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Objective:  This study aimed to search for  Enterococcus faecalis  in children’s deep carious dentine and characterize their virulence traits. Material and Methods:  Eight isolates from 15 carious molars identified by 16S rDNA species-specific PCR as  E. faecalis  were included. These eight isolates were subject to identification by MALDI‐TOF and characterized regarding: (i) bacterial aggregation and biofilm formation on polystyrene and glass, with/without saliva, as single or dual-species (associated to  Streptococcus mutans ); (ii) environmental pH measurement before and after 24 h incubation; (iii) acidogenicity; (iv) gelatinase production; (v) macrophage adherence; and (vi) toxicity towards  Caenorhabditis elegans . Statistical analyses were performed using two-way ANOVA/Tukey or Fisher’s exact tests. Results:  All isolates initially identified as  E. faecalis  by PCR were correctly identified as  Lactobacillus  by MALDI-TOF, being designated as  Lactobacillus  misidentified as  Enterococcus  (LME). These isolates produced biofilm in the presence of saliva and in the dual-species assays. Bacterial aggregation was only observed in the dual-species model. After 24 h, environmental pH dropped from 7.5 to 4.5 for seven of eight isolates, and to 4.0 in all dual-species models. LME isolates were acidogenic, none of them produced gelatinase or adhered to macrophages, but all presented toxicity towards  C. elegans . Conclusions:  No  E. faecalis  were identified in the children’s caries lesions. All LME isolates presented important virulence traits, including biofilm formation and high acidogenicity, which cause enamel demineralization, that might increase the risk of dental caries in children carrying LME. Thus, the correct identification and in-depth virulence characterization of microorganisms isolated from dental caries are important to understand the dynamics of this disease.

Virulence of Lactobacillus spp. misidentified as Enterococcus faecalis from children’s carious dentine