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

Irradiation Sensitivity of Planktonic and Biofilm-Associated Escherichia coli O157:H7 Isolates Is Influenced by Culture Conditions▿

Ionizing radiation effectively inactivates Escherichia coli O157:H7, but the efficacy of the process against biofilm cells versus that against free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm cells was determined for three isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894). Biofilms were formed on sterile glass slides incubated at 37°C for either 24 h, 48 h, or 72 h. The biofilm and planktonic cultures were gamma irradiated at doses ranging from 0.0 (control) to 1.5 kGy. The dose of radiation value required to reduce the population by 90% (D10) was calculated for each isolate, culture, and maturity based on viable populations at each radiation dose. For each of the times sampled, the D10 values of isolate 43894 planktonic cells (0.454 to 0.479 kGy) were significantly (P < 0.05) higher than those observed for biofilm cells (0.381 to 0.385 kGy), indicating a significantly increased sensitivity to irradiation for cells in the biofilm habitat. At the 24-h sampling time, isolate C9490 showed a similar pattern, in which the D10 values of planktonic cells (0.653 kGy) were significantly higher than those for biofilm cells (0.479 kGy), while isolate 35150 showed the reverse, with D10 values of planktonic cells (0.396 kGy) significantly lower than those for biofilm cells (0.526 kGy). At the 48-h and 72-h sampling times, there were no differences in radiation sensitivities based on biofilm habitat for C9490 or 35150. Biofilm-associated cells, therefore, show a response to irradiation which can differ from that of planktonic counterparts, depending on the isolate and the culture maturity. Culture maturity had a more significant influence on the irradiation efficacy of planktonic cells but not on biofilm-associated cells of E. coli O157:H7

Pasteurization of Foods with Ultrasound: The Present and the Future

In the last two decades, much research has been carried out using ultrasound as an alternative for pasteurization. Cavitation, the main effect of ultrasound, can disrupt and perforate cell membranes, generate free radicals, and produce sonoluminescence. Ultrasound in combination with additional hurdles such as temperature, pressure, or antimicrobials can achieve a 5-log reduction. Pathogens, spoilage microorganisms, yeast, and molds have been successfully inactivated by this novel technology. Currently, ultrasound is investigated as an option to reduce the content of aflatoxins during pasteurization. Ultrasound can inactivate those enzymes related to the stability of pasteurized food products, extending the shelf-life of the products. New uses of sonication are surging; for example, ultrasound has been studied as an option for pasteurizing plant-based foods. An important area of research is ultrasound’s effect on food’s bioactive compounds. Results exhibit an increase in the concentration of phenolics, carotenoids, anthocyanins, and other nutrients after the use of ultrasound because of an extractive effect. Finally, an area of concern in the early ages of ultrasound has been studied, food quality. In most cases, sonicated products have similar quality parameters to raw products. Lastly, there are some areas of opportunity in ultrasound’s future, such as the equipment improvement, regulation, and toxicology of sonicated products

Pasteurization of Foods with Ultrasound: The Present and the Future

In the last two decades, much research has been carried out using ultrasound as an alternative for pasteurization. Cavitation, the main effect of ultrasound, can disrupt and perforate cell membranes, generate free radicals, and produce sonoluminescence. Ultrasound in combination with additional hurdles such as temperature, pressure, or antimicrobials can achieve a 5-log reduction. Pathogens, spoilage microorganisms, yeast, and molds have been successfully inactivated by this novel technology. Currently, ultrasound is investigated as an option to reduce the content of aflatoxins during pasteurization. Ultrasound can inactivate those enzymes related to the stability of pasteurized food products, extending the shelf-life of the products. New uses of sonication are surging; for example, ultrasound has been studied as an option for pasteurizing plant-based foods. An important area of research is ultrasound&rsquo;s effect on food&rsquo;s bioactive compounds. Results exhibit an increase in the concentration of phenolics, carotenoids, anthocyanins, and other nutrients after the use of ultrasound because of an extractive effect. Finally, an area of concern in the early ages of ultrasound has been studied, food quality. In most cases, sonicated products have similar quality parameters to raw products. Lastly, there are some areas of opportunity in ultrasound&rsquo;s future, such as the equipment improvement, regulation, and toxicology of sonicated products

