Linoleic Acids Overproducing Lactobacillus casei Limits Growth, Survival, and Virulence of Salmonella Typhimurium and Enterohaemorrhagic Escherichia coli.

Mengfei Peng,Zajeba Tabashsum,Puja Patel,Debabrata Biswas,Cassandra Bernhardt

doi:10.3389/fmicb.2018.02663

Abstract

Probiotics, particularly lactic acid bacteria, are biologic agents which limit the growth, virulence, and survival/colonization of various enteric bacterial pathogens and serve as potential alternatives to antibiotics. Mechanisms that contribute to this antimicrobial effect include producing bioactive metabolites/acids, increasing nutrient and receptor-mediated competition, and modulating gut microbiome ecology. However, these functions of common probiotic strains are limited due to the finite quantity of metabolites they produce and their total number in the gut ecosystem. Conjugated linoleic acids (CLAs), critical metabolites of Lactobacillus, have multiple beneficial effects on human health including anti-carcinogenesis, anti-inflammation, anti-oxidation, and anti-pathogenicity. In this study, we aim to overexpress the myosin cross-reactive antigen gene (mcra) in Lactobacillus casei (LC) to enhance the production of CLA and investigate its effectiveness against enteric bacterial pathogens, specifically Salmonella enterica serovar Typhimurium (ST) and enterohaemorrhagic Escherichia coli (EHEC). By inserting mcra in L. casei, we generated LC-CLA and found the total linoleic acid production by an individual bacterial cell was raised by 21-fold. The adherence ability of LC-CLA on human epithelial cells increased significantly and LC-CLA competitively excluded both ST and EHEC in a mixed-culture condition. Furthermore, LC-CLA significantly altered the physicochemical properties, biofilm formation abilities, interactions with host cells of both ST and EHEC, and triggered anti-inflammatory activities of host cells. These findings offer insights on applying a genetically engineered probiotic to control gut intestinal infections caused by ST and EHEC and prevent foodborne enteric illness in human.

Highlights

Human enteric microbial infections are principally characterized by diarrhea with or without other complications/consequences, which causes approximately 4–6 million deaths annually and possesses huge economic burden worldwide (Viswanathan et al, 2009; Christou, 2011)
In comparison with Lactobacillus casei (LC)-WT, LC-Conjugated linoleic acids (CLAs) maintained their in vivo growth/survival rate during exponential, stationary and death phases up to 96 h (Figure 2A) and remarkably (p < 0.05) improved their host cell adhesion ability onto human epithelial (INT-407) cells ex vivo (Figure 2B)
The genetically engineered probiotic strain LC-CLA induced significant (p < 0.05) up-regulation on mcra mRNA level expression identified by qPCR; with HPLCMS/MS analysis, we detected fold increment in relative total linoleic acids per 1 mL overnight cultural supernatant as well as even higher fold boost in relative total linoleic acids per bacterial cell (Table 3)

Summary

Introduction

Human enteric microbial infections are principally characterized by diarrhea with or without other complications/consequences, which causes approximately 4–6 million deaths annually and possesses huge economic burden worldwide (Viswanathan et al, 2009; Christou, 2011). The dominant causative agents of enteric bacterial diseases include Salmonella, enterohaemorrhagic Escherichia coli (EHEC), Campylobacter, Listeria monocytogenes, and Shigella (Viswanathan et al, 2009; Mor-Mur and Yuste, 2010; Forsythe, 2016; Huang et al, 2016). These enteric bacterial pathogens are typically acquired through contaminated foods and water; risk is always associated with these foodborne diseases for everyone living on this planet. The mixed concentration of by-produced lipid molecules in human colon is approximately 50–150 mM, and these beneficial lipid molecules are active and help modulate the host’s immune responses (Louis et al, 2014)

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