1 Inhibitory effects of lactic and malic organic acids on autoinducer type 2 (AI-2) quorum 2 sensing of Escherichia coli O157:H7 and Salmonella Typhimurium 3 4 Ahmad Almasoud1, Navam Hettiarachchy1*, Srinivas Rayaprolu1, Dinesh Babu2, Young Min 5 Kwon3 and Andy Mauromoustakos4 6 1 Department of Food Science, University of Arkansas, Fayetteville, AR 72704, Department of 2 7 Department of Toxicology, University of Louisiana, Monroe, LA 71209, USA 3 8 9 4 Poultry Science, University of Arkansas, Fayetteville, AR 72701 Department of Agricultural Statistics, University of Arkansas, Fayetteville, AR 72701 10 *Author for correspondence: Navam S. Hettiarachchy 11 Department of Food Science & Institute of Food Engineering, 12 University of Arkansas, 13 2650 N. Young Avenue, 14 Fayetteville, 15 AR 72704 16 Phone: (479) 575-4779 17 Fax: (479) 575-6936 18 Email: nhettiar@uark.edu 19 20 21 22 Short version of title (Effect of organic acids on quorum sensing) 1 © 2015. This manuscript version is made available under the Elsevier user license http://www.elsevier.com/open-access/userlicense/1.0/ 23 Abstract 24 Several organic acid based antimicrobials are reported to reduce bacterial populations but 25 studies showing inhibition of autoinducer-2 (AI-2) activity or quorum sensing are limited. The 26 effect of lactic and malic acids on autoinducer activity of selected strains of E. coli O157:H7 and 27 Salmonella Typhimurium is tested in this study. The strains were screened for AI-2 like activity 28 on spinach and cantaloupe homogenates using autoinducer sensing V. harveyi biosensor strains. 29 The ED 14 strain of E. coli O157:H7 and the SD 10 strain of Salmonella showed highest AI-2 30 like activity of 55 and 53 Relative Light Units respectively. These two strains were used to 31 evaluate the AI-2 inhibitory activities of lactic and malic acids at 1 to 4% concentrations (alone 32 or in combinations). Lactic acid at 4.0% had the highest inhibition of 80% on ED 14 E. coli 33 strain while the combination treatment of lactic acid + malic acid at 4.0% each had the highest 34 inhibition of 80% on SD 10 Salmonella strain. Results from the study indicate that the quorum 35 sensing ability of the E. coli O157:H7 and Salmonella Typhimurium strains can be effectively 36 inhibited by antimicrobial organic acids. 37 Key words: Quorum sensing, auto inducers, pathogenic bacteria, produce, organic acids. 2 38 Introduction 39 Quorum sensing is a cell-cell signaling process used by certain bacteria to coordinate 40 virulence gene expression and survival. During quorum sensing, certain bacteria are known to 41 modulate the cellular functions through signaling compounds known as autoinducers (Silagyi et 42 al., 2009). Both gram-positive and gram-negative bacteria use this cell density dependent system 43 as a response to environmental stresses such as lack of nutrients, inhibitory effects of 44 temperature and host defense responses (Waters and Bassler 2005). The autoinducer (AI) 45 molecules affect the bacterial expression of various genes involved in virulence, toxicity, 46 sporulation, plasmid transformation, antibiotic production and biofilm formation (Bainton et al., 47 1992; Bassler 1999; Davies et al., 1998; Luo and Farrand 2001; Sperandio et al., 2001; Oger and 48 Farrand 2002; Kendall and Sperandio 2014). Certain bacteria exhibit quorum-sensing behavior 49 as a regulatory process to ensure the presence of sufficient cell density before a specific gene 50 product is made. This process allows them to multiply exponentially and initiate to express a 51 certain phenotype such as biofilm production. Excess concentrations of autoinducer compounds 52 beyond a threshold within the cells are known to activate (some repress) a regulatory protein that 53 binds to specific DNA sequence regions and activates transcription mechanism resulting in the 54 production of biofilm (Bassler 1999; Moriera et al., 2006). Quorum sensing bacteria such as E. 55 coli O157:H7 and Salmonella Typhimurium are known to respond to two types