P2-119 Ampicillin Selection of Listeria monocytogenes Mutants Unable to Replicate on Rind of Fresh Cantaloupe

Tuesday, August 2, 2016
America's Center - St. Louis
Victor Jayeola, North Carolina State University, Raleigh, NC
Cameron Parsons, North Carolina State University, Raleigh, NC
William Miller, U.S. Department of Agriculture-ARS, Albany, CA
Lisa Gorski, U.S. Department of Agriculture-PSMRU-WRRC-ARS, Albany, CA
Sophia Kathariou, North Carolina State University, Raleigh, NC
Introduction: Several outbreaks of listeriosis have implicated fresh produce. However, Listeria monocytogenes (LM) genes required for growth on produce remains poorly understood. Such genes can be identified via localization of transposon insertion in transposon mutants unable to grow on produce. We hypothesized that such mutants will be selected from mutant libraries since ampicillin (β-lactam antibiotic) kills only growing cells.

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.