (Fig. 1B). To study the rapid antimicrobial effect of BIT at higher concentration on Salmonella, a BIT treatment at 2000 μg/ml for 1 h contact time with bacteria was included. The presence of the membrane-damaged dead cells was also observed by confocal microscopy images when Salmonella was treated with 2000 μg/ml BIT for 1 h (Fig. 1C). The TEM micrographs of S. Braenderup treated with DMSO and BIT are shown in Fig. 2. Following DMSO treatment, the bacterial cell membranes slightly wrinkled, but the cytoplasm remained uniform and evenly distributed (Fig. 2D). The treatment with BIT at 200 μg/ml for 24 h or 2000 μg/ml for 1 h caused disruption of the outer membrane, large membrane protrusions on the cell surface, uneven cytoplasmic content with dark deposits around the perimeter of the cell, and cell lysis leading to release of cytoplasmic content (Fig. 2E and F). Overall, TEM examination revealed that Salmonella treated with 200 μg/ml BIT
2.7. Statistical analysis All the experiments had duplicate samples for each treatment and control, and the experiments were repeated three times. The data were analyzed using PROC-GENMOD procedure of SAS version 9.4 (SAS Institute, Cary, NC). Antimicrobial activity tests (zone of inhibition) were presented as mean ± SD (standard deviation). Motility and Shiga toxin production were presented as mean ± SEM (standard error of the mean). Differences among the means were analyzed at P < 0.05 using Fisher's least significance difference test. 3. Results 3.1. Antibacterial activity The inhibition zones by seven glucosinolate hydrolysis compounds against E. coli O157:H7 and Salmonella are shown in Table 1. No inhibition zone was observed in the discs with DMSO (negative control). The inhibition pattern varied with isothiocyanates and bacterial strains. The inhibitory effect of six isothiocyanates (butyl-, ethyl-, isopropyl-, methyl-, phenethyl-, and allyl-) were marginal against bacterial pathogens and lower than the positive control (Gentamicin at 10 μg/disc).
Table 1 Antimicrobial activity of seven glucosinolate hydrolysis compounds against E. coli O157:H7 and Salmonella enterica serovars by disc diffusion assaya. Bacteria E. coli O157:H7
Salmonella enterica
Strain
BIT
1918 4406 4407 4688 5279 S. Braenderup S. Negev S. Newport S. Thompson S. Tennessee
B
BIC ab
13 ± 6 9 ± 3 ab B 12 ± 1 ab B 14 ± 3 ab B 10 ± 3 a A 43 ± 11 a A 64 ± 12 a A 35 ± 0 a A 45 ± 1 a A 39 ± 5 a B
A
EIT b
10 ± 1 11 ± 1 ab A 10 ± 1 c A 11 ± 1 ab A 10 ± 0 a A 10 ± 0 c A 10 ± 1 c A 10 ± 0 c B 6 ± 1 ab B 8±1c A
A A A A A A A A A A
9 8 7 9 6 6 7 9 5 9
IIT ± ± ± ± ± ± ± ± ± ±
b
2 1 ab 1c 2b 6a 5c 2c 2c 1b 2c
A A A A A A A A A A
MIT b
9±2 9 ± 2 ab 4±4c 9 ± 2b 9±2a 7±6c 9±2c 9±2c 8 ± 0b 4±3c
A
PIT b
8±2 4 ± 4b A 7±1c A 9 ± 2b A 8±2a AB 7±6c AB 7 ± 6 bc AB 6±5c B 0±0c AB 5±4c A
A A A A A A A A A A
AIT b
9±1 9 ± 3 ab 8 ± 3 bc 10 ± 2 ab 9±3a 10 ± 1 c 10 ± 1 c 9±2c 9 ± 0b 9±1c
A A A A A A A A A A
Gentamicin b
10 ± 0 9 ± 1 ab 9 ± 1 bc 10 ± 1 ab 9±1a 10 ± 1 c 9±1c 8±2c 11 ± 0 b 10 ± 0 c
A A A A A A A A A A
18 18 19 18 17 15 16 19 17 16
± ± ± ± ± ± ± ± ± ±
2a 6a 5a 4a 5a 2b 3b 2b 0a 1b
a The tested hydrolysis glucosinolate compounds included benzyl isothiocyanate (BIT), butyl isothiocyanate (BIC), ethyl isothiocyanate (EIT), isopropyl isothiocyanate (IIT), methyl isothiocyanate (MIT), phenethyl isothiocyanate (PIT), and allyl isothiocyanate (AIT). A DMSO (diluent) and antibiotic (Gentamicin at 10 μg/disc) control were also included. Dara are presented as mean ± SD (mm) (n = 3). Means with different superscripts a-c in a row and A−B in a column differ significantly (P < 0.05).