of auto-inducers 56 called acylated homoserine lactones (AI-1) and furanosyl borate diester (AI-2) (Lu et al., 2005; 57 Reading et al., 2007). Marine bacterium Vibrio harveyi is commonly utilized as a bioluminescent 58 reporter strain to detect these autoinducer molecules (Figure-1). Inhibition of autoinducer 59 molecules or their activity may affect quorum-sensing behavior or biofilm formation by certain 60 bacteria. Various natural compounds have been studied to demonstrate their role as AI-2 3 61 inhibitors. For example, fatty acids such as stearic acid, palmitic acid, oleic acid, and linoleic 62 acids isolated from poultry meat (have shown to inhibit AI-2 activity (Widmer et al., 2007). 63 Other examples of natural compounds that are reported to inhibit bacterial quorum sensing are 64 vanilla extracts (Choo et al., 2006) and traditional Chinese medicinal plant extracts (Koh and 65 Tham 2001). Pure compounds like p-coumaric acid have also been found to provide inhibitory 66 effect against cell-cell communication in bacteria (Bodini et al., 2009). Interestingly, the 67 antimicrobial effects of several organic acids against bacteria have been studied, but the studies 68 on their inhibitory effects on biofilm formation and autoinducer-2 activities are limited. 69 Considering the importance of quorum sensing in biofilm formation by foodborne 70 pathogens such as E. coli O157:H7 and Salmonella Typhimurium that often contaminate 71 minimally processed produce, it would be important to inhibit the autoinducer molecules of these 72 strains so that their survival or persistence can be hindered on these foods. In this study the effect 73 of lactic and malic acids on autoinducer-2 (quorum sensing molecule) activity of E. coli 74 O157:H7 strains on spinach and Salmonella Typhimurium strains on cantaloupes was 75 demonstrated. Reporter strain, Vibrio harveyi (BB170), was used to detect quorum sensing based 76 on its ability to produce bioluminescence in response to AI-2 activity. 77 78 Materials and Methods 79 We used the Vibrio harveyi reporter strain to detect the autoinducer-2 (AI-2) activity of 80 E. coli O157:H7 and Salmonella Typhimurium strains inoculated in minimally processed 81 produce samples (spinach and cantaloupe). The methodology of culture preparation, inoculation 82 and autoinducer measurements using the reporter strain are indicated below. 83 Culture preparation of E. coli O157:H7, Salmonella Typhimurium and Vibrio harveyi. 4 84 The procedure for this method was obtained from previous research using Vibrio harveyi 85 for detecting quorum sensing (Kim et al., 2009). The reporter strain, Vibrio harveyi BB170, 86 produces light in response to AI-2 (due to luxS gene) and is useful in biological assays for 87 detecting and quantifying AI-2 production by bacterial cultures (Taga, and Xavier, 2005). Agar 88 slants with pure cultures of the food pathogenic bacteria E. coli O157:H7 and Salmonella 89 Typhimurium were obtained from the University of Georgia, Center for Food Safety, Griffin, 90 GA. The bacterial strains were; E. coli O157:H7 green fluorescent protein (GFP)-labeled ED 14 91 (CV267); ED15 (6980-2); ED16 (6982-2); MD58 (CV261), MD46 (F4546); MD47 (K4492), 92 Salmonella Typhimurium -GFP-labeled SD 10 and SD 11. Working cultures from the frozen 93 stocks (-70 C) were prepared by transferring aseptically to 10mL of brain heart infusion broth 94 media (BHI) (Becton Dickinson Microbiology Systems, Sparks, MD, U.S.A.) and incubated at 95 37 C for 24h with shaking (200-rpm) in a New Brunswick Scientific (Edison NJ, U.S.A.) 96 agitating-incubator. Second-day inoculum of each strain was prepared by transferring 10µl of 97 first-day culture into 10mL of fresh BHI and incubated for 24h in a shaker maintained at 37 C 98 and this was used to inoculate (109 log CFU/mL) spinach and cantaloupe for the study. The 99 Vibrio harveyi BB170 (ATCC BBA-1117) was used as a reporter strain to detect AI-2 molecule 100 while V. harveyi BB152 (ATCC BBA-1119) that produces AI-1 and AI-2, was used as a positive 101 control. The Vibrio strains were grown in the auto-inducer bioassay (AB) medium. The AB 102 medium was prepared as follows: A solution consisting of NaCl (17.5g/L), MgSO4 (12.3g/L), 103 and vitamin-free cas-amino acids (2g/L) was dissolved in 1L of water with final pH 7.5 and 104 sterilized by autoclaving (15min, 121 °C). When the solution was cooled, sterile 1 M potassium 105 phosphate solution (pH 7.0, 10mL/L), 50% glycerol (20mL/L) and filter-sterilized 0.1 M L- 106 arginine (10mL/L) were added. 