Juan M. Cevallos-Cevallos

Internal Colonization of Salmonella enterica Serovar Typhimurium in Tomato Plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 109 CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 104 CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 106 CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.

Dispersal of Salmonella Typhimurium by rain splash onto tomato plants.

Outbreaks of Salmonella enterica have increasingly been associated with tomatoes and traced back to production areas, but the spread of Salmonella from a point source onto plants has not been described. Splash dispersal by rain could be one means of dissemination. Green fluorescent protein-labeled, kanamycin-resistant Salmonella enterica sv. Typhimurium dispensed on the surface of plastic mulch, organic mulch, or soil at 10⁸ CFU/cm² was used as the point source in the center of a rain simulator. Tomato plants in soil with and without plastic or organic mulch were placed around the point source, and rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Dispersal of Salmonella followed a negative exponential model with a half distance of 3 cm at 110 mm/h. Dispersed Salmonella survived for 3 days on tomato leaflets, with a total decline of 5 log and an initial decimal reduction time of 10 h. Recovery of dispersed Salmonella from plants at the maximum observed distance ranged from 3 CFU/g of leaflet after a rain episode of 110 mm/h for 10 min on soil to 117 CFU/g of leaflet on plastic mulch. Dispersal of Salmonella on plants with and without mulch was significantly enhanced by increasing rain duration from 0 to 10 min, but dispersal was reduced when rainfall duration increased from 10 to 30 min. Salmonella may be dispersed by rain to contaminate tomato plants in the field, especially during rain events of 10 min and when plastic mulch is used.

Organically managed soils reduce internal colonization of tomato plants by Salmonella enterica serovar Typhimurium.

A two-phase experiment was conducted twice to investigate the effects of soil management on movement of Salmonella enterica Typhimurium in tomato plants. In the first phase, individual leaflets of 84 tomato plants grown in conventional or organic soils were dip inoculated two to four times before fruiting with either of two Salmonella Typhimurium strains (10(9) CFU/ml; 0.025% [vol/vol] Silwet L-77). Inoculated and adjacent leaflets were tested for Salmonella spp. densities for 30 days after each inoculation. Endophytic bacterial communities were characterized by polymerase chain reaction denaturing gradient gel electrophoresis before and after inoculation. Fruit and seed were examined for Salmonella spp. incidence. In phase 2, extracted seed were planted in conventional soil, and contamination of leaves and fruit of the second generation was checked. More Salmonella spp. survived in inoculated leaves on plants grown in conventional than in organic soil. The soil management effect on Salmonella spp. survival was confirmed for tomato plants grown in two additional pairs of soils. Endophytic bacterial diversities of tomato plants grown in conventional soils were significantly lower than those in organic soils. All contaminated fruit (1%) were from tomato plants grown in conventional soil. Approximately 5% of the seed from infested fruit were internally contaminated. No Salmonella sp. was detected in plants grown from contaminated seed.

GC-MS based metabolomics for rapid simultaneous detection of Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Muenchen, and Salmonella Hartford in ground beef and chicken.

A metabolomic-based method for rapid detection of Escherichia coli O157:H7, Salmonella Hartford, Salmonella Typhimurium, and Salmonella Muenchen in nonselective media was developed. All pathogenic bacteria were grown in tryptic soy broth (TSB) at 37 °C followed by metabolite quantification at 2-h intervals for 24 h. Results were compared with the metabolite profiles similarly obtained with E. coli K12, Pseudomonas aeruginosa, Staphylococcus aureus, Saccharomyces cereviseae, and Aspergillus oryzae grown individually or as a cocktail under the same conditions. Principal component analysis (PCAS) discriminated pathogenic microorganisms grown in TSB. Metabolites responsible of PCAS classification were dextrose, cadaverine, the aminoacids L-histidine, glycine, and L-tyrosine, as well as the volatiles 1-octanol, 1-propanol, 1butanol, 2-ethyl-1-hexanol, and 2,5-dimethyl-pyrazine. Partial least square (PLS) models based on the overall metabolite profile of each bacteria were able to detect the presence of Escherichia coli O157:H7 and Salmonella spp. at levels of approximately 7 ± 2 CFU/25 g of ground beef and chicken within 18 h.

Ingress of Salmonella enterica Typhimurium into tomato leaves through hydathodes.

Internal contamination of Salmonella in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars “Florida Lanai”, “Crown Jewel”, “Ailsa Craig” and the transgenic line Sp5 of “Ailsa Craig” were inoculated with 1 µl GFP-labeled Salmonella Typhimurium through guttation droplets at concentrations of 109 or 107 CFU/ml. Survival of Salmonella on/in tomato leaves was detected by both direct plating and enrichment methods. Salmonella cells survived best on/in the inoculated leaves of cultivar “Ailsa Craig” and decreased fastest on/in “Florida Lanai” leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of Salmonella into leaves and the colonization on the surface of tomato leaves. Internalization of Salmonella Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants.

Untargeted metabolite analysis of healthy and Huanglongbing-infected orange leaves by CE-DAD

Huanglongbing (HLB) is considered the most destructive bacterial citrus disease worldwide. Early detection of HLB is crucial for minimizing its spread. CE was used for the discovery of potential biomarkers for HLB. Optimization of extraction and separation allowed resolving 24 compounds of which 6 were present in significantly higher (p<0.05) concentrations in HLB‐infected samples collected monthly for 6 months during the 2007–2008 season. Three of these compounds were identified by mobility and UV spectra as hesperidin, naringenin, and quercetin with mean increase in concentration of 154, 555, and 467%, respectively, above that in healthy leaves. Results support the potential of CE‐DAD for untargeted plant metabolomic analysis. CZE, NACE, and MEEKC were compared for metabolic differentiation of healthy and HLB‐infected citrus leaves. CZE in a semi‐aqueous BGE solution consisting of 8.5 mM of sodium borate (pH 9.3), 15% ACN, and 9% 1‐butanol yielded the best peak separation with detection at 190 nm.

