Cavitation Bubbles Remove and Inactivate Listeria and Salmonella on the Surface of Fresh Roma Tomatoes and Cantaloupes

Raw produce has frequently been identified as the source of bacterial pathogens that can cause human illnesses, including listeriosis and salmonellosis. Microbial pathogens may attach and form biofilms on raw fruit surfaces and can be difficult to remove. A cavitation process (injection of bubbles into water) was studied for its effectiveness for removal and inactivation of Listeria monocytogenes and Salmonella Newport from the surfaces of fresh Roma tomatoes and cantaloupes. Individual fruit were separately inoculated with each pathogen, then submerged in a water tank and treated with a bubble flow through an air stone using one airflow rate (0–14 liters/min.) for up to 60 s. As airflow increased, L. monocytogenes reduction on tomato and cantaloupe surfaces increased up to 1.2 and 0.8 log CFU/fruit greater than with water alone (no bubbles), respectively. With a 14 L/min flow rate, Salmonella reduction on tomato and cantaloupe surfaces increased up to 0.9 and 0.7 log CFU/fruit greater than when no bubbles applied, respectively. Also, with the bubble treatments, additional pathogen reduction (detached organisms) was observed in the tank water. Therefore, these bubble streams can be used to enhance the detachment of bacteria from fruit surfaces and to inactivate a proportion of these detached microorganisms. Additionally, recoveries of Salmonella from inoculated Roma tomatoes and cantaloupe were determined for treatment water that contained 50 or 150 ppm sodium hypochlorite. Combining both cavitating bubbles and 150 ppm chlorine in the tank water resulted in greater efficacy of removing or inactivating S. Newport from the surface of cantaloupe (2.9 log CFU) than with cavitation (2.5 log CFU) or chlorine (1.9 log CFU) alone. The physical force of a bubble stream on raw produce can effectively detach and inactivate surface bacteria, and has the potential to reduce antimicrobial chemical use and water use in post-harvest packing operations

Gamma Irradiation Influences the Survival and Regrowth of Antibiotic-Resistant Bacteria and Antibiotic-Resistance Genes on Romaine Lettuce

Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) occurs during production. Post-harvest interventions are emplaced to mitigate pathogens, but could also mitigate ARB and ARGs on vegetables. The objective of this research was to determine changes to lettuce phyllosphere microbiota, inoculated ARB, and the resistome (profile of ARGs) following washing with a sanitizer, gamma irradiation, and cold storage. To simulate potential sources of pre-harvest contamination, romaine lettuce leaves were inoculated with compost slurry containing antibiotic-resistant strains of pathogenic (Escherichia coli O157:H7) and representative of spoilage bacteria (Pseudomonas aeruginosa). Various combinations of washing with sodium hypochlorite (50 ppm free chlorine), packaging under modified atmosphere (98% nitrogen), irradiating (1.0 kGy) and storing at 4°C for 1 day versus 14 days were compared. Effects of post-harvest treatments on the resistome were profiled by shotgun metagenomic sequencing. Bacterial 16S rRNA gene amplicon sequencing was performed to determine changes to the phyllosphere microbiota. Survival and regrowth of inoculated ARB were evaluated by enumeration on selective media. Washing lettuce in water containing sanitizer was associated with reduced abundance of ARG classes that confer resistance to glycopeptides, β-lactams, phenicols, and sulfonamides (Wilcoxon, p < 0.05). Washing followed by irradiation resulted in a different resistome chiefly due to reductions in multidrug, triclosan, polymyxin, β-lactam, and quinolone ARG classes (Wilcoxon, p < 0.05). Irradiation followed by storage at 4°C for 14 days led to distinct changes to the β-diversity of the host bacteria of ARGs compared to 1 day after treatment (ANOSIM, R = 0.331; p = 0.003). Storage of washed and irradiated lettuce at 4°C for 14 days increased the relative abundance of Pseudomonadaceae and Carnobacteriaceae (Wilcoxon, p < 0.05), two groups whose presence correlated with detection of 10 ARG classes on the lettuce phyllosphere (p < 0.05). Irradiation resulted in a significant reduction (∼3.5 log CFU/g) of inoculated strains of E. coli O157:H7 and P. aeruginosa (ANOVA, p < 0.05). Results indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4°C are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and relative abundance of various ARG classes

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012–2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech’s Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate – TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields

Microbubbles Remove <i>Listeria monocytogenes</i> from the Surface of Stainless Steel, Cucumber, and Avocado

