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Abstract
Lactococcus lactis is used as cell-factory and strain selections are regularly performed to improve production processes. When selection regimes only allow desired phenotypes to survive, for instance by using antibiotics to select for cells that do not grow in a specific condition, the presence of more resistant subpopulations with a wildtype genotype severely slows down the procedure. While the food grade organism L. lactis is not often exposed to antibiotics we characterized its response to ampicillin in more detail, to better understand emerging population heterogeneity and how this might affect strain selection procedures. Using growth-dependent viability assays we identified persister subpopulations in stationary and exponential phase. Growth-independent viability assays revealed a 100 times larger subpopulation that did not grow on plates or in liquid medium, but had an intact membrane and could maintain a pH gradient. Over one third of these cells restored their intracellular pH when we induced a temporary collapse, indicating that this subpopulation was metabolically active and in a viable but non-culturable state. Exposure of L. lactis MG1363 to ampicillin therefore results in a heterogeneous population response with different dormancy states. These dormant cells should be considered in survival-based strain selection procedures.
Highlights
Lactococcus lactis is an important organism in the dairy industry and it is increasingly explored as a cell factory for food ingredients and as a delivery vehicle for bioactive molecules[1,2]
When cells were not treated with ampicillin all cells restored their pH gradient after the lactate exposure. Overall these results indicate that around 1.2% (36% of 3.5%) of the ampicillin-treated cells were metabolically active while only 0.029 ± 0.004% of the cells could grow, confirming that ampicillin-treated L. lactis MG1363 cultures contain viable but non-culturable (VBNC) cells
In this study we analyzed the response of L. lactis MG1363 to ampicillin using four different viability assays, which led to the identification of several subpopulations (Fig. 6)
Summary
Lactococcus lactis is an important organism in the dairy industry and it is increasingly explored as a cell factory for food ingredients and as a delivery vehicle for bioactive molecules[1,2]. We initially sought to select mutants that do not acidify the growth medium below a certain pH and intended to achieve this by exposing a partly acidified culture to ampicillin, thereby killing all cells that can grow in these conditions. Dormant, non-growing cells can survive treatments with antibiotics that target growth-related processes[8,9]. Persisters were first described in 1944 by Joseph Bigger[16] He showed that penicillin could not completely kill growing staphylococcal cultures and that surviving cells were as sensitive to penicillin as the parent culture. Persister subpopulations have been identified in several species, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Enterococcus faecalis and Mycobacterium tuberculosis[12,20,21,22]
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