Screening probiotic strains for safety: Evaluation of virulence and antimicrobial susceptibility of enterococci from healthy Chinese infants

The aim of this study was to evaluate the safety of enterococci isolated from Chinese infants and screen out potential probiotic candidates. One hundred eight strains were isolated from feces of 34 healthy infants, and 38 strains of Enterococcus spp. were categorized as follows: E. faecalis (22), E. faecium (10), E. hirae (3), E. durans (2), and E. casseliflavus (1). Of these, 72.7% of E. faecalis came from infants delivered by cesarean and 62.5% of E. faecium from infants delivered vaginally. For safety evaluation of strains, we determined presence of virulence genes; production of hemolysin, gelatinase, and biofilm; and antimicrobial susceptibility of enterococci. Six out of 14 virulence genes were detected with a distribution of gelE (26.3%), cylA (39.4%), esp (15.8%), efaA (63.2%), asa1 (50.0%), and ace (50.0%). In phenotype analysis, 36.8% of the strains exhibited positive hemolytic activity and 17.5% were positive for production of gelatinase. Results of antimicrobial susceptibility showed that different percentages of the strains were resistant to ciprofloxacin (5.2%), vancomycin (7.8%), rifampicin (10.5%), erythromycin (52.6%), and gentamycin (52.6%); remarkably, none of the strains were resistant to ampicillin or chloramphenicol. In total, 10 strains, including 6 E. faecium, which are free of virulence determinants and sensitive to common antimicrobial agents (e.g., ampicillin and vancomycin), were further assessed for their probiotic properties. All strains survived well in simulated gastric fluid and intestinal tract, with maximum reductions of 0.600 and 0.887 log cfu/mL, respectively. Six strains of E. faecium could resist 0.3 to 1.0% bile salt, of which E. faecium WEFA23 presented the highest growth (75.06%) at 1.0% bile salt. All strains showed bile salt hydrolase activity on glycodeoxycholic acid, but only 3 of E. faecium showed activity on taurodeoxycholic acid. These results deliver useful information on the safety of enterococci in infants in China, and provide a protocol to screen probiotics for absence of virulence and antimicrobial susceptibility of enterococci.