Listeria monocytogenes is a facultative intracellular bacterium that causes foodborne disease in humans. L. monocytogenes invade the gut mucosa and then disseminate, causing systemic infections associated with high mortality rates in immunocompromised individuals. It is unknown how L. monocytogenes traffic to the mesenteric lymph nodes, which represent an important bottleneck for systemic spread. In addition, little is known about the gastrointestinal stage of infection due to the general resistance of mice to oral infection with L. monocytogenes. Our laboratory developed a novel foodborne mouse model of listeriosis utilizing a murinized strain of L. monocytogenes to investigate the gastrointestinal stage of infection. First, we found that the majority of L. monocytogenes isolated from the intestinal tissue and MLN were extracellular; however, the minimal fraction of intracellular L. monocytogenes was vital for persistence in the gut and spread to the MLN. The vast majority of cell-associated L. monocytogenes in the MLN were adhered to inflammatory monocytes, but these cells did not support the intracellular growth of L. monocytogenes. A minor proportion of L. monocytogenes were associated with migratory dendritic cells in the intestinal lamina propria and MLN, but like monocytes, these cells did not appear to serve as an intracellular growth niche for L. monocytogenes. Lastly, extracellular L. monocytogenes were observed migrating in mesenteric lymphatic vessels that drain from the intestine to the MLN, suggesting that L. monocytogenes can spread beyond the intestinal mucosa independent of migratory immune cells. Overall, these studies are the first to characterize the interaction of L. monocytogenes with immune cells in the intestine and MLN following foodborne infection and suggest that extracellular, and not cytosolic L. monocytogenes, primarily drive innate immune responses in the gut.

<div>Fresh produce has been the leading source of foodborne illness outbreaks in the US, surpassing typical pathogen carriers such as meat, dairy, and seafood. Among the fresh produce popular to the consumers, cantaloupe and sprouts are mostly susceptible to pathogen contaminations and outbreaks. However, it has been a challenge to address the key factor in the contamination - the biofilms formed by pathogens are highly resistant to conventional washing and cleaning procedures. For cantaloupe, the net-like and porous surface forms a barrier for washing. For sprouts, the fragile texture of seedlings prevents aggressive cleaning operation and biofilm removal.</div><div><br></div><div>In this study, innovative interventions were developed to improve microbial safety of fresh produce, using cantaloupe and alfalfa sprouts as models. For cantaloupe, abrasive brushing was designed to remove pathogen biofilm from cantaloupe. Our research found pathogens could form biofilm at cantaloupe rind surface as the residence time of pathogens increased. Biofilm formed on cantaloupe rind was imaged by cryo-scanning electron microscopy (cryo-SEM), and its resistance to sodium hypochlorite and lauroyl arginate ethyl (LAE) was confirmed. Furthermore, abrasive brushing with peroxyacetic acid (PAA) could effectively remove biofilm formed at cantaloupe rind. The efficacy of this novel cleaning technique was highly desirable, which could achieve 3 log reduction in pathogen population. Mechanism of abrasive brushing to remove biofilm at cantaloupe rind surface was also proposed. Conceivably, brushing with diatomaceous earth (DE) and PAA could be an innovative and cost-effective method to remove pathogen biofilm from cantaloupe rind.</div><div><br></div><div>For alfalfa sprouts, since most of the outbreaks are linked to the sprouting seeds, seed disinfection treatments are considered to be the most effective method to improve microbial safety of sprouts. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. The calcium alginate coating in the presence of 2.5% lactic acid (CA-LA coating) reduced foodborne pathogens inoculated on alfalfa seeds to an undetectable level on day 1 during 28 day-seed storage, while chlorine (20,000 ppm) or lactic acid (2.5%) treatment took longer time to reach the same level. With sprouts, CA-LA coating resulted in > 2.5 log reduction for pathogen cells. In contrast, log reduction was < 0.6 for either chlorine (20,000 ppm) or lactic acid (2.5%) treatment. In general, this study indicated the effect of calcium alginate coating on reducing bacterial load of alfalfa seeds and sprouts, however, the germination rate of treated seeds was compromised due to the addition of lactic acid in the seed coating. Further study is needed to select antimicrobial compounds with minimum impact on germination rate of seeds.</div><div><br></div>

Cronobacter sakazakii is a gram-negative opportunistic pathogen known to survive in dry environments and food matrices, such as infant formula. This foodborne bacterium can cause fatal human infections of the blood, central nervous system, and gastrointestinal tract; it is also problematic in wounds and urinary tract infections. Preterm infants and immunocompromised individuals are in higher risk categories related to necrotizing enterocolitis, neonatal sepsis, and meningitis due to this organism. Therefore, there is a need for increased understanding of how this bacterium is able to persist in thermally treated low-moisture products that do not support growth. The objective of this research is to identify genes and mechanisms in C. sakazakii that contribute to its resistance to desiccation and survival in low-moisture food matrices, including powdered infant formula. C. sakazakii sequence type 4 (ST4) is of particular interest as it is often the cause of neonatal infections originating from contaminated feedings of powder infant formula. The method chosen to explore these genetic patterns is massively parallel transposon insertion sequencing (Tn-seq). The E. coli strain MFDpir was used to facilitate transposon insertional mutagenesis to create a library of mutated C. sakazakii. Three different C. sakazakii ST4 isolates of different origins (clinical, environmental, and infant formula-derived) were selected for this study. Once transposon mutagenesis occurred with the aid of E. coli MFDpir, the three mutant libraries were subjected to desiccation stress in a closed system equilibrated to 11.3% relative humidity. The surviving mutant genomes were analyzed with Tn-seq. The sequencing data revealed that, while transposition events did occur successfully within the genomes of each of the selected C. sakazakii isolates, these events were not dense enough to draw biological conclusions nor statistical inferences concerning which genes contribute to this organism’s uncanny desiccation tolerance. However, we concluded that the Tn-seq method is a promising tool with this organism of interest, despite incomplete results in this first round of experimentation.

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