Development of an Avirulent Salmonella Surrogate for Modeling Pathogen Behavior in Pre- and Postharvest Environments

Abstract

Recurrent outbreaks of bacterial gastroenteritis linked to the consumption of fresh fruits and vegetables highlight the paucity of understanding of the ecology of Salmonella enterica under crop production and postharvest conditions. These gaps in knowledge are due, at least in part, to the lack of suitable surrogate organisms for studies for which biosafety level 2 is problematic. Therefore, we constructed and validated an avirulent strain of Salmonella enterica serovar Typhimurium. The strain lacks major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 as well as the virulence plasmid pSLT. Deletions and the absence of genomic rearrangements were confirmed by genomic sequencing, and the surrogate behaved like the parental wildtype strain on selective media. A loss-of-function (phoN) selective marker allowed the differentiation of this strain from wildtype strains on a medium containing a chromogenic substrate for alkaline phosphatase. Lack of virulence was confirmed by oral infection of female BALB/c mice. The strain persisted in tomatoes, cantaloupes, leafy greens, and soil with the same kinetics as the parental wild-type and selected outbreak strains, and it reached similar final population levels. The responses of this strain to heat treatment and disinfectants were similar to those of the wild type, supporting its potential as a surrogate for future studies on the ecology and survival of Salmonella in production and processing environments. IMPORTANCEThere is significant interest in understanding the ecology of human pathogens in environments outside of their animal hosts, including the crop production environment. However, manipulative field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks. Therefore, we constructed an avirulent strain of S. enterica serovar Typhimurium and characterized it extensively. O utbreaks of human disease caused by nontyphoidal Salmonella enterica have been linked to sprouts, fruits, vegetables, and nuts that were contaminated at various points in the production cycle (1-8). Generic Escherichia coli indicator levels are currently monitored to ensure the safety of produce; however, the persistence of Salmonella on the surfaces of peaches, cantaloupes, lettuce, and bell peppers has been shown to be very different from that of E. coli (9, 10). In general, Salmonella survives better outside of its animal hosts than E. coli (11) and can persist in processed organic waste and amended soils better than fecal coliforms, although its persistence appears to be strain and/or site dependent (12-14).There is significant interest in understanding the ecology of specific pathogens in the environment outside of their animal hosts. However, field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks associated with environmental exposure. Surrogate organisms have been used to better understand how human pathogens survive in environments and respond to...

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing

“…can proliferate within red ripe tomato pericarps, reaching ϳ10 7 CFU per fruit (9,19,21,25,35). To represent the entire transposon insertion library, the screening needed to be done using an inoculum titer very close to the carrying capacity of tomatoes.…”

“…induced plant immune responses at lower levels than specialized plant pathogens, like Pseudomonas syringae (24). Some studies reported that Salmonella strains lacking SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 are not defective for the colonization of tomato and cantaloupe fruits (9,25). Furthermore, there is no direct evidence that Salmonella can translocate effectors into plant cells via SPI-encoded T3SS, calling into question the requirement for the corresponding genes during plant colonization (unless the T3SS apparatus performs other noncanonical functions during plant colonization, a hypothesis that has not yet been ruled out).…”

Application of chitosan microparticles for mitigation of Salmonella in agricultural water

“…Typhimurium strain BAA‐14028S (de Moraes et al . ) was provided by Dr. Max Teplitski (University of Florida) and derived from the parent strain of Typhimurium BAA‐14028 by deleting Salmonella pathogenicity islands (SPI1, SPI2, SPI3, SPI4, SPI5) and plasmid (pSLT). Newport C6·3 was isolated from a tomato field on the Eastern Shore of Virginia (de Moraes et al .…”

“…Newport C6·3 was isolated from a tomato field on the Eastern Shore of Virginia (de Moraes et al . ), and UFL‐1352 was isolated from an irrigation pond in Georgia in a prior study (Luo et al . ).…”

“…Typhimurium strain BAA-14028S (de Moraes et al 2016) was provided by Dr. Max Teplitski (University of Florida) and derived from the parent strain of Typhimurium BAA-14028 by deleting Salmonella pathogenicity islands (SPI1, SPI2, SPI3, SPI4, SPI5) and plasmid (pSLT). Newport C6Á3 was isolated from a tomato field on the Eastern Shore of Virginia (de Moraes et al 2016), and UFL-1352 was isolated from an irrigation pond in Georgia in a prior study (Luo et al 2014). CM sensitivity of additional isolates was also examined for strains listed in Table 2 and obtained from both environmental (Luo et al 2014) and clinical sources (Dr. Carina Blackmore, Florida Department of Health).…”