The Effect of Lactiplantibacillus plantarum from Motal Cheese on the Adhesion of Cronobacter sakazakii to Epithelial Cells

Cronobacter sakazakii is an opportunistic pathogen, which has been linked to the contamination of powdered infant formula, and associated with outbreaks leading to fatalities in neonatal intensive care units. Few studies have explored the direct interaction between probiotics and C. sakazakii. In this study, the effect of a Lactiplantibacillus plantarum strain (M17) along with the standard strain Lactobacillus plantarum (ATCC 8014) and the well-characterized probiotic strain Lactobacillus rhamnosus GG on the adhesion of C. sakazakii to intestinal epithelial cells was analyzed.

Acid and bile tolerance of M17 was evaluated in the presence of pepsin and pancreatin. L-arginine hydrolysis was investigated using an arginine-including medium. Auto-aggregation and co-aggregation assays were performed by absorbance measurement. Minimum inhibitory concentrations of the antimicrobials recommended by the European Food Safety Authority were established. Total lactic acid and the ratio of D/L lactate isomers were determined with a Megazyme enzymatic kit. The ability of the isolate to produce biogenic amines was tested by qualitative and quantitative monitoring. Hemolysis was assessed phenotypically on MRS agar enriched with sheep blood. The strain was tested for its capability to adhere to mucin and Caco-2 cells. The antagonistic effects of the strain against C. sakazakii were further evaluated in vitro on mucin and cultured Caco-2 cells. The LAB strain was added simultaneously with, before, and after C. sakazakii to Caco-2 cells for competition, exclusion and displacement assays, respectively. Data analysis was performed in R using one-way analysis of variance, and the experimental groups were compared with the controls using Tukey’s test. P values <0.05 were considered statistically significant.

There was no significant difference in the survival rate of M17 and L. plantarum ATCC 8014 at pH = 4. After 2 h of incubation at pH = 2.5, the survival rate of L. plantarum ATCC 8014 was estimated to be higher than strain M17, but this difference was not significant. After 4 hours of incubation at pH = 8, M17 showed a higher survival rate than L. plantarum ATCC 8014, and this difference was significant after transfer from pH = 4. These results confirm the appropriate viability of M17 in the gastrointestinal tract. Both M17 and L. plantarum ATCC 8014 developed the color yellow in the L-arginine hydrolysis assay, which confirms the safety of these strains. The percentage of auto-aggregation for M17, L. plantarum ATCC 8014, and Lactobacillus rhamnosus LGG was estimated at 24.38, 25.28, and 32 after 6 hours, respectively, and no statistically significant difference between the two isolates were noticed. Given the auto-aggregation and co-aggregation parameters of M17, this strain may constitute a defense mechanism against C. sakazakii. Strain M17 showed resistance to kanamycin and clindamycin antibiotics. With intrinsic resistance, the risk of transferring resistance genes is not only speculative, but practically impossible. Intrinsic resistance of lactic acid bacteria may be considered desirable because it ensures their survival when the host is treated with antibiotics. Both D and L isomers of lactic acid were produced by the studied strains. In humans, D(-)-lactic acidosis is a rare metabolic complication that has only been reported in individuals with short bowel syndrome). Clinical studies have shown that the consumption of probiotic bacteria producing D(-)-lactic acid is safe for children and does not cause a long-term increase in blood D(-)-lactic acid. The reference L. plantarum strain and M17 did not produce biogenic amine precursors, and had no ß-hemolytic activity. Mucin adhesion assay exhibited that M17 has less adhesion (12.10 ± 1.14 %) than L. plantarum ATCC 8014 (13.33 ± 2.30 %) and LGG (15.93 ± 2.06 %) although these differences were not statistically significant. However, the amount of adhesion for the positive control sample Escherichia coli K12 (25.19 ± 4.40 %) was significantly higher than those of the other strains. Compared to the positive control, M17 had a significantly lower adhesion rate (6.8 ± 1.41) to CaCo-2 cells. This value was estimated at 13.77 ± 3.53 % for the reference strain and 21.6 ± 7.54 % for Lactobacillus fermentum PCC (positive control). In antagonistic assays, M17 was able to reduce the adhesion of C. sakazakii to mucin and CaCo-2 cells in all three methods of exclusion/inhibition, competition and displacement. Statistical analysis of the results does not show a significant difference between M17 and LGG. Therefore, the performance of M17 is similar to that of the standard probiotic LGG.

Lactic acid bacteria with acceptable ability to adhere to epithelial cells can be suitable for colonization in the intestine. They can act as a barrier to fight pathogens through various competitive mechanisms, such as co-aggregation with pathogens and adhesion. The M17 strain has an acceptable immune profile and probiotic properties because it shows an acceptable antagonistic activity against C. sakazakii invasion.