3
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Fig. 1. Confocal microscopic images of Salmonella Braenderup treated with benzyl isothiocyanate (BIT). Salmonella Braenderup was treated with (A) DMSO for 24 h, (B) BIT at 200 μg/ml for 24 h, or (C) 2000 μg/ml for 1 h at 37 °C, stained by the Live/Dead assay, and imaged using a confocal microscope. Panels show the merge of the green and red channel. Scale bar is valid for all panels.
4. Discussion
for 24 h showed more pronounced ultrastructural cell damages as compared to the treatment with 2000 μg/ml BIT for 1 h.
This study was conducted to evaluate the in vitro antimicrobial activities of seven isothiocyanates against Salmonella and E. coli O157:H7 and the potential inhibitory effect of BIT on the virulence factors of these bacteria. The preliminary screening for antibacterial activity of seven isothiocyanates against Salmonella and E. coli O157:H7 revealed that BIT was the most potent antibacterial among these compounds. The results of the current study are in the agreement with previous studies (Aires et al., 2009; Jang et al., 2010), where authors reported that BIT exerted the highest antimicrobial activity among the isothiocyanates against pathogenic bacteria in vitro. The antimicrobial activities of isothiocyanates can be attributed to their ability to nonenzymatically react with thiol groups such as glutathione and amino acid in proteins forming dithiocarbamates and thioureas, respectively (Holst and Williamson, 2004; Juge et al., 2007). These reactions can lead to bacterial death because of the increased oxidation and the inhibition of essential proteins and enzymes. Role of glucosinolate hydrolysis compounds against plant pathogens and plant diseases have been previously reported but still not yet clear in foodborne pathogens (Becker and Juvik, 2016). In this study, the antimicrobial effect of BIT at the cellular level on Salmonella was visualized by Live/Dead staining and TEM technique. Confocal microscopic images of Salmonella treated with BIT and stained with Live/
3.4. Bacterial motility assay The effect of BIT on motility of Salmonella and E. coli O157:H7 is shown in Fig. 3. Motility zones of DMSO-treated E. coli O157:H7 were ~75 mm, whereas exposure to BIT at 25 μg/ml (below SIC) reduced the motility zones by 42% to less than 40 mm and BIT completely inhibited the bacterial motility at the SIC levels (P < 0.05). Similarly, BIT concentrations at 25 μg/ml (below SIC) and 125 μg/ml (SIC) significantly reduced motility zone of Salmonella by 60% and 96% to less than 30 mm and 3 mm, respectively (P < 0.05). 3.5. Shiga toxin production The efficacy of BIT in reducing Shiga toxin production by E. coli O157:H7 is shown in Fig. 4. The Shiga toxin production by E. coli O157:H7 in 0.2% DMSO control measured at absorbance 450nm was considered as 100%. The BIT at 150 μg/ml (SIC) significantly inhibited Shiga toxin production by 32% and 36% in E. coli O157:H7 strains ATCC43895 and EDL933, respectively. Similarly, a lower concentration of BIT at 125 μg/ml significantly reduced (≤18%) Shiga toxin production by E. coli O157:H7.
