Safety of the surrogate microorganism Enterococcus faecium NRRL B-2354 for use in thermal process validation

Abstract

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

FIG 1

Hierarchical clustering of 127 E. faecium genomes. Genome comparisons were based on the presence/absence of 7,017 orthologs found in the E. faecium pangenome. Nonclinical strains are labeled in blue, clinical strains in red, and strains with unidentified origins in black. An artificial outlier was included. Strain NRRL B-2354 is indicated by an asterisk. Red and green values in certain nodes indicate approximately unbiased and biased probabilities for the bootstrapping, respectively.

FIG 2

Alignment of 127 E. faecium genomes. (A) Circular alignment of E. faecium genomes. The outside of the circle is the NRRL B-2354 reference genome to which the other genomes are aligned. Genes identified as potential VF in NRRL B-2354 are indicated. Loci unique to NRRL B-2354 are also indicated (SP1 to SP5). (B) Key to circular alignment map. Gene types and strain origins are depicted in the same order (outwards to inwards) in the circular map. AR, antibiotic resistance genes; VF, virulence factors; ME, mobile genetic elements.

FIG 3

Numbers of AR, VF, and ME genes in strains with or without cas genes. Three types of genes, AR (A), VF (B), and ME (C), that have important roles in virulence of E. faecium were counted and compared statistically between CL and NC E. faecium strains (Student's t test). Strains with unidentified origins (UN) were also shown for reference. The arrow indicates the value of NRRL B-2354 in each panel.

FIG 4

NRRL B-2354 acm gene insertion and impaired collagen adherence. (A) Schematic diagram of the acm gene in E. faecium NRRL B-2354 and clinical strain E. faecium TX0082. The genes are 99% identical in the coding regions (gray); however, a 1,059-bp insertion sequence (hatched lines) is located 52 bp from the start codon in NRRL B-2354. (B) E. faecium binding to type I collagen. Absorbance was significantly higher for TX0082 than for the other two strains (Tukey's honestly significant difference [HSD], P < 0.05). The averages ± SD of three replicates per strain are shown.

FIG 5

Ebp locus and biofilm formation in E. faecium NRRL B-2354. (A) Schematic diagram of the ebp operon in E. faecium NRRL B-2354 and TX0082. (B) E. faecium biofilm formation on polystyrene. Absorbance was significantly higher for TX0082 than for the other two strains (Tukey's HSD, P < 0.05). The averages ± SD of three replicates per strain are shown.

FIG 6

Stress responses to low pH, heat, and ethanol. (A) Survival at pH 2.4. Numbers of viable cells of E. faecium NRRL B-2354, TX 0082, and 1,231,502 were determined at intervals of 10 min during incubation in physiological saline adjusted to pH 2.4. Detection limit was 20 CFU/ml. The averages ± SD of three replicates per strain are shown. (B) Thermal survival of E. faecium in physiological saline. Numbers of viable cells of E. faecium NRRL B-2354, TX 0082, and 1,231,502 were determined at 10-min intervals during incubation at 50°C (solid lines) and 60°C (dashed lines). Detection limit (black dashed line) was 20 CFU/ml. The averages ± SD of three replicates per strain are shown. (C) Ethanol tolerance of E. faecium. Growth of E. faecium strains NRRL B-2354, TX 0082, and 1,231,502 was monitored during incubation in 8% ethanol for 24 h with absorbance at OD₆₀₀ measured every 15 min. The averages ± SD of three replicates per strain are shown.