Gamma irradiation influences the survival and regrowth of antibiotic-resistant bacteria and antibiotic-resistance genes on Romaine lettuce

Authors: DHARMARHA, VAISHALI - Virginia Tech; GURON, GISELLE - Virginia Tech; BOYER, RENEE - Virginia Tech; Niemira, Brendan; PRUDEN, AMY - Virginia Tech; STRAWN, LAURA - Virginia Tech; PONDER, MONICA - Virginia Tech

Submitted to: Frontiers in Sustainable Food Systems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2019
Publication Date: N/A
Citation: N/A

Interpretive Summary: Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria, and the genes conferring antibiotic resistance occurs during production. Therefore, post-harvest interventions are needed, not only to mitigate pathogens, but also to control the spread of antibiotic resistance. The objective of this research was to determine how post-harvest treatments including sanitizer washes and/or gamma irradiation change the bacterial populations on lettuce leaves, and the antibiotic resistance genes that might be present. To simulate real-world sources of contamination such as contact with compost in soil, romaine lettuce leaves were inoculated with a compost slurry containing antibiotic-resistant strains of the human pathogen Escherichia coli O157:H7 and the non-pathogenic spoilage bacteria Pseudomonas aeruginosa. Inoculated lettuce leaves were first washed with sodium hypochlorite at 50 ppm (a commonly used sanitizer solution), then packaged under modified atmosphere (98% nitrogen), irradiated (1.0 kGy), and stored at 4ºC for up to 14 d. The surviving bacteria were counted on selective growth media. The genetic effects of these post-harvest treatments on the populations of bacteria present, and on the collection of antibiotic resistance genes, were analyzed by gene sequencing. The chlorine-based sanitizer reduced the abundance of genes that confer resistance to certain key antibiotics, including glycopeptide, ß-lactam, phenicol, and sulfonamide. The combination treatment of sanitizing followed by irradiation reduced a different set of antibiotic resistance genes, including those related to triclosan, polymyxin, ß-lactam, quinolones, and multidrug resistance. During storage after irradiation, the genetic diversity of the antibiotic resistant bacteria changed. This was observed as the lettuce was stored 4C for 1 day up to 14 days. Cold storage of washed and irradiated lettuce changed the relative abundance of bacterial species known to harbor antibiotic resistance genes. Overall, irradiation reduced pathogenic and non-pathogenic bacteria by 99.97%. Results of this study indicate that washing, irradiation and cold storage of modified atmosphere packaged lettuce are effective strategies to reduce the spread of antibiotic-resistant pathogens, and the genes that confer antibiotic resistance.

Technical Abstract: Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria (ARB), and the genes conferring antibiotic resistance (ARGs) occurs during production. Therefore, post-harvest interventions are needed to mitigate pathogens, but also ARB, and ARGs on vegetables. The objective of this research was to determine changes to lettuce phyllosphere microbiota, and resistome (collection of ARGs) following post-harvest treatments including washing in a sodium hypochlorite solution and/or gamma irradiation. To simulate real-world sources of contamination such as contact with compost in soil, romaine lettuce leaves were inoculated with compost slurry containing antibiotic-resistant strains of pathogenic (Escherichia .coli O157:H7) and spoilage bacteria (Pseudomonas aeruginosa). Inoculated lettuce leaves were washed with sodium hypochlorite (50 ppm free chlorine), packaged under modified atmosphere (98% nitrogen), irradiated (1.0 kGy), and stored at 4ºC for up to 14 d. 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 glycopeptide, ß-lactam, phenicol, and sulfonamide (Wilcoxon, p<0.05). Washing followed by irradiation resulted in a different resistome chiefly due to reductions in ARG classes that confer resistance to: multidrug, triclosan, polymyxin, ß-lactam, and quinolones (Wilcoxon, p<0.05). Irradiation followed by storage at 4ºC for 14d lead to distinct changes to the ß-diversity of the host bacteria of ARGs compared to 1d after treatment (ANOSIM, R=0.331; p=0.003). Storage of washed and irradiated lettuce at 4 ºC for 14 d 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 of this study indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4ºC are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and some other ARBs and ARGs..