5 107 108 Preparation of antimicrobial solutions. 109 Separate stock solutions of organic acids were prepared in 10mL of sterile water by 110 dissolving malic acid powder and lactic acid to a concentration of 10%. The stock solutions were 111 diluted to prepare 1.0, 2.0, 3.0, and 4.0% of both organic acid test solutions one day before the 112 study (Massey et al., 2013). The solutions were vacuum filtered through Whatman no. 4 filter 113 paper to remove insoluble particles that may interfere with the spraying. Deionized water was 114 measured (similar to the weight of the organic acid solution) and the pH was adjusted to that of 115 the test solutions with 1N hydrochloric acid (HCl) to prepare the control solutions for the organic 116 acids. 117 118 Spinach/cantaloupe homogenate preparation for auto-inducer assay. 119 Spinach and cantaloupe were purchased fresh from a local grocery store on the day of the 120 experiment. Spinach leaves and cantaloupe rinds were rinsed with water, and submerged in 121 sodium hypochlorite solution (6.25mL/liter) for 3min to reduce the microbial background. The 122 leaves/rinds were submerged again in sterile water for 3 min, removed and left under the 123 biological safety cabinet for 2h to dry. After drying, the leaves/rinds were placed in separate 124 sterile bags and weighed. Sterile water was added at a volume of twice the weight of the sample 125 to each bag and stomached using a lab-scale masticator (Neutec Group Inc., Farmingdale, NY) 126 for 3min to prepare homogenates. Ninety microliter samples of the homogenates 127 (spinach/cantaloupe) were dispensed into separate 96-well plates (Becton Dickinson and Co. 128 Franklin lakes, NJ) before adding 10µL of the second day culture of E. coli O157:H7 or 129 Salmonella Typhimurium. The 96-well plates were incubated for 24h at 25 C. After incubation 6 130 the homogenates with the pathogen cultures were transferred to sterile 1.5mL Eppendorf tubes 131 and centrifuged at 3,000g for 5min at 25 C to separate the bacterial cells. The cell-free 132 supernatants (CFS) were collected and stored at –20 C for autoinducer activity assay. 133 134 Auto-inducer activity assay 135 Autoinducer (AI-2) detection assay in minimally processed produce was conducted as 136 described previously (Silagyi et al., 2009). The overnight cultures of the Vibrio reporter and 137 positive control strains were separately diluted (1:5000) in fresh AB medium and the diluted 138 cells (90µl) were dispensed into a 96-well plate. For the auto-inducer activity assay the CFS of 139 spinach/cantaloupe homogenates that were earlier exposed to E. coli O157:H7 or Salmonella 140 Typhimurium strains, potentially containing AI-2 molecules, were added to the reporter strain 141 cultures previously dispensed into a 96-well plate. The wells with V. harveyi BB152 strain were 142 used as positive control. The plates were incubated for 3h at 30 C with shaking and 143 luminescence measurements were made using a plate reader at 490nm. This assay measured the 144 uninhibited or the highest relative AI-2 activity among the stains of E. coli O157:H7 and 145 Salmonella Typhimurium. 146 147 Assay to measure the effect of organic acids on the AI-2 activity 148 The inhibitory effects of malic (MA) and lactic (LA) acids on AI-2 activity by E. coli 149 O157:H7/ and Salmonella Typhimurium were tested by adding the organic acid solutions at 150 different concentrations (1.0, 2.0, 3.0, 4.0%). As described above, the CFS of treated 151 spinach/cantaloupe homogenates were added to 96-well plates containing the reporter strain V. 152 harveyi BB170. Combinations of LA and MA (LA1% + MA1%, LA4% + MA4%) were also 7 153 investigated. The plates were incubated at 30 C for 3h with shaking. Luminescence readings 154 were measured at 490nm using a plate reader. 