Antimicrobial activity of oregano oil against antibiotic-resistant Salmonella enterica on organic leafy greens at varying exposure times and storage temperatures

The objective of this study was to evaluate the effectiveness of oregano oil on four organic leafy greens (Iceberg and Romaine lettuces and mature and baby spinaches) inoculated with Salmonella Newport as a function of treatment exposure times as well as storage temperatures. Leaf samples were washed, dip inoculated with S. Newport (6-log CFU/ml) and dried. Oregano oil was prepared at 0.1, 0.3, and 0.5% concentrations in sterile phosphate buffered saline (PBS). Inoculated leaves were immersed in the treatment solution for 1 or 2 min, and individually incubated at 4 or 8 °C. Samples were taken at day 0, 1, and 3 for enumeration of survivors. The results showed that oregano oil was effective against S. Newport at all concentrations. S. Newport showed reductions from the PBS control of 0.7–4.8 log CFU/g (Romaine lettuce), 0.8–4.8 log CFU/g (Iceberg lettuce), 0.8–4.9 log CFU/g (mature spinach), and 0.5–4.7 log CFU/g (baby spinach), respectively. The antibacterial activity also increased with exposure time. Leaf samples treated for 2 min generally showed greater reductions (by 1.4–3.2 log CFU/g), than those samples treated for 1 min; however, there was minimal difference in antimicrobial activity among samples stored under refrigeration and abuse temperatures. This study demonstrates the potential of oregano oil to inactivate S. Newport on organic leafy greens.     

Antimicrobial activity of different copper alloy surfaces against copper resistant and sensitive Salmonella enterica

Copper has shown antibacterial effects against foodborne pathogens. The objective of this study was to evaluate the antibacterial activity of copper surfaces on copper resistant and sensitive strains of Salmonella enterica. Six different copper alloy coupons (60–99.9% copper) were tested along with stainless steel as the control. The coupons were surface inoculated with either S. Enteritidis or one of the 3 copper resistant strains, S. Typhimurium S9, S19 and S20; stored under various incubation conditions at room temperature; and sampled at various times up to 2 h. The results showed that under dry incubation conditions, Salmonella only survived 10–15 min on high copper content alloys. Salmonella on low copper content alloys showed 3–4 log reductions. Under moist incubation conditions, no survivors were detected after 30 min–2 h on high copper content alloys, while the cell counts decreased 2–4 logs on low copper content coupons. Although the copper resistant strains survived better than S. Enteritidis, they were either completely inactivated or survival was decreased. Copper coupons showed better antimicrobial efficacy in the absence of organic compounds. These results clearly show the antibacterial effects of copper and its potential as an alternative to stainless steel for selected food contact surfaces.     

Antimicrobial activity of olive oil, vinegar, and various beverages against foodborne pathogens

The survival of foodborne pathogens in aqueous extracts of olive oil, virgin olive oil, vinegar, and several beverages was evaluated. Vinegar and aqueous extracts of virgin olive oil showed the strongest bactericidal activity against all strains tested. Red and white wines also killed most strains after 5 min of contact, black and green tea extracts showed weak antimicrobial activity under these conditions, and no effect was observed for the remaining beverages (fruit juices, Coca-Cola, dairy products, coffee, and beer). The phenolic compound content of the aqueous olive oil and virgin olive oil extracts could explain their antibacterial activity, which was also confirmed in mayonnaises and salads used as food models. Virgin olive oil in mayonnaises and salads reduced the counts of inoculated Salmonella Enteritidis and Listeria monocytogenes by approximately 3 log CFU/g. Therefore, olive oil could be a hurdle component in certain processed foods and exert a protective effect against foodborne pathogens when contaminated foods are ingested.     

