Purpose: To assess the capacity of ampicillin selection to identify LM transposon mutants unable to grow on cantaloupe rind.
Methods: L1E4 is a cold-sensitive, erythromycin-resistant mutant of the erythromycin-susceptible strain 2858, implicated in the 2011 cantaloupe outbreak of listeriosis. Rind fragments of fresh cantaloupe were spot-inoculated with L1E4, 2858 and a 1:1 mixture of the two strains. Inoculated fragments were placed in Petri dishes with 10 ml sterile H2O with or without ampicillin (100 µg/ml) so that inoculated rind surfaces remained immersed in the solution and incubated for 14 d at 4°C, a temperature that permits growth of 2858 but not L1E4. LM populations were enumerated on selective media (MOX) at 0, 7 and 14 d. Populations from fragments inoculated with mixed cultures were screened for erythromycin resistance to determine L1E4: 2858 ratios.
Results: Populations of the cold-sensitive transposon mutant L1E4 remained unchanged on the rind at 4°C with or without ampicillin. However, the proportion of L1E4 from fragments inoculated with mixed cultures increased from 47% at day 0 to 58 and 73% at 7 and 14 d, respectively (P< 0.001). Findings suggested that growing 2858 cells on cantaloupe were killed with ampicillin while non-replicating L1E4 cells were uninhibited.
Significance: Findings support value of the ampicillin selection protocol in screening mixed transposon mutant libraries to isolate non-replicating mutants on produce. Identification of LM genes essential for growth on produce will contribute to science-based strategies to enhance produce safety.