Repression of Staphylococcus aureus and Escherichia coli by Lactiplantibacillus plantarum Strain AG10 in Drosophila melanogaster In Vivo Model

Abstract

Probiotic bacteria exhibiting antagonistic activities against pathogenic bacteria are widely considered as potential options for the prevention and treatment of various infectious diseases and represent potential substitutes of antibiotics. Here we show that the L. plantarum AG10 strain represses the growth of Staphylococcus aureus and Escherichia coli in vitro and diminishes their negative effects in vivo in a Drosophila melanogaster model of survival on embryonic (larvae) and pupa stages. In an agar drop diffusion test, L. plantarum AG10 exhibited antagonistic properties against Escherichia coli, Staphylococcus aureus, Serratia marcescens and Pseudomonas aeruginosa, and repressed the growth of E. coli and S. aureus during milk fermentation. In a Drosophila melanogaster model, L. plantarum AG10 alone did not provide any significant effect, either during the embryonic stage or during further development of the flies. Despite this, it was able to restore the viability of groups infected with either E. coli and S. aureus, almost to the level of non-treated control at all stages of development (larvae, pupa and adult). Moreover, in the presence of L. plantarum AG10, pathogens-induced mutation rates and recombination events reduced 1.5-2-fold. The genome of L. plantarum AG10 was sequenced and deposited at NCBI under the accession number PRJNA953814 and consists of annotated genome and raw sequence data. It consists of 109 contigs and is 3,479,919 bp in length with a GC content of 44.5%. The analysis of the genome has revealed considerably few putative virulence factors and three genes responsible for the biosynthesis of putative antimicrobial peptides, with one of them exhibiting a high probability of antimicrobial properties. Taken together, these data allow the suggestion that the L. plantarum AG10 strain is promising for use in both dairy production and probiotics as a preservative from foodborne infections.

Keywords: Drosophila melanogaster; antimicrobial peptides; in vivo antagonistic activity; lactic acid bacteria.

Figure 1

Antimicrobial activity against E. coli (A,B) and S. aureus (C,D)and repression of pathogenic bacteria by either L. plantarum AG10 (A,C) or L. plantarum 8PA3 (B,D) during co-cultivation in fermented milk and subsequent storage at a temperature of 4 degrees for 21 days with control points on 1, 3, 7 and 21 days by the CFU counting method. Median values with interquartile ranges are shown.

Figure 2

Fluorescence microscopy of gut of third stage D. melanogaster larvae of L. plantarum strains developed on media with either E. coli or S. aureus. Representative images of gut samples taken from five larvae in each group in three independent experiments are shown.

Figure 3

Antagonistic effects of L. plantarum strains against pathogens (E. coli and S. aureus) at embryo development (A), pupa morphogenesis (B) and fertility (C) stages of D. melanogaster development. Insects were grown on medium either without or with solely Lactobacilli (greys), solely pathogens (reds) or pathogens with Lactobacilli (greens) as indicated on the left. Asterisks indicate statistically significant differences (p < 0.05) determined using one-factor analysis of variance (one-way ANOVA) statistical test with Holm-Sidak correction for multiple testing between flies grown either in the presence of solely pathogen or with additional presence of L. plantarum strain as indicated (n = 10).

Figure 4

Suppression of E. coli and S. aureus genotoxicity by L. plantarum strains against D. melanogaster. (A) frequency of spots (SMART-test), (B) DNA damage index (DNA comet test). Insects were grown on medium either without or with solely Lactobacilli (greys), solely pathogens (reds) or pathogens with Lactobacilli (greens), as indicated on the left. Asterisks show statistically significant differences (p < 0.05) determined using one-factor analysis of variance (one-way ANOVA) statistical test with Holm–Sidak correction for multiple testing between flies grown in presence of solely pathogen and in addition of L. plantarum strain as indicated ((A) n = 10, (B) n = 30–50).