Inactivation of Escherichia coli O157:H7, Salmonella enterica serotype enteritidis,and Listeria monocytogenes on lettuce by hydrogen peroxide and lactic acid and by hydrogen peroxide with mild heat

Iceberg lettuce is a major component in vegetable salad and has been associated with many outbreaks of foodborne illnesses. In this study, several combinations of lactic acid and hydrogen peroxide were tested to obtain effective antibacterial activity without adverse effects on sensory characteristics. A five-strain mixture of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis, and Listeria monocytogenes was inoculated separately onto fresh-cut lettuce leaves, which were later treated with 1.5% lactic acid plus 1.5% hydrogen peroxide (H2O2) at 40 degrees C for 15 min, 1.5% lactic acid plus 2% H2O2 at 22 degrees C for 5 min, and 2% H2O2 at 50 degrees C for 60 or 90 s. Control lettuce leaves were treated with deionized water under the same conditions. A 4-log reduction was obtained for lettuce treated with the combinations of lactic acid and H2O2 for E. coli O157:H7 and Salmonella Enteritidis, and a 3-log reduction was obtained for L. monocytogenes. However, the sensory characteristics of lettuce were compromised by these treatments. The treatment of lettuce leaves with 2% H2O2 at 50 degrees C was effective not only in reducing pathogenic bacteria but also in maintaining good sensory quality for up to 15 days. A < or = 4-log reduction of E. coli O157:H7 and Salmonella Enteritidis was achieved with the 2% H2O2 treatment, whereas a 3-log reduction of L. monocytogenes was obtained. There was no significant difference (P > 0.05) between pathogen population reductions obtained with 2% H2O2 with 60- and 90-s exposure times. Hydrogen peroxide residue was undetectable (the minimum level of sensitivity was 2 ppm) on lettuce surfaces after the treated lettuce was rinsed with cold water and centrifuged with a salad spinner. Hence, the treatment of lettuce with 2% H2O2 at 50 degrees C for 60 s is effective in initially reducing substantial populations of foodborne pathogens and maintaining high product quality.     

过氧化氢处理中鼠伤寒沙门氏菌VBNC态形成及其机制解析

为确定过氧化氢的有效杀菌浓度,以鼠伤寒沙门氏菌为目标微生物,借助分子生物学手段,探究过氧化氢对自来水中鼠伤寒沙门氏菌活性和可培养性的影响及其活的非可培养状态(viable but non-culturable,VBNC)的形成原因。结果表明:较低过氧化氢浓度(0～15 mmol/lL)处理可达到部分杀菌的效果,1 mol/L以上过氧化氢可完全灭菌。而15 mmol/L过氧化氢处理导致4.08 lg(CFU/mL)鼠伤寒沙门氏菌全部进入VBNC状态。该状态的沙门氏菌形态和结构改变,过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶几乎完全被钝化,处理中产生的自由基强度在0～30 min内与VBNC发生系数呈正相关。     

Antimicrobial efficacy of gaseous chlorine dioxide against Salmonella enterica Typhimurium on grape tomato (Lycopersicon esculentum)

The aim of this study was to investigate the effects of gas level and treatment condition on antimicrobial efficacy of ClO₂ gas against Salmonella enterica Typhimurium on grape tomato (Lycopersicon esculentum). Grape tomatoes were dip‐inoculated with inoculum of S. enterica Typhimurium TISTR 292 (ATCC 13311) (9.79 ± 0.39 log CFU mL^?1) and allowed to dry before treatments. The pre‐inoculated samples were exposed to 0.15–0.85 mg of ClO₂ gas, for up to 58 min. The treatments at 4 and 25 °C resulted in population reductions of S. enterica Typhimurium of up to 3.95 ± 0.22 and 7.37 ± 0.39 log CFU per fruit, respectively. The population reduction results were used to construct statistical models to predict efficacy of ClO₂ gas. The second‐order equations for treatments at 4 and 25 °C had R² of 0.87 and 0.95, respectively, and indicated that efficacy of ClO₂ gas was significantly influenced by ClO₂ gas level, exposure time and treatment temperature.     

