Ampicillin-treated Lactococcus lactis MG1363 populations contain persisters as well as viable but non-culturable cells

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.

A Novel Method for Long-Term Analysis of Lactic Acid and Ammonium Production in Non-growing Lactococcus lactis Reveals Pre-culture and Strain Dependence

“…When prolonged translation inhibition is applied, cell death is inevitable and can be responsible to the decline in product formation to some extent. Antibiotic exposure to Lactococcus lactis has also been reported to induce heterogenous population response regarding dormancy states and the corresponding death rates and metabolic activity ( van Tatenhove-Pel et al, 2019 ). To characterize the effects of erythromycin in our experiments, a combination of CFU counting and live-dead staining measurements was employed to determine how cellular viability and integrity relate to the observed decline in acidification.…”

Microbial competition reduces metabolic interaction distances to the low µm-range

“…All the strains that were used are listed in Table 1 . L. lactis NZ9000 strains PTSman_GFP, PTScel_GFP, and GlcU_GFP were obtained by a single-crossover integration of vector pSEUDO:: Pusp45-gfp [ 22 ] into the pseudo 10 locus on the chromosome of L. lactis NZ9000∆ ptcC ∆ GlcU , NZ9000∆ ptnABCD ∆ GlcU , and NZ9000∆ ptnABCD ∆ ptcC [ 23 ], respectively. Integration was performed as previously described [ 24 ].…”

“… [ 28 ] MG1363_GFP L. lactis MG1363 carrying pSEUDO:: P usp45 -gfp . [ 22 ] NZ9000_PTSman_GFP Ery r , NZ9000 ∆ptcC∆GlcU carrying pSEUDO:: P usp45 -gfp . This work NZ9000_PTScel_GFP Ery r , NZ9000 ∆ptnABCD∆GlcU carrying pSEUDO:: P usp45 -gfp .…”

“… This work Plasmids Description Reference pSEUDO:: P usp45 -gfp Ery r , integration vector, pSEUDO:: P usp45 -sfgfp(Bs) derivative, carrying the gene coding for the green fluorescent protein (Dasher-GFP). [ 22 ] …”

Lifestyle, metabolism and environmental adaptation inLactococcus lactis

“…In addition, the contribution of VBNC cells to bioconversion of raw materials has been demonstrated, where for example, it was shown that VBNC Lactobacillus hilgardii can contribute to malolactic fermentation occurring during apple cider production (Quirós et al 2009). Recently, it was described that exposure of L. lactis MG1363 to ampicillin leads to the emergence of both VBNC and persister subpopulations (van Tatenhove-Pel et al 2019). Ampicillininduced persister cells represented only a minor subpopulation (0.3%) when exponentially growing cultures were used, but increased substantially (7.6%) when cells were obtained from the stationary phase of growth (van Tatenhove-Pel et al 2019).…”