Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion

Abstract

Background: One of the working mechanisms of probiotic bacteria is their ability to compete with pathogens. To define the probiotic properties of seven Lactic Acid Bacteria (LAB) strains, we tested them for survival in simulated gastro-intestinal conditions, antimicrobial activities, co-aggregative abilities, and interferences studies against five human intestinal pathogens (Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644, Escherichia coli O157: H7 ATCC 35150, Cronobacter sakazakii ATCC 29544 and Campylobacter jejuni ATCC 33291).

Results: The LAB strains were able to survive the stomach simulated conditions, and varied in their abilities to survive the small intestinal-simulated conditions. The strains showed antibiotic susceptibility profiles with values equal or below the breakpoints set by the European Food and Safety Authority. The LAB cell-free cultures supernatants showed antimicrobial activities, with inhibition zones ranging from 10.0 to 17.2 mm. All the LAB strains showed moderate auto-aggregation abilities while the greatest co-aggregation abilities were observed for Bifidobacterium bifidum W23, Lactobacillus plantarum W21 and Lactobacillus rhamnosus W71. The individual LAB strains showed strain-specific abilities to reduce the invasion of intestinal pathogens in an interference model with Caco-2 cells. Increased invasion inhibition was found when different combinations of LAB strains were used in the interference tests.

Conclusion: The LAB strains examined in this study may protect the intestinal epithelium through a series of barriers (antimicrobial activity, co-aggregation with pathogens, adherence) and interference mechanisms. Consequently, these LAB strains may be considered candidates for prophylactic use to prevent intestinal infections.

Keywords: Gut pathogens; Interference; Lactic acid bacteria; Probiotic properties.

Fig. 1

In vitro GI survival data of LAB strains. Each lyophilized strain (2 g, 10⁹ CFU/gr) was rehydrated in 100 ml of demineralized water for 15 min at room temperature (baseline). The rest of the experiment was performed at 37 °C. The stomach was simulated by adding 1 ml of porcine pepsine solution and decreasing the pH in four steps of 15 min to 4.8, 4.5, 3.5 and 2.5. After 75 min (stomach), the entry into the proximal duodenum was mimicked by increasing the pH to 6.5 by adding 0.1 N NaOH and, after 90 min, 10 ml of porcine bile extract solution and 2 ml of porcine pancreatin solution were added. After 3 h (duodenum), bile salts were deactivated by adding 11.5 mM of calcium chloride. The pH was maintained at 6.5 until 6 h (ileum) which was the end of the experiment. The experiments have been performed in triplicate

Fig. 2

Adhesion ability of the different LAB strains to the human Caco-2 cell monolayers. Caco-2 monolayers were incubated for 1 h with a LAB strain. Thereafter the supernatants were discarded and the monolayers were washed to remove the non-attached bacteria. Then the monolayers were trypsinized to release the eukaryotic cells and adhered bacteria and the bacteria were plated on MRS plates. Results are expressed as the percentage of bacteria adhered with respect to the amount of bacteria added (% CFU bacteria adhered/CFU bacteria added). The experiments have been performed in triplicate

Fig. 3

Invasion inhibition of human intestinal pathogens by single LAB strains and their four combinations. a S. enteritidis ATCC 13076, b L. monocytogenes ATCC 7644, c E. coli O157: H7 ATCC 35150, d C. sakazakii ATCC 29544, e C. jejuni ATCC 33291. Asterisks represented values statistically significant (p < 0.05) in comparison to the control group (Kruskal–Wallis non parametric test)