In situ evaluation of Paenibacillus alvei in reducing carriage of Salmonella enterica serovar Newport on whole tomato plants

Abstract

Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of Salmonella enterica serovar Newport and other Salmonella serovars. Long-term intervention measures to reduce Salmonella prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as Paenibacillus alvei was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against Salmonella and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of S. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After P. alvei strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of S. Newport declined significantly (P ≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of Salmonella by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of Salmonella Newport on whole tomato plants. The application of P. alvei strain TS-15 is a promising approach for reducing the risk of Salmonella contamination during tomato production.

FIG 1

In vitro inhibition of food-borne pathogens and tomato bacterial phytopathogens by Paenibacillus alvei A6-6i and TS-15 on tryptic soy agar. The inhibition zones (mm) were measured against strains of Salmonella spp., Escherichia coli, Cronobacter sakazakii (CS), Listeria monocytogenes (LM), Shigella dysenteriae (SD), methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Ralstonia solanacearum race 5, Pseudomonas syringae pv. tomato strain dc3000, and Erwinia carotovora subsp. carotovora. The plot represents the lowest, highest, and average measurements for each of the species listed above. The experiment was repeated twice.

FIG 2

Growth inhibition of major food-borne pathogens in P. alvei A6-6i cell-free culture supernatants. Brain heart infusion (BHI) broth was used as a control. Bacterial growth of L. monocytogenes (LM), S. dysenteriae (SD), E. coli O157, C. sakazakii (CS), and S. Newport strains (A) and methicillin-resistant S. aureus (MRSA) strains (B) in P. alvei A6-6i CFCSs and BHI was determined in five replicates by measuring the OD₆₀₀ at 20-min intervals for 24 h. The experiment was repeated twice.

FIG 3

Recovery of S. Newport from intact tomato fruit surfaces after treatment with antagonist inoculations over 24 h at 30°C in a humidity chamber. (A) Recovery of S. Newport with S. Newport inoculated first. (B) Recovery of S. Newport with the antagonist inoculated first. Error bars represent the standard deviations of the means of two experiments, each with 10 replicates (n = 20).

FIG 4

Recovery of an attenuated S. Newport strain from tomato plants, including leaves, blossoms, and tomato fruits. In the high-tunnel study, S. Newport was recovered from leaves, blossoms, and tomato fruits at 0, 1, 2, 3, and 5 dpi (for blossoms) or 6 dpi (for leaves and tomato fruits) with inoculation with S. Newport only or coinoculation with S. Newport plus the antagonist. The results were tallied for each combination of plant location, antagonist, plant, and day. The estimated recovery of S. Newport from each sample point from log-transformed data in control (−) and antagonist treatment (+) panels was scatter plotted for leaf (A), tomato fruit (B), and blossom (C).

FIG 5

Rate of decrease in the S. Newport concentration postinoculation on leaves, blossoms, and tomato fruits. In a high-tunnel setting, leaves, blossoms, and tomato fruits were harvested at 0, 1, 2, 3, and 5 dpi (for blossoms) or 6 dpi (for leaves and tomato fruits) to recover any remaining S. Newport bacteria. The rates of decrease in the S. Newport concentration in the control and antagonist treatment groups were calculated and compared. Results are shown as means ± 2 standard errors. An asterisk indicates that the rate of decrease in the S. Newport concentration was significantly higher (P < 0.05) than that in the control group.