Antimicrobial activity of plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on fresh lettuce

Plant extracts have been found to be effective in reducing microorganisms. This study evaluated antimicrobial activity of 12 plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes by using a disk diffusion assay, and Syzygium aromaticum (clove) showed the highest inhibitory effect. To investigate the efficacy of clove extract that inactivates pathogens on lettuce, inoculated lettuce with S. Typhimurium, E. coli O157:H7, and L. monocytogenes was treated with diluted clove extracts or distilled water for 0, 1, 3, 5, and 10 min. Clove extract treatment significantly reduced populations of the 3 tested pathogens from the surface of lettuce. Practical Application: This result indicated that clove extract is a useful antimicrobial agent to reduce the microbial level of foodborne pathogens on fresh lettuce. It also might be a natural antimicrobial for reducing or replacing chemical sanitizers in food preservation.     

Use of organic acids to inactivate Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh apples and lettuce

Abstract: This study was undertaken to investigate the antimicrobial effect of organic acids against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on whole red organic apples and lettuce. Several studies have been conducted to evaluate organic acids as sanitizers. However, no studies have compared antimicrobial effects of various organic acids on organic fresh produce, including evaluation of color changes of produce. Apples and lettuce were inoculated with a cocktail of 3 strains each of 3 foodborne pathogens provided above and treated with 1% and 2% organic acids (propionic, acetic, lactic, malic, and citric acid) for 0, 0.5, 1, 5, and 10 min. With increasing treatment time and acid concentration, organic acid treatments showed significant reduction compared to the control treatment (distilled water), and differences in antimicrobial effects between organic acids were observed. After 10 min of treatment with 1% and 2% organic acids in apples, propionic (0.92 to 2.75 log reduction), acetic (0.52 to 2.78 log reduction), lactic (1.69 to >3.42 log reduction), malic (1.48 to >3.42 log reduction), and citric acid (1.52 to >3.42 log reduction) exhibited significant (P < 0.05) antibacterial effects against 3 foodborne pathogens compared to the control treatment. In lettuce, propionic (0.93 to 1.52 log reduction), acetic (1.13 to 1.74 log reduction), lactic (1.87 to 2.54 log reduction), malic (2.32 to 2.98 log reduction), and citric acid (1.85 to 2.86 log reduction) showed significant (P < 0.05) effects compared to the control treatment. Changes in sample color subjected to organic acids treatment were not significant during storage. Practical Application: It is suggested that organic acids have a potential as sanitizers for organic fresh produce. These data may help the organic produce industry provide safe fresh produce for consumers.     

Electrostatic spraying of food-grade organic and inorganic acids and plant extracts to decontaminate Escherichia coli O157:H7 on spinach and iceberg lettuce

The prevalence of foodborne illnesses is continually on rise. In the U.S.A., Escherichia coli O157:H7 (E. coli) has been associated with several outbreaks in minimally processed foods. Spinach and lettuce pose higher food safety risks and recurring food recalls suggest the insufficiency of current disinfection strategies. We aimed at offering a natural antimicrobial alternative using organic acids (malic, tartaric, and lactic acids [MA, TA, and LA, respectively]) and grape seed extract (GSE) and a novel application method using electrostatic spraying to evenly distribute the antimicrobials onto produce. Spinach and lettuce samples were washed, sanitized with sodium hypochlorite solution (6.25 mL/L), dip inoculated in water containing E. coli (7.0 log CFU/mL) for 24 h, and rewashed with sterile water to remove nonadhered pathogens. The samples were sprayed electrostatically with MA, LA, and GSE alone and in combinations and for comparison, with phosphoric acid (PA) and pH controls with deionized water adjusted to 1.5/2.3/3.6 and stored at 4 °C. When combined with LA (3%), MA (3%) showed 2.1 to 4.0 log CFU/g reduction of E. coli between the days 1 and 14 on spinach and 1.1 to 2.5 log CFU/g reduction on lettuce. Treatment with PA (1.5%) and PA (1.5%)-GSE (2%) exhibited 1.1 to 2.1 log CFU/g inhibition of E. coli on spinach during the 14-d storage. Our findings demonstrated the efficacy of electrostatic spraying of MA, LA, and GSE on fresh produce to improve the safety and lower the public health burden linked to produce contamination. PRACTICAL APPLICATION: Electrostatic spraying is an emerging technique that can be adopted to improve the distribution and application of antimicrobials during fresh produce sanitation. Relatively simple and quick, the process can access most/all parts of produce surface and offer protection from food pathogens. The use of malic and lactic acids with or without grape seed extract can serve as effective antimicrobials when sprayed electrostatically, lowering the risk from postcontamination issues with spinach and iceberg lettuce. This application technology can be extended to improve the commercial food safety of other produce, fruits, poultry, and meat.     