Occurrence,Characterization, and Antimicrobial Susceptibility of Salmonella enterica in Slaughtered Pigs in Sardinia

The aim of this study was to determine Salmonella occurrence in slaughtered finishing pigs and piglets and in slaughterhouse environment in order to characterize the isolates with phenotypical (antimicrobial testing) and molecular (PFGE, MLVA) methods. Nine slaughterhouses located in Sardinia were visited. Six hundred and eight samples collected from 106 pigs and 108 environmental samples were collected and analyzed. Salmonella was isolated in 65 of 504 (12.9%) samples from finishing pigs, with an occurrence of 15.1% in colon content, 12.7% in lymph nodes and liver, and 11.1% in carcass surface samples. Salmonella was never detected in piglets. The combined results of serotyping and PFGE showed a possible self‐contamination in 71.5% of Salmonella positive carcasses of lymph nodes and/or colon content carriers, pointing out the role of healthy pigs for carcass contamination. A significantly higher (P < 0.05) occurrence was detected in finishing pigs of EC countries origin (23%) than in pigs of local farms (8%). Salmonella was also detected in 3.7% of environmental samples. The most prevalent serovar was S. Anatum, followed by S. Rissen, S. Derby, and monophasic S. Typhimurium. Resistance to at least 3 antimicrobial was observed in 97.1% of strains and 7 different patterns of multiple resistance were identified. The most common resistance was detected against sulphonamide compounds. A strict slaughterhouse application of hygiene standards is essential to control the risk of Salmonella contamination.     

Reductions of Salmonella enterica on chicken breast by thymol, acetic acid, sodium dodecyl sulfate or hydrogen peroxide combinations as compared to chlorine wash

Poultry products are important vehicles for Salmonella transmission to humans and have been incriminated in several Salmonella outbreaks. Thymol (THY) from thyme oil has wide inhibitory effects against foodborne pathogens including Salmonella, and has shown great potential as a natural alternative to chlorine. In order to improve the cost-effectiveness of thymol-based washing solutions, formulas of THY with combination of organic acid or surfactant were developed and their efficacies to reduce Salmonella on chicken breast were investigated in the current study. Surface-inoculated chicken breasts were washed with the two thymol-based washing solutions: 0.2 mg/mL THY+5% (w/v) sodium dodecyl sulfate (SDS)+2 mg/mL acetic acid (AA) or 0.2 mg/mL THY+2 mg/mL AA for 2 min. Both solutions achieved around 2.2 log reductions of Salmonella on chicken breast and their efficacy was comparable to log reduction obtained by 200 ppm chlorine washing. Addition of SDS did not result in more log reduction of Salmonella on chicken meat samples. More than 3.3 log reduction in the used THY washing solutions was determined and it was similar to log reduction from the spent chlorine solution. None of these antimicrobial agents changed the pH and texture values of chicken breasts. Therefore, 0.2 mg/mL THY+2 mg/mL AA has great potential to be a natural alternative to chlorine-based washing solution for reducing Salmonella contamination on chicken breast meat.     

Antimicrobial activity of chitosan‐based films against Salmonella typhimurium and Staphylococcus aureus

The antibacterial effect of polyamide 6/66‐chitosan blend and chitosan‐coated plastic films was compared to chitosan films and chitosan solution, against Salmonella typhimurium and Staphylococcus aureus. The chitosan films did not show inhibition halos; however, contact inhibition was observed. During the tests, the films absorbed moisture, increasing of the diameter discs in relation to the concentration of the chitosan for both microorganisms at least 31%. Chitosan plastic film coating (bilayer system) showed only contact inhibition, without increase in contact area. The films from polyamide 6/66‐chitosan blend had no antibacterial activity. The highest inhibitory effect resulted from a chitosan solution at 1000 and 2000 ppm for S. typhimurium and S. aureus, respectively. The results of this study showed that the antimicrobial activity was lessened when the chitosan was combined with the plastic matrix because of the absence of contact inhibition, and the best activity was obtained with films prepared by casting of chitosan solution.     

