Abstract
The aim of the work is to assess the antimicrobial activities of Cell Free Supernatants (CFS) and Membrane Vesicles (MVs), produced by Lactobacillus reuteri DSM 17938, versus Gram-positive and Gram-negative bacteria and investigate their metabolic profiles. The Minimum Inhibitory Concentration was determined through the broth microdilution method and cell proliferation assay while the Minimum Bactericidal Concentration was determined by Colony Forming Units counts. The characteristics of the antimicrobial compounds were evaluated by pH adjustments, proteinase treatment, and size fractionation of the CFS. The cytotoxicity of CFS was tested on two human cell lines. A detailed snapshot of the L. reuteri metabolism was attained through an untargeted metabolic profiling by means of high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with Electrospray Ionization Source (ESI). The results showed (i) a greater efficacy of CFS and its fractions towards Gram-negative compared to Gram-positive bacteria; (ii) an antimicrobial effect related to pH-dependent compounds but not to MVs; (iii) a molecular weight < 3 KDa as well as an a non-proteinaceous nature of the antimicrobial compounds; and (iv) more than 200 and 500 putative metabolites annotated in MVs and supernatants, covering several classes of metabolites, including amino acids, lipids, fatty and organic acids, polyalcohols, nucleotides, and vitamins. Some putative compounds were proposed not only as characteristic of specific fractions, but also possibly involved in antimicrobial activity.
Highlights
Probiotics have been defined by the World Health Organization as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”
Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured on Mueller-Hinton agar (MHA; Oxoid Ltd.) for 24 h in aerobic conditions; Fusobacterium nucleatum was cultured on Fastidious Anaerobe Agar (FAA; Lab M, Heywood, UK) and 5% (v/v) of defibrinated horse sterile blood (Oxoid Ltd.) for 48 h in anaerobiosis (Anaerogen Pak Jar, Oxoid Ltd.); and Streptococcus mutans was cultured on Columbia agar (CA; Oxoid Ltd.) and 5% (v/v) of defibrinated horse sterile blood (Oxoid Ltd.) for 24 h at 5% CO2
Membrane Vesicles (MVs) may deliver beneficial nutritional compounds such as proteins, vitamins, neurotransmitters precursors, as well as antimicrobial substances that reach host cells and play several roles that include the modulation of the immune host system, the protection of pathogens colonization, the interaction with the nervous systems, and other organs affecting the hosts in an efficient manner
Summary
Probiotics have been defined by the World Health Organization as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”. Lactobacillus reuteri, recently reclassified as Limosilactobacillus reuteri [5], colonizes the gastrointestinal tract of mammals and birds and are transmitted from one generation to another [6]. It has co-evolved with humans over millions of years [7]. As a member of gut microbiota, L. reuteri strains interact with the host in different ways, such as the stimulation of the immune system and the production of molecules or compounds that modulate the microbiota composition and prevent pathogen colonization [6]. Many studies demonstrated that several L. reuteri strains release various antimicrobial compounds capable of inhibiting the growth of pathogens in vitro [12,13]. It has been demonstrated that during its growth L. reuteri DSM 17938 produces Membrane Vesicles (MVs), defined extracellular vesicles, in both planktonic (p) and biofilm (b) phenotypes [15,18,19]
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