Antimicrobial-Loaded Polymeric Micelles Inhibit Enteric Bacterial Pathogens on Spinach Leaf Surfaces During Multiple Simulated Pathogen Contamination Events

Abstract

Protecting fresh-packed produce microbiological safety against pre- and post-harvest microbial pathogen contamination requires innovative antimicrobial strategies. Although largely ignored in the scientific literature, there exists the potential for gross failure in food safety protection of fresh fruits and vegetables leading to opportunity for multiple produce contamination events to occur during production and post-harvest handling of food crops. The primary objective of this research was to determine the efficacy of plant-derived antimicrobial-loaded nanoparticles to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium on spinach leaf surfaces whilst simulating multiple pathogen contamination events (pre-harvest and post-harvest). Spinach samples were inoculated with a blend of E. coli O157:H7 and S. Typhimurium, each diluted to ~8.0 log10 CFU/mL. The inoculated samples were then submerged in solutions containing nanoparticles loaded with geraniol (GPN; 0.5 wt.% geraniol), unencapsulated geraniol (UG; 0.5 wt.%), or 200 ppm chlorine (HOCl; pH 7.0), with untreated samples serving for controls. Following antimicrobial treatment application, samples were collected for surviving pathogen enumeration or were placed under refrigeration (5°C) for up to 10 days, with periodic enumeration of pathogen loads. After 3 days of refrigerated storage, all samples were removed, aseptically opened and subjected to a second inoculation with both pathogens. Treatment of spinach surfaces with encapsulated geraniol reduced both pathogens to non-detectable numbers within 7 days of refrigerated storage, even with a second contamination event occurring 3 days after experiment initiation. Similar results were observed with the UG treatment, except that upon recontamination at day 3, a higher pathogen load was detected on UG-treated spinach vs. GPN-treated spinach. These data fill a research gap by providing a novel tool to reduce enteric bacterial pathogens on spinach surfaces despite multiple contamination events, a potential food safety risk for minimally processed edible produce.

Encapsulated Plant-Derived Antimicrobial Reduces Enteric Bacterial Pathogens on Melon Surfaces during Differing Contamination and Sanitization Treatment Scenarios

“…GNP treatment achieved the greatest numerical reductions in the numbers of both organisms, though survivor numbers did not statistically differ from those obtained from HOCl samples (Figure 2). The reductions observed in the current study for GNP treatment were significantly less than those we recently reported on spinach [20]. A lack of more impressive reductions in microbial counts may be at least partially explained by the second inoculation/contamination event, where the numbers of the organisms were refreshed on sample surfaces.…”

“…Additionally, our group reported geraniol-loaded nanoparticles outperformed unencapsulated geraniol and 200 mg/L HOCl on spinach surfaces, reducing E. coli O157:H7 and S. Typhimurium to non-detectable numbers (<0.5 log 10 CFU/cm 2 ) when applied multiple days prior to pathogen application. Similar effects were observed when E. coli O157:H7or S. Typhimurium-inoculated spinach was sanitizer-treated and then re-inoculated 3 days later, simulating pathogen contamination of the food crop during pre-and post-harvest operations [20]. These findings data indicated encapsulated geraniol afforded longer-lasting food safety protection versus other conventional chemical sanitizing treatments, even under conditions of gross failures in good agricultural practices (GAPs).…”

“…Melon samples were inoculated with a mixture of S. enterica serovar Typhimurium ATCC 700720 and E. coli O157:H7 ATCC 700728, both naturally resistant to 100 µg/mL rifampicin. Stock cultures of these microorganisms were revived and prepared with methods previously described [20]. Each microorganism was extracted from frozen storage, passed twice in tryptic soy broth (TSB; Becton, Dickinson and Co., Sparks, MD, USA) incubating at 35 • C for 24 h, and then washed twice by centrifugation (15 min, 2191× g, 25 • C) and resuspended in 0.1% (w/v) peptone diluent (Becton, Dickinson and Co.) prior to mixing to prepare the inoculum.…”

“…Samples assigned to refrigerated storage were covered with an oxygen-permeable low-density polyethylene film, and stored at 5 ± 1 • C for either 3, 5, 7, or up to 10 days. For samples that were subjected to scenario 2, all samples were removed at day 3 of refrigerated storage and re-inoculated with a mixture of organisms prepared to a target of 7.0 log 10 CFU/mL per previous assessment of post-harvest contamination in commercial fruit and vegetable packinghouses [20,21]. Reinoculated samples were then either returned for further refrigerated storage or prepared for microbiological analysis.…”

Reduction of Bacterial Enteric Pathogens and Hygiene Indicator Bacteria on Tomato Skin Surfaces by a Polymeric Nanoparticle-Loaded Plant-Derived Antimicrobial

“…Tomato samples were inoculated by spot-inoculation of cocktailed E. coli O157:H7 and S. typhimurium cells using the same method previously reported [9,10]. Briefly, 0.1 mL total inoculum fluid was applied by evenly distributing ten 10.0 µL spots over the three combined tomato discs within a sample.…”