New Insights into the Virulence Traits and Antibiotic Resistance of Enterococci Isolated from Diverse Probiotic Products

The GRAS (generally recognized as safe) status of Enterococcus has not yet been authenticated, but enterococci, as probiotics, have been increasingly applied in human healthcare and animal husbandry, for instance as a dietary supplement, feed additive, or growth promotor. The food chain is the important route for introducing enterococci into the human gut. The pathogenicity of Enterococcus from probiotic products requires investigation. In the study, 110 commercial probiotic products used for human, animal, aquaculture, and plants were examined, among which 36 enterococci were identified, including 31 from Enterococcus faecium, 2 from E. faecalis, 2 from E. casseliflavus, and 1 from E. gallinarum. Strikingly, 28 of the 36 enterococci isolated from probiotics here did not mention the presence of Enterococcus in the labeled ingredients, and no Enterococcus isolates were found from 5 animal probiotics that were labeled with the genus. In total, 35 of the 110 products exhibited hemolysis, including 5 (10.6%) human probiotics, 14 (41.2%) animal probiotics, 8 (57.1%) aquaculture probiotics, and 8 (53.3%) plant probiotics. The detection rates of virulence factors associated with adhesion, antiphagocytosis, exoenzyme, biofilm, and other putative virulence markers (PVM) in 36 enterococci were 94.4%, 91.7%, 5.6%, 94.4% and 8.3%. Twenty-six of the 36 isolated strains exhibited biofilm formation ability, where 25 strains (69.4%) and one (2.8%) were strong and weak biofilm producers, respectively. We analyzed the resistance rates against erythromycin (97%), vancomycin and ciprofloxacin (8%), tetracycline (3%), and high-level aminoglycosides (0%), respectively. High detection rates of msrC/lsaA (86%) and aac(6′)-Ii (86%) were observed, followed by vanC (8%), tetM (3%). The Tn5801-tetM-like integrative conjugative element (ICE) was identified in E. gallinarum, exhibiting resistance to tetracycline (64 μg/mL). Seven probiotic E. faecalis and E. faecium, as active ingredients in human probiotics, shared the same STs (sequence types) and were distinct from the STs of other contaminated or mislabeled enterococci, indicating that two particular STs belonged to native probiotic isolates. These findings advocate appropriate assessments of enterococci when used in probiotics.


Human
Fuyang, Anhui Note: (a) Probiotic products exhibiting hemolysis were in bold and underlined. (b) -, the active ingredient was not identified in the product; Ingredients of product contained Enterococcus spp. were in bold and underlined. Table S2. Brief introduction of virulence factor function.

Related factors Function
Ace Mediating binding to immobilized collagen type I, collagen type IV, and mouse laminin.

Acm
Interacts with collagen type I and to a lesser extent with collagen type IV.
Bee or Srt Biofilm enhancer.

BopD
Homologous to a sugar-binding transcriptional regulator involved in biofilm production; The actual role is unknown, but the association of enhanced biofilm formation in the presence of glucose and the possible involvement of a sugar-binding transcriptional regulator suggest a linkage to increased biofilm production in E. faecalis in the presence of specific carbohydrates.

CdsA
Phosphatidate cytidylyltransferase catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin, contributes to capsule synthesis and daptomycin resistance.

Ebp
Ebp pili are important for adherence to host extracellular matrix proteins, including fibrinogen and collagen and play a role in biofilm formation.

EcbA
Binds to collagen type V.

EfaA
Might be functioning as an adhesion in endocarditis; A solute binding-protein receptor for manganese transport system.

Esp
Contributes to colonization and persistence of E. faecalis in urinary tract infections and also associates with promotion of primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Fsr
The Fsr quorum sensing system is an important regulator with both positive and negative effects, regulating gelE, sprE and bopD expression that are important for biofilm formation, along with genes implicated in several metabolic pathway.
Fss microbial surface components recognizing adhesive matrix molecules.

GelE
Capable of degrading a broad spectrum of substrates, including casein, hemoglobin, collagen, fibrin, gelatin, certain E. faecalis sex-pheromone-related peptides and polymerized fibrin; May functioning as clearing the bacterial cell wall of misfolded proteins, and disruption of gelE gene has been shown to increase the bacterial chain length; May play a role in increasing dissemination of bacteria in high density environments; A gelE knockout has shown reduced virulence in models of mouse peritonitis, rabbit endophthalmitis, and in a Caenorhabditis elegans virulence model.

IS16
Transposase enriched in hospital-associated strains; contributes to the genomic plasticity of E. faecium.
Hyaluronidase (Hyl) An important pathogenic bacterial spreading factor, and cleave hyaluronan, which is a constituent of the extracellular matrix of connective tissues; May also pave the way for deleterious effects of other bacterial toxins, thus increasing the magnitude of the damage; Another role may be to supply nutrients for the bacteria, since the degradation products of its target substrates are disaccharides that can be transported and metabolized intracellularly be bacteria.
orf2514 & orf2515 pseudogene encoding a surface B-type Cna protein, enhance adherence to extracellular matrix molecules.
PGC-1 PGC-1 is unique among Efm pilus loci as it also contains a housekeeping class A sortase and may play a role during colonization or pathogenesis in the mammalian host.
PGC-2 A pili cluster similar to Ebp from E. faecium.
PGC-3 may play a role during colonization or pathogenesis in the mammalian host but their biological functions remain to be determined.
PGC-4 putative role in biofilm formation and adhesion. Note: (a) The MICs over the breakpoint were in bold and italic; no Enterococcus of high-level gentamicin resistance (HLGR) or high-level streptomycin resistance (HLSR) was found.