155 156 Assay to measure the effect of organic acids on V. harveyi strain BB170 bacteria 157 Overnight AB medium cultures of V. harveyi BB170 were diluted (1:5000) with fresh AB 158 medium. The subculture of V. harveyi BB170 and organic acids that showed the highest AI-2 159 inhibition activity were added to individual sterilized tubes at the ratio of 9:1 v/v (V. harveyi 160 BB170: organic acids solution sample), and incubated at 30 C for a similar time period as the 161 AI-2 bioassay. Serial dilutions were made for enumeration of viable cells of V. harveyi BB170 162 on Marine agar (Becton Dickinson) and log counts were recorded. 163 164 Statistical analysis 165 The JMP 10.0 software (SAS Institute, Cary, NC) was used for one way analysis of 166 variance to determine the effect of organic acids on AI-2 like activities, activity inhibitions, and 167 the growth of reporter strain V. harveyi BB170. A factorial design was used in the study for the 168 organic acid treatments. Analysis of variance (ANOVA) was performed and significant 169 difference was determined at P < 0.05. Tukey HSD was used to compare means and all 170 experiments were conducted in triplicates and repeated for reproducibility. 171 172 Results and discussion 173 Evaluation of the relative AI-2 activity by E.coli and Salmonella on spinach and cantaloupe 174 The pathogenic bacteria, E. coli and Salmonella were selected for study on spinach and 175 cantaloupe respectively since they have been found to contaminate these produce causing 8 176 numerous cases of illness. Quorum sensing and biofilm formation in pathogenic bacteria have 177 been shown to enhance their survival rate while affecting the severity of the contamination 178 problem (Gandhi and Chikindas, 2007; Annous et al., 2009; Silgayi et al., 2009). The 179 autoinducer molecules AI-1 and AI-2 were previously detected to induce quorum sensing 180 (Surette and Bassler 1998; Surette et al., 1999) and were used by researchers in studying quorum 181 sensing and biofilm formation (Lu et al., 2004; Uckoo et al., 2015). To investigate the quorum 182 sensing ability of a pathogen the relative AI-2 activity was determined as the ratio of the average 183 value of the sample against that of the negative control. 184 The results of relative AI-2-like activity in spinach homogenate inoculated with six stains 185 of E. coli followed by incubation for 12h are given in Table.1. The ED 14 strain produced the 186 highest relative AI-2-like activity of 55 Relative Light Units (RLU, the units of measurement for 187 the chemiluminescence or bioluminescence) which was significantly high in comparison to other 188 tested strains (p <0.05). Hence, ED 14 strain of E. coli O157:H7 was selected to investigate the 189 effect of various organic acid concentrations, alone and/or in combination. Table.2 shows the 190 relative AI-2 activity produced by two Salmonella Typhimurium strains (SD 10 and SD 11) in 191 cantaloupe homogenate after 12 h incubation, where the SD 10 strain showed higher (53%) 192 relative AI-2 activity in comparison to SD 11 (21%). Hence, SD 10 strain was chosen to further 193 investigate the effect of organic acids on biofilm formation mechanism. 194 195 Determination of the effect of organic acids on the AI-2 activity 196 Lactic and malic organic acids at concentrations of 1.0, 2.0, 3.0, and 4.0% (alone and in 197 combination) were tested for their ability to inhibit AI-2 activity in spinach and cantaloupe 198 homogenates inoculated with E.coli O157:H7 (ED 14) and Salmonella Typhimurium (SD 10) 9 199 respectively. These concentrations were selected based on previous studies on effective pathogen 200 inhibition by the organic acids on produce (Ganesh et al., 2010; Ganesh et al., 2012; Massey et 201 al., 2013). 