Effect of acidified sodium chlorite, chlorine, and acidic electrolyzed water on Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes inoculated onto leafy greens

Recent foodborne outbreaks implicating spinach and lettuce have increased consumer concerns regarding the safety of fresh produce. While the most common commercial antimicrobial intervention for fresh produce is wash water containing 50 to 200 ppm chlorine, this study compares the effectiveness of acidified sodium chlorite, chlorine, and acidic electrolyzed water for inactivating Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes inoculated onto leafy greens. Fresh mixed greens were left uninoculated or inoculated with approximately 6 log CFU/g of E. coli O157:H7, Salmonella, and L. monocytogenes and treated by immersion for 60 or 90 s in different wash solutions (1:150, wt/vol), including 50 ppm of chlorine solution acidified to pH 6.5, acidic electrolyzed water (pH 2.1 +/- 0.2, oxygen reduction potential of 1,100 mV, 30 to 35 ppm of free chlorine), and acidified sodium chlorite (1,200 ppm, pH 2.5). Samples were neutralized and homogenized. Bacterial survival was determined by standard spread plating on selective media. Each test case (organism x treatment x time) was replicated twice with five samples per replicate. There was no difference (P > or = 0.05) in the time of immersion on the antimicrobial effectiveness of the treatments. Furthermore, there was no difference (P > or = 0.05) in survival of the three organisms regardless of treatment or time. Acidified sodium chlorite, resulted in reductions in populations of 3 to 3.8 log CFU/g and was more effective than chlorinated water (2.1 to 2.8 log CFU/g reduction). These results provide the produce industry with important information to assist in selection of effective antimicrobial strategies.     

Enhanced bactericidal effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against Salmonella enterica in apple juice

The effect of the broad spectrum cyclic antimicrobial peptide enterocin AS-48 combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode) was tested on Salmonella enterica CECT 915 in apple juice. A response surface methodology was applied to study the bactericidal effects of the combined treatment. The process variables were AS-48 concentration, temperature, and HIPEF treatment time. While treatment with enterocin AS-48 alone up to 60 microg/ml had no effect on the viability of S. enterica in apple juice, an increased bactericidal activity was observed in combination with HIPEF treatments. Survival fraction was affected by treatment time, enterocin AS48 concentration and treatment temperature. The combination of 100 micros of HIPEF treatment, 30 microg/ml of AS-48, and temperature of 20 degrees C resulted in the lowest inactivation, with only a 1.2-log reduction. The maximum inactivation of 4.5-log cycles was achieved with HIPEF treatment for 1000 micros in combination with 60 microg/ml of AS-48 and a treatment temperature of 40 degrees C. Synergism between enterocin AS-48 and HIPEF treatment depended on the sequence order application, since it was observed only when HIPEF was applied in the presence of previously-added bacteriocin. The combined treatment could improve the safety of freshly-made apple juice against S. enterica transmission.     

Antimicrobial susceptibility of Salmonella from organic and conventional dairy farms

The objective of this study was to compare antimicrobial susceptibility of Salmonella isolated from conventional and organic dairy farms in the Midwest and Northeast United States. Environmental and fecal samples were collected from organic (n = 26) and conventional (n = 69) farms in Michigan, Minnesota, New York, and Wisconsin every 2 mo from August 2000 to October 2001. Salmonella isolates (n = 1,243) were tested using a broth microdilution method for susceptibility to amoxicillin-clavulanic acid, ampicillin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Herd-level logistic regression and logistic proportional hazards multivariable models were used to examine the association between farm management type and susceptibility to antimicrobial agents. For most antimicrobial agents tested, susceptibility of Salmonella isolates was similar on organic and conventional herds when controlling for herd size and state. Conventional farms were more likely to have at least one Salmonella isolate resistant to streptomycin using logistic regression (odds ratio = 7.5; 95% confidence interval = 1.7-5.4). Conventional farms were more likely to have Salmonella isolates with greater resistance to streptomycin (odds ratio = 5.4; 95% confidence interval = 1.5-19.0) and sulfamethoxazole (odds ratio = 4.2; 95% confidence interval = 1.2-14.1) using logistic proportional hazards models. Although not statistically significant, conventional farms tended to be more likely to have at least one Salmonella isolate resistant to 5 or more antimicrobial agents when compared with organic farms.     

