AUTHOR=Liu Gang , Kragh Martin Laage , Aabo Søren , Jensen Annette Nygaard , Olsen John Elmerdahl 

TITLE=Inhibition of Virulence Gene Expression in Salmonella Dublin, Escherichia coli F5 and Clostridium perfringens Associated With Neonatal Calf Diarrhea by Factors Produced by Lactic Acid Bacteria During Fermentation of Cow Milk

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.828013

DOI=10.3389/fmicb.2022.828013

ISSN=1664-302X

ABSTRACT=<p>Diarrhea is a major health problem in neonatal and young calves worldwide. It can be caused by a variety of infectious agents, including the bacteria <italic>Salmonella enterica</italic> serovar Dublin (<italic>S</italic>. Dublin), enterotoxigenic <italic>Escherichia coli</italic> (ETEC), and <italic>Clostridium perfringens</italic>. Preventive alternatives to antibiotic treatment should be identified. As a first step toward this, the aim of the current study was to examine whether cell-free supernatants from cow milk fermented by lactic acid bacteria affects virulence-gene expression in strains of <italic>S</italic>. Dublin, ETEC <italic>E. coli</italic> F5 and <italic>C. perfringens</italic>. pH-neutralized, cell-free, spent medium of milk (nCFSM) fermented by 61 different lactic acid bacteria (LAB) and non-LAB starter cultures belonging to 17 genera was assayed for their effect on expression of important virulence factors (<italic>S</italic>. Dublin <italic>hilA, ssrB, ssaG, flhD, prgI, fliC</italic>; ETEC <italic>E. coli</italic> F5 <italic>fanC, estA, fim41a</italic>; <italic>C. perfringens cpa</italic>), when the bacteria were grown in the nCFSM. Screening was done using either a promoter-reporter expression system or RT-qPCR. nCFSM from <italic>Bifidobacterium longum</italic> BL-15955 and <italic>Limosilactobacillus reuteri</italic> LR-33016 downregulated the expression of <italic>fanC, fim41a</italic> and <italic>estA</italic> genes in the four tested ETEC <italic>E. coli</italic> F5 strains without affecting their growth, while mainly <italic>B. longum</italic> BL-15955 downregulated expression of <italic>cpa</italic> in the four tested strains of <italic>C. perfringens</italic>. nCFSM from the mixed cultures; NU-TRISH<sup>®</sup> BY-Mild (<italic>Lactobacillus delbrueckii</italic> subsp. <italic>bulgaricus, Streptococcus thermophilus</italic> and <italic>Bifidobacterium</italic> BL-15954) and COMBO4 (<italic>Lactobacillus delbrueckii</italic> subsp. <italic>bulgaricus</italic> and <italic>Streptococcus thermophilus</italic>), as well as <italic>Lactobacillus helveticus</italic> CNRZ32 downregulated the tested virulence genes in the three tested strains of <italic>S</italic>. Dublin. To enable possible downregulation of the expression of virulence genes in all three target bacteria simultaneously, nCFSM was prepared from NU-TRISH<sup>®</sup> By-Mild in combination with <italic>B. longum</italic> BL-15955 (i.e. a four-strain combination). The nCFSM from this combination downregulated the virulence genes expression in all the three species. In the future, NU-TRISH<sup>®</sup> By-Mild and <italic>B. longum</italic> BL-15955 in combination could potentially be used for prevention of neonatal calf diarrhea caused by <italic>S</italic>. Dublin, <italic>E. coli</italic> F5, and <italic>C. perfringens</italic>, reducing the need for antimicrobial treatment, however, field studies are needed to prove that.</p>