Impact of co-cultivation with Enterococcus faecalis over growth, enterotoxin production and gene expression of Staphylococcus aureus in broth and fresh cheeses

Abstract

Biocontrol of Staphylococcus aureus by lactic acid bacteria can be considered as a feasible alternative to the use of chemicals in foods, but the mechanisms underlying this antagonistic interaction remains poorly understood. This study aimed to evaluate the impact of a LAB species (Enterococcus faecalis) over the growth, enterotoxin production and gene expression of S. aureus under experimental conditions. E. faecalis 41FL1 and S. aureus ATCC 29213 were inoculated isolated and together in brain heart infusion (BHI) at 30 °C and in a fresh cheese model at 15 °C: microbial populations were monitored by culture plating, production of classical staphylococcal enterotoxins (SEs) was verified by an ELISA assay, expression of S. aureus genes (virulence, transcriptional regulation and central carbon metabolism) was investigated by quantitative real-time PCR, and pH and contents of water-soluble metabolites in both matrices were measured. S. aureus growth was inhibited in co-cultures assays, with a 2.02-log reduction in BHI and a 3.39-log reduction in cheese model compared to respective single cultures. Classical SEs were detected in S. aureus single culture assays (BHI and cheese), in BHI inoculated with both strains after 48 h of incubation, but not detected in co-inoculated cheeses. pH in all matrices containing E. faecalis reached lower values than in matrices containing S. aureus alone, due to lactate production by E. faecalis. Expression of genes coding for transcription regulators (ccpA and rex) and enzymes involved in central carbon metabolism (alsD and citZ) was mostly upregulated in co-inoculated cheeses, whereas expression of several virulence determinants (agrC, hld, hla, entA and spa) was strongly downregulated. This study provides relevant data on the behaviour of S. aureus in the presence of competing microbiota and support the use of controlled population dominance by LAB as an effective biopreservation strategy to ensuring food safety.

Keywords: Foodborne pathogen; HPLC; Lactic acid bacteria; Metabolism; Microbial interaction; Virulence; qPCR.