Plant-Microbe and Abiotic Factors Influencing Salmonella Survival and Growth on Alfalfa Sprouts and Swiss Chard Microgreens

Abstract

Microgreens, like sprouts, are relatively fast-growing products and are generally consumed raw. Moreover, as observed for sprouts, microbial contamination from preharvest sources may also be present in the production of microgreens. In this study, two Salmonella enterica serovars (Hartford and Cubana), applied at multiple inoculation levels, were evaluated for survival and growth on alfalfa sprouts and Swiss chard microgreens by using the most-probable-number (MPN) method. Various abiotic factors were also examined for their effects on Salmonella survival and growth on sprouts and microgreens. Community-level physiological profiles (CLPPs) of sprout/microgreen rhizospheres with different levels of S. enterica inoculation at different growth stages were characterized by use of Biolog EcoPlates. In the seed contamination group, the ability of S. enterica to grow on sprouting alfalfa seeds was affected by both seed storage time and inoculation level but not by serovar. However, the growth of S. enterica on Swiss chard microgreens was affected by serovar and inoculation level. Seed storage time had little effect on the average level of Salmonella populations in microgreens. In the irrigation water contamination group, the growth of Salmonella on both alfalfa sprouts and microgreens was largely affected by inoculation level. Surprisingly, the growth medium was found to play an important role in Salmonella survival and growth on microgreens. CLPP analysis showed significant changes in the microbial community metabolic diversity during sprouting for alfalfa sprouts, but few temporal changes were seen with microgreens. The data suggest that the change in rhizosphere bacterial functional diversity was dependent on the host but independent of Salmonella contamination.IMPORTANCE Sprouts and microgreens are considered "functional foods," i.e., foods containing health-promoting or disease-preventing properties in addition to normal nutritional values. However, the microbial risk associated with microgreens has not been well studied. This study evaluated Salmonella survival and growth on microgreens compared to those on sprouts, as well as other abiotic factors that could affect Salmonella survival and growth on microgreens. This work provides baseline data for risk assessment of microbial contamination of sprouts and microgreens. Understanding the risks of Salmonella contamination and its effects on rhizosphere microbial communities enables a better understanding of host-pathogen dynamics in sprouts and microgreens. The data also contribute to innovative preventive control strategies for Salmonella contamination of sprouts and microgreens.

Keywords: CLPP; Salmonella enterica; Swiss chard microgreens; alfalfa sprouts; growth.

FIG 1

Salmonella enterica growth on alfalfa sprouts via irrigation water contamination. Sprouts were grown in a Conviron E7/2 climate-controlled growth chamber for the duration of the experiment. Before germination, growth chamber temperatures were maintained at 24°C (daytime) and 22°C (nighttime) with no light. Temperatures were maintained for 2 days at a constant 22°C day and night, with light, after germination. Growth chambers were kept at a constant relative humidity of 65%. At harvest, the mean log MPN per gram of fresh weight (y axis) was calculated against 4 different inoculation levels (x axis) for S. enterica serovars Cubana (gray) and Hartford (black). Numbers in parentheses show the numbers of replicated samples examined at each inoculation level. The detection limit for Salmonella in this experiment was 0.3 MPN/g.

FIG 2

Salmonella enterica growth on Swiss chard microgreens via irrigation water contamination. Microgreens were grown in a Conviron E7/2 climate-controlled growth chamber for the duration of the experiment. Before germination, growth chamber temperatures were maintained at 24°C (daytime) and 22°C (nighttime) with no light. Temperatures were maintained for 4 days at a constant 24°C day and night, with light, after germination. Growth chambers were kept at a constant relative humidity of 65%. At harvest, the mean log MPN per gram of fresh weight (y axis) was calculated against various inoculation levels (x axis) for S. enterica serovars Cubana (gray) and Hartford (black) for different growth media, including hydroponic growth, soil A, and soil B. Numbers in parentheses show the numbers of replicated samples examined at each inoculation level. The detection limit for Salmonella in this experiment was 0.3 MPN/g.

FIG 3

Average well color development (AWCD) (A and B) and richness (R) (C and D) of metabolized substrates in Biolog EcoPlates after inoculation with different levels of S. enterica pregermination, after light exposure, and at the harvest stage. Data for sprouts are presented in panels A and C, and data for microgreens are presented in panels B and D. Each sample had three replicates. The data shown here are the means of 31 substrate well absorbance values at day 6. Error bars represent standard deviations (n = 3), and different letters indicate a significant difference (P < 0.05).

FIG 4

Principal coordinate analysis (PCoA) of bacterial community metabolic profiles for different rhizosphere samples from alfalfa sprouts (A) and Swiss chard microgreens (B). The percentage of total variance explained by each axis is shown parenthetically. All values are based on AWCD data. The various samples from different growth stages are represented by symbols of different colors. Ellipses were drawn with a confidence limit of 0.95.