Salmonella can reach tomato fruits on plants exposed to aerosols formed by rain

Outbreaks of Salmonella enterica have been associated with tomatoes and traced back to production areas but the spread of Salmonella in agricultural fields is still poorly understood. Post-rain Salmonella transfer from a point source to the air and then to tomato plants was evaluated. GFP-labeled kanamycin-resistant S. enterica serovar Typhimurium (10(8)CFU/mL) with and without expression of the rdar morphotype (rough colonies; cells with fimbriae and cellulose) was used as the point source in the center of a rain simulator. Rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Petri dishes with lactose broth and tomato plants with fruit (50-80 cm high) were placed in the simulator after the rain had ceased. Salmonella recovery from air was maximum (300 CFU/plate) after a rain episode of 60 mm/h for 10 min at distances of at least 85.5 cm above the source and when the rdar morphotype strain was used. Small scale experiments showed that the smooth-colony strain without fimbriae precipitated from the air in significantly higher numbers than the rdar strain. Transfer of aerial Salmonella with the rdar morphotype to tomato fruits on plants followed a beta distribution (2.5950, 4.7393) within the generalized range from 0 to 30 min of rain. Results show for the first time that Salmonella may transfer from rain to the air and contaminate tomato fruits at levels that could possibly be infectious to humans.

Adhesion and splash dispersal of Salmonella enterica Typhimurium on tomato leaflets: effects of rdar morphotype and trichome density.

Salmonella enterica strains with rdar (red dry and rough) and saw (smooth and white) morphotypes have previously been associated with tomato outbreaks but the dispersal mechanisms of these morphotypes are still poorly understood. In this study, Salmonella adhesion was distinguished from attachment by comparing different contact periods. Initial adhesion of rdar and saw morphotypes of Salmonella was compared in relation to tomato plants with different leaf trichome densities. Trichome densities were increased or reduced by treatment with jasmonic or salicylic acid, respectively. The overall effect of Salmonella morphotype and trichome density on splash dispersal was assessed in a rain simulator and correlated to cell hydrophobicity and initial adhesion. The presence of the rdar morphotype increased initial adhesion at high trichome densities but not at low trichome densities. Attachment of the rdar strain occurred after 30s contact time regardless of trichome density. Splash dispersal was slightly further for the saw morphotype than the rdar morphotype of S. enterica at all trichome densities. Salmonella cells of both morphotypes survived significantly better on the surface of high trichome density leaflets.

Pleiotropy and its dissection through a metabolic gene Miniature1 (Mn1) that encodes a cell wall invertase in developing seeds of maize

The Mn1-encoded endosperm-specific cell wall invertase is a major determinant of sink strength of developing seeds through its control of both sink size, cell number and cell size, and sink activity via sucrose hydrolysis and release of hexoses essential for energy and signaling functions. Consequently, loss-of-function mutations of the gene lead to the mn1 seed phenotype that shows ∼70% reduction in seed mass at maturity and several pleiotropic changes. A comparative analysis of endosperm and embryo mass in the Mn1 and mn1 genotypes showed here significant reductions of both tissues in the mn1 starting with early stages of development. Clearly, embryo development was endosperm-dependent. To gain a mechanistic understanding of the changes, sugar levels were measured in both endosperm and embryo samples. Changes in the levels of all sugars tested, glc, fru, suc, and sorbitol, were mainly observed in the endosperm. Greatly reduced fru levels in the mutant led to RNA level expression analyses by q-PCR of several genes that encode sucrose and fructose metabolizing enzymes. The mn1 endosperm showed reductions in gene expression, ranging from ∼70% to 99% of the Mn1 samples, for both suc-starch and suc--energy pathways, suggesting an in vivo metabolic coordinated regulation due to the hexose-deficiency. Together, these data provide evidence of the Mn1-dependent interconnected network of several pathways as a possible basis for pleiotropic changes in seed development.

Survival of Salmonella enterica Typhimurium in Water Amended with Manure

Outbreaks of Salmonella enterica have been associated with water sources. Survival of S. enterica in various environments has been studied but survival in water has rarely been attempted. In two separate experiments, we examined the survival of S. enterica Typhimurium in clean spring water at various eutrophication levels and temperatures. In the first experiment, lasting for 135 days, survival of S. enterica (10(10) CFU/ml) in water with 0, 50, 100, 500, and 1,000 mg/liter of added carbon at 7, 17, and 27°C was monitored weekly. In the second experiment, lasting for 3 weeks, survival of S. enterica in water at 0, 100, and 200 mg/ liter of added carbon and 27°C was studied daily. Each experiment had four replicates. Dissolved organic carbon was measured daily in each experiment. At the beginning, midpoint, and end of the survival study, microbial communities in both experiments were assessed by denaturing gradient gel electrophoresis (DGGE). Even at minimal carbon concentrations, S. enterica survived for at least 63 d. Survival of Salmonella was highly dependent on eutrophication levels (as measured by dissolved organic carbon) and temperature, increasing at high eutrophication levels, but decreasing at high temperatures. Survival was also strongly affected by microbial competition or predation.