Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce

Authors: JAYEOLA, VICTOR - North Carolina State University; PARSONS, CAMERON - North Carolina State University; Gorski, Lisa; KATHARIOU, SOPHIA - North Carolina State University

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2019
Publication Date: 4/12/2019
Citation: Jayeola, V., Parsons, C., Gorski, L.A., Kathariou, S. 2019. Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce. FEMS Microbiology Letters. 366(7):fnz076. https://doi.org/10.1093/femsle/fnz076.
DOI: https://doi.org/10.1093/femsle/fnz076

Interpretive Summary: Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. In this study we tested whether we could select for mutants of L. monocytogenes that had growth defects on produce. Ampicillin selection is a method that is based on the bacteriostatic effects of ampicillin in that bacteria are not killed by the antibiotic but fail to grow in its presence. Once ampicillin is removed the bacteria continue growth. To validate this protocol, we examined relative recovery of L. monocytogenes strain 2011L-2858, implicated in the 2011 listeriosis outbreak via whole cantaloupe, and its erythromycin resistant, cold-sensitive mutant L1E4, following inoculation of cantaloupe rind with 1:1 mixtures of the strains and incubation with or without ampicillin (100µg ml-1) for 14 d at 4oC. L. monocytogenes colonies from cantaloupe rind fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh cantaloupe rinds for a second round of enrichment. In the presence of ampicillin, the proportion of the cold-sensitive mutant L1E4 increased from 55% on day 0 to 69 and 78% on days 7 and 14, respectively, with even higher recovery (85% after 14 days) in the second round of enrichment. The data suggested that the non-growing mutant L1E4 was enriched on cantaloupe rind while growing cells of 2858 were killed by ampicillin. Application of this protocol to transposon mutant libraries from three different produce-associated L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.

Technical Abstract: Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. Based on the ability of ß-lactam antibiotics to specifically kill growing bacterial cells, we hypothesized that ampicillin selection can enrich for mariner-based transposon mutants unable to grow on cantaloupe rind. To validate the protocol, we examined relative recovery of L. monocytogenes strain 2011L-2858, implicated in the 2011 listeriosis outbreak via whole cantaloupe, and its erythromycin resistant, cold-sensitive mutant L1E4, following inoculation of the rind with 1:1 mixtures of the strains and incubation with or without ampicillin (100µg ml-1) for 14 d at 4oC. L. monocytogenes colonies from fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh cantaloupe rinds for a second round of enrichment. In the presence of ampicillin, the proportion of the cold-sensitive mutant L1E4 increased from 55% on day 0 to 69 and 78% on days 7 and 14, respectively, with even higher recovery (85% after 14 days) in the second round of enrichment. The data suggested that the non-growing mutant L1E4 was enriched on cantaloupe rind while growing cells of 2858 were killed by ampicillin. Application of this protocol to transposon mutant libraries from three different produce-associated L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.