202 Inhibition of E. coli O157:H7, ED 14 AI-2 activity in spinach homogenate is shown in 203 Table 3. Overall results show that lactic acid was more effective than malic acid in inhibiting AI- 204 2 activities. At its highest concentration of 4.0%, malic acid showed 37% inhibition of AI-2 and 205 at the same concentration, lactic acid inhibited the AI-2 activity by 80% which was significant 206 (p<0.05). The combined treatment of lactic and malic acid at 1.0% and 4.0% were not significant 207 in inhibiting AI-2 activity. The treatment LA+MA (1.0% each) resulted in 17% inhibition while 208 LA+MA (4.0% each) inhibited the relative AI-2 activity by 25%. Hence, a combination of 209 organic acids did not have any synergistic or additive effect on E.coli’s ability to produce AI-2. 210 The Table 4 shows the inhibition of Salmonella Typhimurium, SD 10 strain by organic 211 acid treatments. The combined treatment of LA+MA (4.0% each) had the highest inhibition by 212 80%. However, it is not significantly different from that shown by lactic acid alone at 4.0% 213 concentration which resulted in an inhibition of 76%. These results on reduction of quorum 214 sensing ability by organic acids can supplement previous studies which demonstrated their 215 antimicrobial effect on produce (Ganesh et al., 2010). 216 These outcomes can be explained by the fact that the inhibitory properties of the organic 217 acids is due to their concentrations, ability to penetrate the bacterial cell wall and the capacity to 218 alter the microbial cytoplasmic pH (Midolo et al., 1995; Eswaranandam et al., 2004). The 219 differences in efficacy among the organic acids can be attributed to their molecular size (Ganesh 220 et al., 2010). 221 10 222 Inhibitory effect of organic acids on the growth of V. harveyi BB170 223 The influence of organic acids on the multiplication of the reporter strain is shown in 224 Table 5. The treatments selected for this tests were: Lactic acid 4.0%, Malic acid 4.0%, and LA+ 225 MA (4.0% each) since they caused significant inhibition of AI-2. This experiment was conducted 226 to verify if the decline in AI-2 activity was due to the growth inhibition of the reporter strain V. 227 harveyi BB170. For this purpose, a mixture of (1:9) of organic acids and the diluted reporter 228 strain V. harveyi BB170 in AB medium was incubated as mentioned previously. There were no 229 significant differences between the control (growth in AB medium) and the various treatments, 230 suggesting that inhibition of reporter strain growth was not the basis for inhibition of AI-2 231 activity by the organic acids. Enumeration of the V. harveyi BB170 cells was conducted to 232 determine the effect of organic acids on the report bacteria as an alternative to bioluminescence 233 assay. This is due to the fact that organic acids may impact the redox reactions for NAD(P)H 234 formation (energy source for bioluminescence) in the reporter bacteria there by affecting the 235 bacterial metabolism and the response on AI-2 activity. 236 The AI-2 inhibition activity of the organic acids is considered to be lower in comparison 237 to certain chemical food additives reported in the literature. As an example, turkey patties treated 238 with sodium propionate showed inhibition of AI-2 by 99.8% (Lu et al., 2004). However, 239 naturally occurring organic acids have greater potential for application in foods since most 240 consumers are avoiding chemical additives and preservatives in food products. Among other 241 organic acids such as fatty acids including palmitic acid, steric acid, oleic acid, and linoleic acid 242 isolated from poultry meat have also shown inhibition of the AI-2 activity up to 65% (Soni et al., 243 2008). A mixture of steric, palmitic, oleic, and linoleic acids inhibited the AI-2 activity by 59.5% 244 (Widmer et al., 2007). Although these results show significant AI-2 inhibition, they are not 11 245 comparable to the higher inhibition levels of up to 80% shown by the naturally produced organic 246 acids tested in this study. The overall significance of this study was the effect of organic acids in 247 reducing quorum sensing ability of pathogenic bacteria on minimally processed produce. 