Safety of Enterococcus faecium NRRL B-2354 (ATCC 8459) for Use as a Surrogate in Thermal Process Validation

Introduction: Enterococcus faecium strain NRRL B-2354 (ATCC8459) has been used for over 40 years by the food industry. It is now most commonly applied as a surrogate for Salmonella during challenge testing of thermal processing methods used in the production of dry foods, most notably almonds. However, recent increases in nosocomial infections caused by E. faecium and a rise in antibiotic resistance among some strains have raised questions about the safety of strain NRRL B-2354 (ATCC8459).

Purpose: The purpose of this study was to evaluate the safety of E. faecium NRRL B-2354 (ATCC8459) using both genomic and phenotypic approaches.

Methods: The genomes of E. faeciumNRRL B-2354 and the highly related strain ATCC8459 were sequenced and annotated. The strains were also examined and compared to clinical isolates for their capacity to grow in the presence of medically-relevant antibiotics, produce gelatinase and hemolysin, form biofilms on polystyrene, adhere to extracellular matrix proteins, and survive at high temperatures (>45^oC) and at low pH (pH 2.4).

Results: The genomes of E. faecium NRRL B-2354 and ATCC8459 are nearly identical, are approximately 2.8 Mbp, and include one large (>200 kb) extra-chromosomal plasmid. Hierarchical clustering of genome content showed their distinction from clinical isolates. Both strains lack the majority of virulence factors (acm, cyl, ebp, esp, gelE, hyl, and IS16) and antibiotic resistance loci known for this species. Neither strain produced gelatinase, hemolysin, or pili nor were they able to adhere to collagen, fibronectin, or fibrinogen or form biofilms. NRRL B-2354 and ATCC8459 are also tolerant to adverse environmental conditions (heat and pH) and sensitive to antibiotics used to treat Enterococcus infections.

Significance: The genetic and phenotypic characteristics of E. faecium NRRL B-2354 (ATCC8459) are distinct from clinical strains and support its use as a surrogate in thermal challenge tests.