Fresh produce may be contaminated by bacterial pathogens, including Listeria monocytogenes, during harvesting, packaging, or transporting. A low-intensity cavitation process with air being injected into water was studied to determine the microbubbles’ efficiency when detaching L. monocytogenes from stainless steel and the surface of fresh cucumber and avocado. Stainless steel coupons (1″ × 2″), cucumber, and avocado surfaces were inoculated with L. monocytogenes (LCDC strain). After 1, 24 or 48 h, loosely attached cells were washed off, and inoculated areas were targeted by microbubbles (~0.1–0.5 mm dia.) through a bubble diffuser (1.0 L air/min) for 1, 2, 5, or 10 min. For steel, L. monocytogenes (48 h drying) detachment peaked at 2.95 mean log reduction after 10 min of microbubbles when compared to a no-bubble treatment. After 48 h pathogen drying, cucumbers treated for 10 min showed a 1.78 mean log reduction of L. monocytogenes. For avocados, L. monocytogenes (24 h drying) detachment peaked at 1.65 log reduction after 10 min of microbubbles. Microbubble applications may be an effective, economical, and environmentally friendly way to remove L. monocytogenes, and possibly other bacterial pathogens, from food contact surfaces and the surfaces of whole, intact fresh produce

Microbial quality of agricultural water in Central Florida

<div><p>The microbial quality of water that comes into the edible portion of produce is believed to directly relate to the safety of produce, and metrics describing indicator organisms are commonly used to ensure safety. The US FDA Produce Safety Rule (PSR) sets very specific microbiological water quality metrics for agricultural water that contacts the harvestable portion of produce. Validation of these metrics for agricultural water is essential for produce safety. Water samples (500 mL) from six agricultural ponds were collected during the 2012/2013 and 2013/2014 growing seasons (46 and 44 samples respectively, 540 from all ponds). Microbial indicator populations (total coliforms, generic <i>Escherichia coli</i>, and enterococci) were enumerated, environmental variables (temperature, pH, conductivity, redox potential, and turbidity) measured, and pathogen presence evaluated by PCR. <i>Salmonella</i> isolates were serotyped and analyzed by pulsed-field gel electrophoresis. Following rain events, coliforms increased up to 4.2 log MPN/100 mL. Populations of coliforms and enterococci ranged from 2 to 8 and 1 to 5 log MPN/100 mL, respectively. Microbial indicators did not correlate with environmental variables, except pH (<i>P</i><0.0001). The <i>invA</i> gene (<i>Salmonella</i>) was detected in 26/540 (4.8%) samples, in all ponds and growing seasons, and 14 serotypes detected. Six STEC genes were detected in samples: <i>hly</i> (83.3%), <i>fliC</i> (51.8%), <i>eaeA</i> (17.4%), <i>rfbE</i> (17.4%), <i>stx-</i>I (32.6%), <i>stx-</i>II (9.4%). While all ponds met the PSR requirements, at least one virulence gene from <i>Salmonella</i> (<i>invA-</i>4.8%) or STEC (<i>stx-</i>I-32.6%, <i>stx-</i>II-9.4%) was detected in each pond. Water quality for tested agricultural ponds, below recommended standards, did not guarantee the absence of pathogens. Investigating the relationships among physicochemical attributes, environmental factors, indicator microorganisms, and pathogen presence allows researchers to have a greater understanding of contamination risks from agricultural surface waters in the field.</p></div

Pearson product moment correlation coefficients (r) with <i>P</i>-values determined between each of the physical, chemical, and biological water attributes for all ponds combined.

<p>Pearson product moment correlation coefficients (r) with <i>P</i>-values determined between each of the physical, chemical, and biological water attributes for all ponds combined.</p

Most Probable Number of Total coliform (♦), Generic <i>E</i>. <i>coli</i> (■), and enterococci (▲) (Log MPN/100 mL) from six agricultural ponds (Pond 4: D, Pond 5: E, Pond 6: F) with daily rainfall (mm/Day) in Central Florida.

<p><i>invA</i> positive sampling days were shown with “*”.</p

Calculated MWQP values for all ponds for three consecutive growing seasons (MPN/100 mL).

<p>Calculated MWQP values for all ponds for three consecutive growing seasons (MPN/100 mL).</p

Most Probable Number of Total coliform (♦), Generic <i>E</i>. <i>coli</i> (■), and enterococci (▲) (Log MPN/100 mL) from six agricultural ponds (Pond 1: A, Pond 2: B, Pond 3: C) with daily rainfall (mm/Day) in Central Florida.

<p><i>invA</i> positive sampling days were shown with “*”.</p

Physical conditions of the ponds when sampling started in 2012.

<p>Physical conditions of the ponds when sampling started in 2012.</p