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium,and Listeria monocytogenes on Stored Iceberg Lettuce by Aqueous Chlorine Dioxide Treatment

ABSTRACT: Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes in iceberg lettuce by aqueous chlorine dioxide (ClO₂) treatment was evaluated. Iceberg lettuce samples were inoculated with approximately 7 log CFU/g of E. coli O157:H7, S. typhimurium, and L. monocytogenes. Iceberg lettuce samples were then treated with 0, 5, 10, or 50 ppm ClO₂ solution and stored at 4 °C. Aqueous ClO₂ treatment significantly decreased the populations of pathogenic bacteria on shredded lettuce (P < 0.05). In particular, 50 ppm ClO₂ treatment reduced E. coli O157:H7, S. typhimurium, and L. monocytogenes by 1.44, 1.95, and 1.20 log CFU/g, respectively. The D₁₀‐values of E. coli O157:H7, S. typhimurium, and L. monocytogenes in shredded lettuce were 11, 26, and 42 ppm, respectively. The effect of aqueous ClO₂ treatment on the growth of pathogenic bacteria during storage was evaluated, and a decrease in the population size of these pathogenic bacteria was observed. Additionally, aqueous ClO₂ treatment did not affect the color of lettuce during storage. These results suggest that aqueous ClO₂ treatment can be used to improve the microbial safety of shredded lettuce during storage.     

Activity of ε–Polylysine Against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes

ABSTRACT: ε–polylysine is a homopolymer of L-lysine, an essential amino acid, with a reportedly wide antimicrobial spectrum. This study evaluated the antimicrobial activity of ε–polylysine, as compared with known preservatives and organic acids, against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes , in culture broth. The compounds tested included ε–polylysine (0.0025% to 0.05%), sodium diacetate (0.25%), sodium lactate (3.0%), lactic acid (0.1%), and acetic acid (0.1%), alone, as well as in combination with ε– polylysine (0.0025% to 0.03%); all treatments were evaluated in tryptic soy broth supplemented with 0.6% yeast extract. Treatments were inoculated (approximately 2 log colony-forming units [CFU]/mL) with 5-strain ( E. coli O157:H7, S. Typhimurium) or 10-strain ( L. monocytogenes ) mixtures of the pathogens. Survival/growth of the inoculated bacteria was periodically monitored during incubation at 4 °C (30 d) and 24 °C (48 h). Bactericidal effects of ε–polylysine were obtained against E. coli O157:H7 and S. Typhimurium at 4 °C. At the same temperature (4 °C), ε–polylysine alone or in combination with other compounds tested inhibited growth or was bactericidal against L. monocytogenes. All 3 pathogens were inhibited by ε–polylysine at 24 °C; however, L. monocytogenes was the most sensitive and S. Typhimurium the most resistant. The antimicrobial activity of ε–polylysine against E. coli O157:H7 and S. Typhimurium was enhanced ( P < 0.05) when tested in combination with sodium diacetate or acetic acid. Combination treatments with sodium lactate resulted in loss of ε–polylysine activity by the end of the incubation period. Overall, under the conditions of this study, ε–polylysine exhibited antimicrobial effects against the 3 pathogens tested.     

