Lactobacillus acidophilus AG01 and Bifidobacterium animalis AG02 reduce Clostridium perfringens pathogenicity through different and complementary modes of action
Necrotic enteritis is an economically important disease in poultry production caused by the opportunistic pathogen Clostridium perfringens. With the phasing out of antibiotics for prophylaxis and growth promotion, alternative solutions are needed to prevent diseases. We investigated the effect of two potentially beneficial bacteria, Lactobacillus acidophilus AG01 and Bifidobacterium animalis subspecies lactis AG02, on C. perfringens pathogenicity and observed two distinct but complementary modes of action. Live L. acidophilus AG01 cells had a strong deleterious effect on the survival of C. perfringens 25037-CP01 when grown in co-cultures, likely caused by the production of lactate resulting in the acidification of the media. Using our recently developed single cell RNA-seq method for bacteria, ProBac-seq, we observed a strong effect of the cell-free supernatant (CFS) of both bacteria on overall gene expression and the expression of virulence factors in C. perfringens 25037-CP01. Cells exposed to 5% CFS of B. animalis AG02 were enriched in RNA polymerases, and genes involved in oxidative phosphorylation and fatty acid biosynthesis. Cells exposed to 5% B. animalis AG02 or 3% L. acidophilus CFS showed significant expression reduction of netB, the virulence factor associated with necrotic enteritis. Five other virulence factors had significantly reduced expression in cells exposed to 5% B. animalis AG02 CFS. Epithelial HT-29 cells suffered significantly lower cytotoxicity when exposed to C. perfringens 25037-CP01 cells grown during exposure to 5% B. animalis AG02 CFS. These findings show that interactions among bacteria can reduce pathogenicity of an opportunistic pathogen. The two bacteria tested here reduce C. perfringens pathogenicity in different ways and could be used as an alternative to the administration of preventative  antibiotics. Overall design: Before microfluidic encapsulation, bacteria were fixed in 1% paraformaldehyde to preserve transcripts and permeabilized by mild lysozyme treatment. Permeabilized bacteria were then incubated with the corresponding DNA probe library. Non-hybridized probes were removed with repeated washes . Next, the bacteria were run through a 10X Chromium Controller, where the DNA probes were captured and barcoded in a manner analogous to the barcoding of the transcriptome of eukaryotic cells