Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere

Abstract

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22 degrees C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30 degrees C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.

FIG. 1.

Population dynamics of P. chlororaphis CIL12R (▪), P. agglomerans FC1R (•), and Salmonella serovar Thompson (▵) after inoculation of each strain individually onto cilantro plants and incubation of the plants at 22°C. The growth of indigenous bacteria on control plants (○) is also shown. Each data point indicates the mean of the log-transformed bacterial population size for 12 leaves. Each error bar indicates ±1 standard error of the mean.

FIG. 2.

Colonization of cilantro leaves by strains of S. enterica serovar Derby (•), S. enterica serovar Newport (▴), S. enterica serovar Thompson (⋄), and S. enterica serovar Enteritidis (▪) that were linked to outbreaks from turkey, alfalfa sprouts, cilantro, and mayonnaise, respectively. Each data point indicates the mean of the log-transformed population size for a serovar inoculated onto cilantro plants incubated at 24°C. Each error bar indicates ±1 standard error of the mean of the log-transformed bacterial population size for 12 leaves.

FIG. 3.

Visualization by CLSM projected z series of the localization of Salmonella serovar Thompson(pWM1007) in the phyllosphere of inoculated cilantro plants. (A) Microcolonies of Salmonella serovar Thompson (GFP) above a vein of a cilantro leaf 2 days after inoculation. (B) High density of Salmonella serovar Thompson (GFP) cells in the vein area of a senescent cilantro leaf 9 days after inoculation. (C) Large aggregate of Salmonella serovar Thompson (GFP) and P. agglomerans (DsRed) cells on the vein of a cilantro leaf 7 days after inoculation. (D) Heterogeneous bacterial aggregate comprised of Salmonella serovar Thompson (GFP), P. agglomerans (DsRed), and natural microflora (visualized with the stain SYTO 62 and assigned the pseudocolor blue) (arrow) on the cilantro phylloplane. Bars, 10 μm.

FIG. 4.

Visualization by CLSM of Salmonella serovar Thompson (GFP) on the cuticle of a healthy leaf (A) and within the damaged tissue of a naturally diseased leaf (B) after inoculation onto cilantro plants. The top panels show the localization of Salmonella serovar Thompson in a projected z series of xy optical sections. The lower panels show corresponding cross sections which reveal the localization of Salmonella serovar Thompson above (A) and within (B) the plant tissue by a projected series of xz optical sections and by a single xz optical section, respectively. Note the amplification of the red and green signals above saturation which aids in detection of the very dimly fluorescent cuticle layer of the leaf surface. Bars, 10 μm.

FIG. 5.

Effect of temperature on the colonization of cilantro plants by Salmonella serovar Thompson and by the natural bacterial flora. (A) Population dynamics of Salmonella serovar Thompson (solid symbols) on leaves of inoculated plants and population dynamics of the natural bacterial flora (open symbols) on leaves of control plants. Cilantro plants were incubated at 24°C (circles) or at 30°C (squares). (B) Ratio of the population size of Salmonella serovar Thompson to the population size of the total bacteria on individual leaves of inoculated plants incubated at 24°C (•), 30°C (▪), and 37°C (▴). Each error bar indicates ±1 standard error of the mean of the log-transformed bacterial population size (A) or population size ratio (B) for 15 leaves.

FIG. 6.

Tolerance of Salmonella serovar Thompson (▵), P. agglomerans (○), and P. chlororaphis (□) to alternating wet and dry conditions in the cilantro phyllosphere at 26°C. The data points indicate the means of the log-transformed population size of each strain on cilantro leaves during a period of wet conditions, followed by exposure to 60% relative humidity and a recovery period under wet conditions. The dotted line shows the population size of Salmonella serovar Thompson on plants that were incubated under wet conditions throughout the experiment (▴). Each error bar indicates ±1 standard error of the mean of the log-transformed bacterial population size for 14 leaves.