Influence of cell-free extracts of Bifidobacterium bifidum and Lactobacillus reuteri on proliferation and biofilm formation by Escherichia coli and Pseudomonas aeruginosa
Authors: Knysh O. V. , Isayenko O. Y. , Voyda Y. V. , Kizimenko O. O. , Babych Y. M. 
Published: September 2019

The development of new effective preparations for the correction of microecological disorders based on probiotic derivatives requires a comprehensive study of the biological activity of the latter. We studied the proliferative activity and biofilm formation by clinical isolates: Escherichia coli and Pseudomonas aeruginosa under the influence of cell-free extracts containing structural components and metabolites of the Bifidobacterium bifidum and Lactobacillus reuteri probiotic strains. Cell-free extracts were obtained from disintegrates and cultures of probiotics. Disintegrates were prepared by cyclic freezing-thawing of probiotic cell suspensions. The cultures were obtained by cultivating probiotic microorganisms in their own disintegrates. The obtained disintegrates and cultures were filtered. The proliferative activity of the test cultures was studied using the spectrophotometric microtiter plate method after an hour-long exposure in undiluted cell-free extracts and subsequent cultivation in a nutrient medium containing 30%vol of the studied extracts at 37 °C for 24 hours. The biofilm formation of the test cultures was studied with 30% vol content of cell-free extracts in the cultivation medium using the spectrophotometric microtiter plate method. All the studied extracts exerted a similar effect on the proliferative activity and biofilm formation by E. coli and P. aeruginosa. Exposure of the test cultures in all undiluted extracts during an hour led to a significant decrease in the optical density of the test samples: optical density of the test wells ranged from 36.5% to 49.8% of the control wells. The test cultures that were exposed to the extracts: filtrate of L. reuteri disintegrate (L), filtrate of В. bifidum disintegrate (B) and filtrate of В. bifidum culture, grown in В. bifidum disintegrate (MB) after dilution and subsequent cultivation over the next 24 hours completely restored the ability to proliferate. The proliferative activity of the test cultures that were exposed to the extracts: filtrate of L. reuteri culture, grown in L. reuteri disintegrate (ML) and filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose (MLG), was significantly inhibited after dilution and subsequent cultivation. The inhibition indices calculated for the ML extract were: 25.9% (E. coli) and 53.0% (P. aeruginosa). Inhibition indices calculated for the MLG extract were: 62.0% (E. coli) and 96.9% (P. aeruginosa). MLG extract had more pronounced inhibitory effect on the proliferation of the test cultures than ML extract. All the studied extracts exerted significant inhibitory effect on the biofilm formation of the test cultures. Analysis of the results of the study shows that cell-free extracts of L. reuteri culture grown in its disintegrate without supplementation or supplemented with glycerol and glucose have the highest antimicrobial activity and can be used as metabiotics to prevent overgrowth of potentially pathogenic bacteria, as well as inoculation and proliferation of pathogenic gram-negative bacteria in the gastrointestinal tract. They can be used alone or in combination with cellular probiotics to enhance their probiotic action. This study encourages further careful investigation of the biochemical composition of cell-free extracts and clarifying the mechanism of their action.