Antimicrobial activity of epsilon-polylysine against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in various food extracts

ABSTRACT:  This study compared the antimicrobial effects of ɛ-polylysine (ɛ-PL) against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in 6 food extracts and in broth. The food extracts (10% (w/w) in distilled water) evaluated were fat-free and whole fat milk, beef, bologna, rice, and vegetables (50:50 ratio of broccoli and cauliflower). ɛ-PL was tested at 0.005% and 0.02% (w/v) against E. coli O157:H7 and L. monocytogenes , and 0.02% and 0.04% (w/v) against S. Typhimurium . The substrates were inoculated (5 log CFU/mL) and periodically analyzed for surviving populations during storage at 12 °C for 6 d. In general, all 3 pathogens reached 7 to 9 log CFU/mL within 2 d in control substrates (no ɛ-PL). Immediate bactericidal effects ( P < 0.05) following exposure to ɛ-PL were obtained in the rice (all pathogens) and vegetable ( E. coli O157:H7 and S. Typhimurium ) extracts. During storage, antimicrobial effects of ɛ-PL were more pronounced in the food extracts than in the broth medium. The greatest antimicrobial activity for all 3 pathogens was obtained in the rice and vegetable extracts, where counts were reduced ( P < 0.05) to below the detection limit (0.0 log CFU/mL) by one or both ɛ-PL concentrations tested. In the other food extracts (fat-free milk, whole fat milk, beef, and bologna), both ɛ-PL concentrations tested generally resulted in lower ( P < 0.05) pathogen levels at the end of storage compared to initial counts, with better bactericidal effects exerted by the higher of the 2 ɛ-PL concentrations. Additional research is needed to explore the potential antimicrobial effects of ɛ-PL in real food systems.     

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella in juice concentrates

The survival of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella was studied in apple, orange, pineapple, and white grape juice concentrates and banana puree. Pouches of juice concentrate or puree were inoculated with pathogens at a level > or = 10(3) CFU/g and stored at -23 degrees C (-10 degrees F). Pathogen survival was monitored at 6 and 24 h, once a week for four consecutive weeks, and biweekly thereafter until 12 weeks. When pathogens were not detectable by direct plating, samples were enriched in universal preenrichment broth for 72 h and plated on selective media. Results showed that E. coli O157:H7, L. monocytogenes, and Salmonella were recoverable from all five concentrates through 12 weeks of storage at -23 degrees C.     

Electrochemical disinfection: an efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter

The efficacy of an electrochemical treatment in water disinfection, using boron-doped diamond electrodes, was studied and its suitability for the fresh-cut produce industry analyzed. Tap water (TW), and tap water supplemented with NaCl (NaClW) containing different levels of organic matter (Chemical Oxygen Demand (COD) around 60, 300, 550 ± 50 and 750 ± 50 mg/L) obtained from lettuce, were inoculated with a cocktail of Escherichia coli O157:H7 at 10⁵ cfu/mL. Changes in levels of E. coli O157:H7, free, combined and total chlorine, pH, oxidation–reduction potential, COD and temperature were monitored during the treatments. In NaClW, free chlorine was produced more rapidly than in TW and, as a consequence, reductions of 5 log units of E. coli O157:H7 were achieved faster (0.17, 4, 15 and 24 min for water with 60, 300, 500 and 750 mg/L of COD, respectively) than in TW alone (0.9, 25, 60 min and 90 min for water with 60, 300, 600 and 800 mg/L of COD, respectively). Nonetheless, the equipment showed potential for water disinfection and organic matter reduction even without adding NaCl. Additionally, different mathematical models were assessed to account for microbial inactivation curves obtained from the electrochemical treatments.     

Effect of Combined Ozone and Organic Acid Treatment for Control of Escherichia coli O157:H7 and Listeria monocytogenes on Lettuce

ABSTRACT: This study was conducted to determine the effects of ozonated water (1, 3, and 5 ppm) alone with different exposure times (0.5,1,3, or5min), and combinations of 3 ppm ozone with 1% organic acids (acetic, citric, or lactic acids) during 1-min exposure for inactivating Escherichia coli O157:H7 and Listeria monocytogenes on lettuce and to observe the regrowth of these pathogenic bacteria on treated lettuce during storage for 10 d at 15°C. Results showed that 5 ppm ozone treatment for 5 min gave 1.09-log and 0.94-log reductions of E. coli O157:H7 and L. monocytogenes , respectively, indicating insignificant reductions compared with 3 ppm ozone treatment for 5 min. Treatment with 3 ppm ozone combined with 1 % citric acid for 1 min immersing resulted in 2.31 - and 1.84-log reductions ( P < 0.05), respectively. During storage at 15°C for 10 d after combined treatment and packaging, populations of E. coli O157:H7 and L. monocytogenes increased to approximately 9.0-log colony forming unit (CFU) /g, indicating that this treatment did not have a residual antimicrobial effect during storage. Although the storage study did not show control of these pathogens, the combined ozone-organic acid treatment was more effective in reducing population levels of these pathogens on lettuce than individual treatments.     

Inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in orange and tomato juice using ohmic heating

The effects of ohmic heating on reduction of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in orange and tomato juice were investigated. Orange and tomato juice inoculated with E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes were subjected to ohmic heating with selected parameters including electric field strength from 10 to 20 V/cm and treatment times from 0 to 540 s. The number of pathogens was reduced by increasing the electric field strength from 10 to 20 V/cm as well as increasing treatment time. The population of E. coli O157:H7 was reduced more than 5 log after 120, 210, and 540 s of treatment in orange juice with 20, 15, and 10 V/cm electric field strengths, respectively. In tomato juice, levels of E. coli O157:H7 were reduced more than 5 log after 90, 180, and 480 s with the same electric field strengths. Similar phenomena were observed for Salmonella Typhimurium and L. monocytogenes, but E. coli O157:H7 was the most resistant to ohmic heating treatment. These results show that ohmic heating is potentially useful for inactivation of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes and that the effect of inactivation depends on applied electric field strength, treatment time, pathogen species, and type of juice.     

A survey of iceberg lettuce for the presence of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Japan

No information has been available on the prevalence of pathogens in fresh produce in Japan. In the present study, information was collected on the occurrence of contamination by Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in iceberg lettuce in a Japanese retail store. A total of 419 samples of lettuce that had been harvested in different districts and/or by different producers from July 2008 to March 2009 were examined. A multiplex PCR method was used to simultaneously identify the three bacterial pathogens. No pathogenic bacteria, including Salmonella, E. coli O157:H7, and L. monocytogenes, were detected from any of the samples with this highly sensitive and validated procedure. The aerobic bacteria plate counts and coliform bacteria counts in lettuce throughout the examination period did not show any seasonal trends, and the numbers were comparable to those reported by others from around the world. Based on the results of this study, we concluded that none of the three major pathogens were present in this limited survey of iceberg lettuce sold by a retailer in Japan.     

The antimicrobial effect of thiamine dilauryl sulfate in tofu inoculated with Escherichia coli O157:H7, Salmonella Typhimurium,Listeria monocytogenes and Bacillus cereus

Food Science and Biotechnology - Thiamine dilauryl sulfate (TDS) is a food additive that is used as a bactericidal agent. This study examines the antimicrobial effect of TDS on tofu inoculated with...     

Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca sativa cv. Tamburo) grown in an inoculated hydroponic and soil system. The second aim was to quantify the level of contamination with the use of a proper surface sterilization method. Silver nitrate was superior in reducing the number of viable bacteria on leave surfaces compared to sodium hypochlorite and ethanol. With the hydroponic system internal colonization of lettuce only occurred at high densities with S. Typhimurium MAE 119. With the soil system E. coli O157:H7, S. Typhimurium 110 and S. Typhimurium 119 were found at considerable densities in sterilized leaf samples (respectively, 3.95, 2.57 and 2.37 log cfu/g on average) with prevalences of 0.29, 0.23 and 0.15, respectively. No statistical differences were observed between the Salmonella strains. A negative correlation was observed between shoot weight and leaf contamination. The observed presence of the pathogens in lettuce, after thorough surface sterilization, demonstrates the possible presence of human pathogens in locations were they are unlikely to be removed by the actions of consumer washing and therefore pose a serious threat when occurring in field situations.     

Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR

A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved polymerase chain reaction (PCR) detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially contaminated water containing E. coli O157:H7 and S. typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.     

