The effect of bacteriocin-producing Lactobacillus plantarum strains on the intracellular pH of sessile and planktonic Listeria monocytogenes single cells

Abstract

A wide range of lactic acid bacteria (LAB) produce bacteriocins mainly active against other closely related LAB, but some bacteriocins are also active against the food-borne pathogen Listeria monocytogenes. With the aim of increasing food safety it has thus been considered to utilise bacteriocins and/or bacteriocin-producing LAB as "natural" food preservatives in foods such as cheese, meat and ready-to-eat products. Some strains of Lactobacillus plantarum produce bacteriocins termed plantaricins. Using a single-cell based approach, the effect on the intracellular pH as a measure of the physiological state of sessile and planktonic L. monocytogenes (strains EGDe and N53-1) during co-culturing with plantaricin-producing L. plantarum (strains BFE 5092 and PCS 20) was investigated using fluorescence ratio imaging microscopy (FRIM). Mono-cultures of L. monocytogenes were used as control. Expression levels of plantaricin-encoding genes by sessile and planktonic L. plantarum were determined using qRT-PCR. L.plantarum BFE 5092 possesses the genes for plantaricin EF, JK and N, while L. plantarum PCS 20 contains the genes for plantaricin EF, although determination of the nucleotide sequence of the PCS 20 plantaricin E gene showed that this peptide is probably non-functional. When cultured as mono-culture, both L. monocytogenes strains maintained pH(i) at a constant level around 7.2-7.6 throughout the experiment, independently of the matrix. On a solid surface, L. plantarum BFE 5092 strongly affected pH(i) of L. monocytogenes N53-1 with only 20% of the cells being able to maintain pH(i) in the physiological optimal range with pH>7 and 52% of the cells with pH(i) approximately pH(ex,) showing that the cells had no proton gradient towards the environment. The effect on L. monocytogenes EGDe was less pronounced, but still notable. L.plantarum PCS 20 left both strains of L. monocytogenes virtually unaffected when co-cultured on a solid surface. In liquid, both L. plantarum strains strongly affected the physiological state of L. monocytogenes EGDe as judged by pH(i), whereas L. monocytogenes N53-1 was left virtually unaffected after 5h of co-culturing and after 8h 50% of the cells still maintained pH(i)>or=7. Higher concentrations of lactic acid were produced in liquid compared to a solid surface, and the different response of EGDe and N53-1 to the activities of the two L. plantarum strains probably reflect higher susceptibility of L. monocytogenes EGDe to organic acids compared to L. monocytogenes N53-1. Taken together, our results may be explained by the difference in the range of plantaricins produced by the two L. plantarum strains and matrix- and strain-related differences in the susceptibility of L. monocytogenes to plantaricins and organic acids. In conclusion, the present study represents the first demonstration of the ability of a bacteriocin-producing LAB to dissipate the proton gradient of sessile and planktonic L. monocytogenes.