Edible films containing carvacrol and cinnamaldehyde inactivate Escherichia coli O157:H7 on organic leafy greens in sealed plastic bags

The antimicrobial effects of apple-, carrot-, and hibiscus-based edible films containing carvacrol and cinnamaldehyde against Escherichia coli O157:H7 on organic leafy greens in sealed plastic bags were investigated. Fresh-cut Romaine and Iceberg lettuce, and mature and baby spinach leaves were inoculated with E. coli O157:H7 and placed into Ziploc® bags. Edible films were then added to the bags, which were stored at 4°C. The evaluation of samples taken at days 0, 3, and 7 showed that on all leafy greens, 3% carvacrol-containing films had the greatest effect against E. coli O157:H7, reducing the bacterial population by about 5 log CFU/g on day 0. All three types of 3% carvacrol-containing films reduced E. coli O157:H7 by about 5 log CFU/g at day 0. The 1.5% carvacrol-containing films reduced E. coli O157:H7 by 1–4 logs CFU/g at day 7. Films with 3% cinnamaldehyde showed reduction of 0.6–3 logs CFU/g on different leafy greens.     

Antibacterial activity and mechanism of Litsea cubeba essential oil against food contamination by Escherichia coli and Salmonella enterica

Litsea cubeba essential oil (LC-EO) has been reported as antibacterial agents, but there are few studies about its possible antibacterial mechanism. The antibacterial activities and the underlying mechanisms of LC-EO against Escherichia coli O157: H7 and Salmonella enterica were investigated. The results showed that the LC-EO was more effective against gram-negative bacteria. The inhibition zone for E. coli O157: H7 and S. enterica were 3.1 ± 0.8 and 4.5 ± 0.6 mm, respectively. The minimum inhibitory concentration of LC-EO against both bacteria was 0.9 μg/ml, while the minimum bactericidal concentrations were 4.5 and 9 μg/ml. Gas chromatography–mass spectrometry analysis confirmed that citral (86.8%) was the main component of LC-EO. The results of a time–kill analysis illustrated that treatment with LC-EO led to a rapid decrease in viable bacterial cell number. The release of electrolytes and nucleic acids from the bacterial cells increased with the dose of LC-EO. Propidium iodide uptake revealed that LC-EO caused cell membrane damage. Scanning electron and transmission electron microscopy showed that LC-EO caused damage to the cell walls and membranes, resulting in cell deformation, atrophy, and large central voids. Thus, LC-EO may provide the basis for the development of new natural food preservatives.     

Antimicrobial activity of lauric arginate-coated polylactic acid films against Listeria monocytogenes and Salmonella typhimurium on cooked sliced ham