Synergistic effect of steam and lactic acid against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms on polyvinyl chloride and stainless steel

This study was designed to investigate the individual and combined effects of steam and lactic acid (LA) on the inactivation of biofilms formed by Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on polyvinyl chloride (PVC) and stainless steel. Six day old biofilms were developed on PVC and stainless steel coupons by using a mixture of three strains each of three foodborne pathogens at 25°C. After biofilm development, PVC and stainless steel coupons were treated with LA alone (immersed in 0.5% or 2% for 5s, 15s, and 30s), steam alone (on both sides for 5, 10, and 20s), and the combination of steam and LA. The numbers of biofilm cells of the three foodborne pathogens were significantly (p<0.05) reduced as the amount of LA and duration of steam exposure increased. There was a synergistic effect of steam and LA on the viability of biofilm cells of the three pathogens. For all biofilm cells of the three foodborne pathogens, reduction levels of individual treatments ranged from 0.11 to 2.12 log CFU/coupon. The combination treatment of steam and LA achieved an additional 0.2 to 2.11 log reduction compared to the sum of individual treatments. After a combined treatment of immersion in 2% LA for 15s or 30s followed by exposure to steam for 20s, biofilm cells of the three pathogens were reduced to below the detection limit (1.48 log). From the results of this study, bacterial populations of biofilms on PVC coupons did not receive the same thermal effect as on stainless steel coupons. Effectiveness of steam and LA may be attributed to the difference between Gram-negative and Gram-positive characteristics of the bacteria studied. The results of this study suggest that the combination of steam and LA has potential as a biofilm control intervention for food processing facilities.     

Incidence of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcal enterotoxin in two types of Mexican fresh cheeses

Handcrafted fresh cheeses are popular among consumers in Mexico. However, unsafe raw materials and inadequate food safety practices during cheese manufacture and preservation make them a potential public health risk. The incidence of Salmonella, Listeria, Escherichia coli O157:H7, and staphylococcal enterotoxin was analyzed in two types of fresh cheese (panela and adobera) commonly marketed in Mexico. A total of 200 samples, 100 panela and 100 adobera, were acquired from 100 wholesale milk product distributors who supply small retailers in the Guadalajara metropolitan area, Jalisco State, Mexico. Pathogens were identified using culture and immunoassay (miniVidas) methods. The presence of staphylococcal enterotoxin was determined by an immunoassay method. Of the 200 analyzed samples, 92 were positive for at least one of the pathogens. The incidence in the panela samples was 56%: 34% Salmonella, 16% E. coli O157:H7, and 6% L. monocytogenes. In the adobera samples, incidence was 36%: 20% Salmonella, 4% E. coli O157:H7, and 12% L. monocytogenes. Staphylococcal enterotoxin was not detected in any of the 200 samples. Choice of technique had no effect on detection of pathogen incidence, although the immunoassay method identified more Salmonella serotypes than the culture method. Handcrafted panela and adobera fresh cheeses in Mexico frequently contain pathogenic bacteria and therefore pose a public health risk.     

Electrostatic spraying of organic acids on biofilms formed by E. coli O157:H7 and Salmonella Typhimurium on fresh produce

Electrostatic spraying which has an even and retained surface coverage could be an effective novel technique to completely cover the surface of fresh produce to disrupt biofilm formation by pathogenic bacteria. Spinach leaves and cantaloupe rind were spot-inoculated with a bacterial culture and stored at 8 °C for 72 h to allow biofilm formation. Among various green fluorescent protein-labeled strains, ED 14 strain of E. coli O157:H7 and SD 10 strain of Salmonella Typhimurium had the best attachment based on colony counts. The produce samples were electrostatically sprayed with malic (MA) and lactic (LA) acid solutions alone (1.0/2.0/3.0/4.0% w/v) or in combination (0.5 + 0.5/1.0 + 1.0/1.5 + 1.5/2.0 + 2.0% w/v) to test for a reduction in the attached bacteria. A combined treatment of LA 2.0% w/v + MA 2.0% w/v had the highest log reduction (CFU/disk) of 4.14 and 3.6 on the attached E. coli strain ED 14 (spinach) and Salmonella strain SD 10 (cantaloupe), respectively. Crystal violet assay demonstrated the disruptive effect of organic acids on biofilms formed by the pathogenic bacteria. Application of electrostatic spray with a combination of malic and lactic acids resulting in a log reduction (CFU/disk) of 3.6 or higher can improve the microbial safety of spinach and cantaloupe by preventing the pathogenic biofilm formation and bacterial growth.