Fig. 2. Transmission Electron Microscopy (TEM) micrographs of Salmonella Braenderup exposed to benzyl isothiocyanate (BIT). (A–C) Salmonella negatively stained with 1% PTA. (D–F) Cross sections of resin embedded Salmonella. (A, D) DMSO Control, (B, E) Salmonella treated with 2000 μg/ml BIT for 1 h. (C, F) Salmonella treated with 200 μg/ml BIT for 24 h. Scale bars = 500 nm. 4
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cellular contents, and cell lysis when cells were treated with BIT. In the current study, the BIT at SIC values was used to determine the anti-virulence activity on Salmonella and E. coli O157:H7. Determination of SIC is essential to confirm that the attenuation of bacterial virulence is not due to the bacterial growth inhibition (Bhattaram et al., 2017). We observed that BIT concentrations at SIC levels or even below the SICs significantly inhibited bacterial motility and Shiga toxin production, suggesting that the lower concentration could be used to control bacterial infections by inhibiting bacterial virulence factors. Recently, anti-virulence strategies developed for infectious diseases included preventing bacterial adhesion to host tissue, interfering with bacterial toxins and modulating virulence gene expression and bacterial chemical communication (Rasko and Sperandio, 2010). These anti-virulence therapeutic strategies target bacterial virulence factors, thereby preventing bacterial pathogenesis without threatening their existence because most virulence factors are not essential for bacterial survival (Cegelski et al., 2008; Rasko and Sperandio, 2010). In addition, using antimicrobials at SIC levels as an anti-virulence approach may decrease the selection pressure for the development of bacterial drug resistance since SIC levels are neither bacteriostatic nor bactericidal (Hung et al., 2005; Mellbye and Schuster, 2011). Similar studies have been conducted on the efficacy of other plant compounds for inhibiting Salmonella and E. coli O157:H7 virulence factors. Inamuco et al. (2012) observed that carvacrol at lower concentrations completely inhibited the motility of Salmonella Typhimurium. Burt et al. (2007) stated that carvacrol significantly reduced E. coli O157:H7 motility by preventing the synthesis of bacterial flagellin due to the over-production of a heat shock protein. Other studies have also reported role of antimicrobials at their SIC levels in controlling the transcription of specific genes associated with virulence of microorganisms (Bhattaram et al., 2017; Yin et al., 2015). Results of this study confirmed that BIT significantly reduced motility of Salmonella and E. coli O157:H7 as well as inhibited Shiga toxin production by E. coli O157:H7. Bacterial pathogens with reduced motility are less likely to adhere to epithelial cells for the initiation of bacterial infections (Burt et al., 2007; Girón et al., 2002). As Shiga toxins are the key virulence factors that cause the E. coli O157:H7 infection associated diseases such as hemolytic-uremic syndrome (Goldwater and Bettelheim, 2012), therapeutic interventions that can impede toxin production are potential approaches for the treatment of E. coli O157:H7 infections. In conclusion, our results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by inhibiting bacterial virulence factors including bacterial motility and Shiga toxin production. The toxicity and metabolic activities of BIT in human subjects should be evaluated for potential use of BIT as an alternative therapeutic treatment.
Fig. 3. Effect of benzyl isothiocyanate (BIT) on Salmonella spp. and Escherichia coli O157:H7 motility. Two strains of Salmonella (S. Braenderup and S. Tennessee) and E. coli O157:H7 (ATCC 43895 and EDL933) were included. BIT concentrations tested included sub-inhibitory concentrations (SICs) of BIT (125 μg/ml for Salmonella and 150 μg/ml E. coli O157:H7) and an additional lower concentration at 25 μg/ml. Treatments: Control-0.2% DMSO (■); BIT at 25 μg/ml (□); BIT at SIC levels (▨). Zones of motility are presented in cm and data are presented as mean ± SEM (n = 6/group). * Means of the sample are significantly different from the Control (P < 0.05).
Conflicts of interest None. Acknowledgments Fig. 4. Effect of sub-inhibitory concentration of benzyl isothiocyanate (BIT) on E. coli O157:H7 Shiga toxin production. Two strains of E. coli O157:H7 (ATCC 43895 and EDL 933) were included. BIT concentrations tested included subinhibitory concentration (SIC) at 150 μg/ml E. coli O157:H7 and an additional lower concentration at 125 μg/ml. Treatments: Control-0.2% DMSO (■); BIT at 125 μg/ml ( ); BIT at SIC level (□). Data are presented as mean ± SEM (n = 6/group), where Control (DMSO control) was considered as 100%. * Means of the sample are significantly different from the Control (P < 0.05).
This study was funded in part by the Center for Product Safety grant (SCB-11068). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fm.2019.103303. References
Dead staining showed disruption of cell membrane and uptake of propidium iodide dye by dead cells. The TEM images also revealed rough surface morphology, shrinkage of cell in the bacterial cells, leakage of
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