A novel type of environmentally friendly packaging with antibacterial activity was developed from lauric arginate (LAE)-coating of polylactic acid (PLA) films after surface activation using a corona discharge. Scanning electron microscopy (SEM)-based analysis of the LAE/PLA films confirmed the successful coating of LAE on the PLA surface. The mechanical properties of the LAE/PLA films with different levels of LAE-coating (0% to 2.6%[w/w]) were essentially the same as those of the neat PLA film. The antibacterial activity of the LAE/PLA films against Listeria monocytogenes and Salmonella enterica Serovar Typhimurium (S. Typhimurium) was confirmed by a qualitative modified agar diffusion assay and quantitative JIS Z 2801:2000 method. Using the LAE/PLA film as a food-contact antimicrobial packaging for cooked cured ham, as a model system, suggested a potential application to inhibit L. monocytogenes and S. Typhimurium on ham with a 0.07% (w/w) LAE coating on the PLA when high transparency is required, as evidenced from the 2 to 3 log CFU/tested film lower pathogen growth after 7 d storage but even greater antibacterial activity is obtained with a LAE coating level of 2.6% (w/w) but at the cost of a reduced transparency of the finished product. This article shows how we can simply develop functional green packaging of PLA for food with effective and efficient antimicrobial activity by use of LAE coating on the surface via corona discharge. PRACTICAL APPLICATION: The effectiveness of an innovative antimicrobial LAE-coated PLA film against foodborne pathogens was demonstrated. Importantly, the application of the LAE to form the LAE-coated PLA film can be customized within current film manufacturing lines.     

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.     

Survival, elongation, and elevated tolerance of Salmonella enterica serovar Enteritidis at reduced water activity

Growing microorganisms on dry surfaces, which results in exposure to low water activity (a(w)), may change their normal morphology and physiological activity. In this study, the morphological changes and cell viability of Salmonella enterica serovar Enteritidis challenged to low a(w) were analyzed. The results indicated that exposure to reduced a(w) induced filamentation of the cells. The amount of filamentous cells at a(w) 0.94 was up to 90% of the total number of cells. Surviving filamentous cells maintained their membrane integrity after exposure to low a(w) for 21 days. Furthermore, cells prechallenged to low a(w), obtained with an ionic humectant, demonstrated higher resistance to sodium hypochlorite than control cells. These resistant cells are able to survive disinfection more efficiently and can therefore cause contamination of foods coming in contact with surfaces. This points to the need for increased attention to cleaning of surfaces in household environments and disinfection procedures in processing plants.     

Survival and distribution of Escherichia coli on diverse fresh-cut baby leafy greens under preharvest through postharvest conditions

Escherichia coli O157:H7 has been associated in multiple outbreaks linked to the consumption of whole produce and fresh-cut leafy vegetables. However, plant-based foods had not been traditionally recognized as a host for enteric pathogens until the elevated incidence of produce-related outbreaks became apparent. The survival dynamics of two cocktails of generic E. coli (environmental water, plant and soil isolates) and E. coli O157:H7 within the phyllosphere of Mizuna, Red Chard and Tatsoi during their production, harvest, minimal processing, packaging and storage over two greenhouse production cycles were studied. Genotyping of applied generic E. coli strains to evaluate their comparative survival and relative abundance in the phyllosphere by REP-PCR is also reported. The Mizuna, Red Chard and Tatsoi shoots were grown under standard greenhouse conditions and fertility management. Both E. coli cocktails were spray-inoculated separately and determined to result in an initial mean population density of log 4.2 CFU/cm². Leaves were harvested as mini-greens approximating commercial maturity, minimally processed in a model washing system treated with 3 mg/L of ClO₂ and stored for 7 days at 5 °C. Rapid decline of generic E. coli and E. coli O157:H7 populations was observed for all plant types regardless of the leaf age at the time of inoculation and the irrigation type across both seasonal growth cycle trials. The decline rate of the surviving populations for the fall season was slower than for the summer season. The minimal processing with 3 mg/L of ClO₂ was not sufficient to fully disinfect the inoculated leaves prior to packaging and refrigerated storage. Viable populations of E. coli and E. coli O157:H7 were confirmed throughout storage, including the final time point at the end of acceptable visual leaf quality. In this study, the ability of low populations of E. coli to survive during production and postharvest operations in selected mini-greens has been demonstrated. However, further field-based trials are needed to expand understanding of the post-contamination fate of enteric bacterial pathogens on leafy vegetables. In summary, this research work provides baseline data upon which to develop food safety preventive control guidance during the production and minimal processing of these crops.