Virulence assessment of Listeria monocytogenes grown in different foods using a Galleria mellonella model

Various produce including cantaloupe, caramel-coated apples, and packaged salads, have been recognized in recent years as vehicles for listeriosis, a human foodborne disease caused by intracellular pathogen Listeria monocytogenes. Our knowledge regarding the role of these foods in L. monocytogenes virulence, however, is limited. Understanding their role in modulating L. monocytogenes virulence can be useful in risk assessments and for developing control measures. In this study, we employed the Galleria mellonella larvae model to evaluate virulence potential of fifteen clinical, environmental and food isolates of L. monocytogenes, related to three major outbreaks, after growth on different foods. The non-human pathogen Listeria innocua was also included in the panel. Strains were inoculated in parallel in 5ml of brain heart infusion (BHI) broth, and on the surfaces of cantaloupe and apple fragments (5g each) at about 10(5) colony forming units (CFU)/ml/fragment. One set of inoculated broth and food fragments was incubated at 10°C for 5 days while the second set was kept at 25°C for 3 days. L. monocytogenes cells were recovered from the fruits and BHI, washed twice, re-suspended in saline, and used to inoculate G. mellonella larvae at final concentrations of 10(6) and 10(5) CFU/larva. The larvae were incubated at 37°C and monitored for mortality (LT(50)—time taken to kill 50% of the larvae) and phenotypic changes over seven days. L. monocytogenes grown on cantaloupe and apple flesh surfaces resulted in higher virulence than when grown in BHI. L. monocytogenes infection at 10(6) CFU/larvae resulted in an average LT(50) of ≤ 30, 36 and 47 hours on cantaloupe, apples and BHI, respectively. These results represent a 2.5–4-fold increased mortality compared with an LT(50) ≥120 hours in larvae infected with the same doses of L. innocua grown in corresponding matrices. Similar trends were also recorded with doses of about 10(5) CFU /larvae.