Sensitivity of Planktonic and Biofilm-Associated Salmonella spp. to Ionizing Radiation

Salmonella enterica forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation has been used to inactivate Salmonella on a variety of foods and contact surfaces, but the relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm-associated cells was determined for three food-borne-illness-associated isolates of Salmonella. Biofilms were formed on sterile glass slides in a coincubation apparatus, using inoculated tryptic soy broth, incubated at 37°C for 48 h. Resulting biofilms were 18 to 24 μm in height as determined by confocal scanning laser microscopy. The planktonic and biofilm cultures were gamma irradiated to doses of 0.0 (control), 0.5, 1.0, 1.5, 2.0 and 2.5 kGy. The D(10) value (the dose of radiation required to reduce a population by 1 log(10), or 90%) was calculated for each isolate-culture based on surviving populations at each radiation dose. The D(10) values of S. enterica serovar Anatum were not significantly (P < 0.05) different for biofilm-associated (0.645 kGy) and planktonic (0.677 kGy) cells. In contrast, the biofilm-associated cells of S. enterica serovar Stanley were significantly more sensitive to ionizing radiation than the respective planktonic cells, with D(10) values of 0.531 and 0.591 kGy, respectively. D(10) values of S. enterica serovar Enteritidis were similarly reduced for biofilm-associated (0.436 kGy) versus planktonic (0.535 kGy) cells. The antimicrobial efficacy of ionizing radiation is therefore preserved or enhanced in treatment of biofilm-associated bacteria

Transfer of generic Escherichia coli and attenuated Salmonella enterica Typhimurium from the soil to the surface of in-shell pecans during harvest

During harvest pecan nuts are at risk of contamination with foodborne pathogens from extended contact with the ground. The objective of this study was to determine the potential transfer of Escherichia coli and Salmonella from the ground to in-shell pecans during the harvesting process. Plots (2 m2) were sprayed with 1 L of a rifampicin (rif) resistant strain of either E. coli TVS 353 or an attenuated Salmonella Typhimurium inoculum at a low (∼4 log CFU/ml), mid (∼6 log CFU/ml) or high (∼8 log CFU/ml) concentrations. The following day, nuts were mechanically harvested and samples from each plot were collected at 1 min, 4 h, and 24 h. Samples were enumerated for Salmonella and E. coli on tryptic soy agar supplemented with rif. The Salmonella levels in the soil from the inoculated plots were 2.0 ± 0.3, 4.1 ± 0.1, and 6.4 ± 0.2 log CFU/g for the low, mid, and high inocula, respectively. The E. coli levels in the soil from the inoculated plots were 1.5 ± 0.4, 3.7 ± 0.3, and 5.8 ± 0.1 log CFU/g for the low, mid, and high inocula, respectively. There was a significant difference in the average daily rainfall among the three trials. Trial 3 received 23.8 ± 9.2 cm, while trials 1 and 2 received much less (0.1 ± 0.1 0.0 ± 0.0 cm, respectively). Inoculation concentration and trial were significant (P<0.05) factors that influenced the transfer of E. coli and Salmonella to pecans. For the high inoculum treatment, bacterial transfer to pecans ranged from 0.7 ± 0.3 to 4.1 ± 0.2 for E. coli and 1.3 ± 0.7 to 4.3 ± 0.4 log CFU/g for Salmonella. For the medium inoculum treatment, transfer ranged from <0.3 to 1.5 ± 0.1 for E. coli and <0.3 to 1.9 ± 0.2 log CFU/g for Salmonella. For the low treatment, transfer ranged from <0.3 to 0.4 ± 0.2 and <0.3 to 0.5 ± 0.1 log CFU/g for E. coli and Salmonella, respectively. These results show the need for implementing agricultural practices that prevent potential transfer of foodborne pathogens onto the surface of in-shell pecans during harvest