Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Abstract

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.

FIG. 1.

Salmonella serovar Newport strain 96E01153C-TX, SeN(pKT), E. coli O157:H7 strain F4546, and Ec(pKT) levels on sprouting alfalfa seeds. The data points represent the means and standard deviations of the bacterial levels on inoculated seeds, 1-day-old sprouts, and 2-day-old sprouts. The experiment was repeated at least three times for each strain. Each experiment had three internal replicates, and generally, three sprout samples were taken from each replicate. Symbols: ⧫, Salmonella serovar Newport strain 96E01153C-TX; ◊, SeN(pKT); •, E. coli O157:H7 strain F4546; ○, Ec(pKT).

FIG. 2.

E. coli O157:H7 and S. enterica levels on 2-day-old alfalfa sprouts. The bars and lines represent the means and standard deviations of the changes in bacterial levels on inoculated seeds sprouted for 2 days. The experiment was repeated at least three times for each strain. Each experiment had three internal replicates, and generally, three sprout samples were taken from each replicate. The E. coli O157:H7 strains were as follows: A, F4546; B, 86-24; C, C7927; D, H2439; E, 96A13466; and F, EDL933. The S. enterica strains were as follows: G, S. enterica serovar Newport strain 96E01153C-TX; H, S. enterica serovar Mbandaka strain 99A 1668; I, S. enterica serovar Cubana strain 98A 9878; J, S. enterica serovar Havana strain 98A 4399; and K, S. enterica serovar Schwarzengrund strain 96E01152C-TX-1.

FIG. 3.

Numbers of SeN(pKT) and Ec(pKT) CFU per milliliter of irrigation water and per sprout at different inoculum doses and different sprouting temperatures. The data are means from three repetitions. Each experiment had five internal replicates, and one sprout sample and one water sample were taken from each replicate.

FIG. 4.

Numbers of SeN(pKT) and Ec(pKT) CFU per ml of irrigation water and per sprout with and without regular irrigation water replacement. The data are means and standard deviations from three repetitions. Each experiment had five internal replicates, and one sprout sample and one water sample were taken from each replicate.

FIG. 5.

Fluorescence stereoscope micrographs of SeN(pKT) and Ec(pKT) on sprouting alfalfa seeds. (A) SeN(pKT)-colonized seed coat edge of 1-day-old alfalfa sprout. (B) Ec(pKT)-colonized radicle of 1-day-old alfalfa sprout. (C) SeN(pKT)-colonized root in the region where the root hairs develop on 2-day-old alfalfa sprouts. The clumpy appearance of the GFP-labeled cells on the sprout is due to the large SeN(pKT) aggregates on the sprout. (D) Ec(pKT)-colonized root in the region where the root hairs develop on 2-day-old alfalfa sprouts. Cell aggregates are not visible. sc, seed coat; ra, radicle; r, root.

FIG. 6.

CLSM micrographs of SeN(pKT) and Ec(pKT) on sprouting alfalfa seeds. (A) SeN(pKT) aggregates on the root and on the root hairs of 2- or 3-day-old alfalfa sprout. (B) SeN(pKT) aggregates on alfalfa seed coat edge. (C) Individual Ec(pKT) cells on alfalfa root. (D) Individual Ec(pKT) cells on alfalfa seed coat edge. rh, root hair; sc, seed coat.