248 249 Conclusion 250 Foodborne illness due to E.coli O157:H7 and Salmonella outbreaks on minimally 251 processed produce are a consistent problem in the U.S. leading to economical and health 252 consequences. These bacterial pathogens show virulence and sustained survival due to quorum 253 sensing ability that provides a barrier against the effects of antimicrobials. For the first time, this 254 study demonstrated the ability of natural antimicrobials like organic acids to inhibit the 255 expression of autoinducer-2 molecule which thereby hinders the quorum sensing ability of 256 pathogenic bacteria including E.coli O157:H7 and Salmonella Typhimurium. Lactic acid alone at 257 4.0 %, and a combination treatment of malic acid 4.0% + lactic 4.0% were able to inhibit AI-2 258 activity by E.coli O157:H7 and Salmonella by 80% on spinach and cantaloupe respectively. 259 Therefore, food protection and safety in the minimally processed produce industry, dealing with 260 frozen or refrigerated vegetables and fruits, and salad mixes, can be enhanced using these natural 261 antimicrobials. 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Values provided are means ± standard deviation of triplicates and those conducted by the same letter are not significantly different (P<0.05). **Relative AI-2 like activity was calculated as the ratio of luminescence of the test sample to the negative control (AB medium). 1 2 3 4 5 6 11 1 12 13 14 15 16 17 18 19 20 Table 2. Relative AI-2 like in cantaloupe homogenate inoculated with stains of Salmonella for 12 h. Strain* SD 10 SD 11 Relative AI-2 like activity (%)** 53.2 ± 0.9A 21.3 ± 0.7B *Strains of Salmonella Typhimurium studied. Values provided are means ± standard deviation of triplicates and those connected by the same letter are not significantly different (P<0.05). **Relative AI-2 like activity was calculated as the ratio of luminescence by the test sample to the negative control (AB medium). 2 21 Table.3. Reduction in quorum sensing activity of E. coli ED 14 strain based on AI-2 activity 22 inhibition in spinach by organic acids 23 24 25 Treatments (%)* Inhibition %** LA (1.0) 26.2 ± 1.0E LA 2.0 33.1 ± 0.3CD LA 3.0 49.5 ± 1.5B LA 4.0 80.2 ± 2.3A MA 1.0 23.4 ± 0.4F MA 2.0 26.9 ± 0.2F MA 3.0 28.0 ± 0.4DE MA 4.0 37.5 ± 1.4C LA MA 1 17.2 ± 0.3E LA MA 4 25.4 ± 0.8E Values are means ± standard deviations of triplicates and those connected by the same letter are not significantly different (P<0.05). *LA: lactic acid, MA: Malic acid. **Calculated in comparison to positive control (relative activity of AI-2 by V. harveyi BB152) values. 3 26 27 28 29 30 Table 4. Reduction in quorum sensing activity of Salmonella Typhimurium SD 10 strain based on AI-2 activity inhibition in cantaloupe by organic acids Treatments (%)* Inhibition %** LA (1.0) 27.5 ± 0.8F LA 2.0 36.2 ± 0.6DE LA 3.0 58.0 ± 2.1B LA 4.0 76.6 ± 0.2A MA 1.0 16.1 ± 0.4G MA 2.0 26.3 ± 1.2F MA 3.0 28.8 ± 1.8DE MA 4.0 46.7 ± 1.4C LA MA 1 39.9 ± 1.7D LA MA 4 80.4 ± 2.9A Values provided are means ± standard deviation of triplicates and those connected by the same letter are not significantly different (P<0.05). *LA: lactic acid, MA: Malic acid. **Calculated in comparison to positive control (relative activity of AI-2 by V. harveyi BB152) value. 4 31 32 33 34 35 36 37 Table 5. Effect of organic acids on the growth of the reporter strain V. harveyi BB170 Treatment* AB medium LA 4 % MA 4% LA MA 4% Growth (log CFU/mL) 5.4 ± 0.1A 5.3 ± 0.2A 5.3 ± 0.0A 5.2 ± 0.2A Values provided are means ± standard deviation of triplicates and those connected by the same letter are not significantly different (P<0.05). *AB medium: Auto-inducer bioassay medium; LA: Lactic acid; MA: Malic acid. 5 Figure 1 2 3 4 5 6 7 8 9 10 Figure 1. Simplified scheme of the principle for detection of quorum sensing activity. 1