None of the above virulence factors have been reported in enterococci used in probiotics. Despite growing concerns over the pathogenicity of enterococci in hospital settings, no reports have demonstrated virulence factors in enterococcal probiotics. Domann et al. sequenced and compared E. faecalis contained in the probiotic Symbioflor 1^® to clinical VRE isolates. E. faecalis strains in Symbioflor 1 were found to lack gene coding for essential virulence factors, such as cyl and esp. The E. faecium T110, a component of the probiotic BIO-THREE^®, was sequenced completely and compared to pathogenic and non-pathogenic enterococcal strains. The gene encoding virulence factors were not found in the genome of E. faecium T110. The genome was noticeably different from the pathogenic strains found in hospitals, which included VRE and pathogenicity genes. Of the 40 enterococcal virulence genes established in the VFDB database (http://www.mgc.ac.cn/VFs/main.htm (accessed on 30 May 2023), 32 genes were missing in the T110 genome, affirming its safety as a non-pathogenic strain. The eight virulence genes present in the genome were not well-characterized and did not seem to contribute much to its pathogenicity ^[26][62].

2.3. Antibiotic Resistance

Avoparcin, a glycopeptide antibiotic similar to vancomycin, has been used as a growth promoter in livestock feed since the 1970s. The use of avoparcin coincided with the emergence of VRE outbreaks in hospitals. This chain of events suggests the possibility of VRE originating from antibiotic use in livestock, which subsequently spread to humans and hospitals through consumption ^[27][63]. Enterococcal species can be divided into two major categories based on their susceptibility to antibiotics: clade A and clade B. Clade B strains are susceptible to antibiotics (i.e., ampicillin and vancomycin) and, therefore, are not problematic in treatment against enterococci. Clade A strains, on the other hand, are hospital-derived strains that have diverged from Clade B strains and have adapted antibiotic resistance to various antibiotics, such as ampicillin and vancomycin ^[27][28][63,64]. Persistent use of antibiotics in hospitals and veterinary medicine has created multi-drug-resistant strains of enterococci that may be problematic [5]. These multi-drug resistant strains may spread in hospital settings, mainly through the hands of healthcare workers as well as medical equipment, leading to problematic infections ^[7][43]. Of particular concern is the increase in the incidence of vancomycin-resistant strains, with over 30% of all nosocomial enterococcal infections having resistance to this antibiotic as of 2019 (CDC). Vancomycin is often used to treat ampicillin-resistant pathogens ^[6][42]. Therefore, VRE, when pathogenic, has been associated with higher mortality in patients after undergoing hematopoietic cell transplantation ^[29][65]. Vancomycin resistance is encoded by specific gene clusters with nine variants (vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, vanN), which can be identified by PCR or DNA sequencing. ^[28][64]. The different variants display varying levels of resistance to vancomycin and similar glycopeptide antibiotics. VanA and B are commonly linked to high levels of resistance, whereas vanC, D, E, G, L, M, and N typically demonstrate resistance at lower levels. Each variant employs a unique mechanism to confer resistance to vancomycin. For instance, vanA, B, D, E, L, M, and N are associated with the production of modified peptidoglycan, resulting in reduced affinity towards vancomycin. On the other hand, vanC generates an altered target site that vancomycin struggles to bind effectively, while vanG combines the synthesis of modified peptidoglycan precursors with the alteration of the target site. Complete sequencing of two probiotic strains, E. faecium T110 (BIO-THREE^®) and E. faecalis (Symbioflor 1), showed the absence of antibiotic resistance genes ^[26][27][62,63]. Linezolid, daptomycin, and tigecycline are medications that can treat VRE. However, resistance to these three agents has emerged. According to the global Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) linezolid surveillance monitoring program, the number of linezolid-resistant enterococci (LRE) isolates has increased from 420 in 2002 to 813 in 2014. Similarly, the US Linezolid Experience and Accurate Determination of Resistance (LEADER) surveillance monitoring program reported that LRE faecium increased from 428 in 2004 to 589 in 2014, while LRE faecalis isolates increased from 196 to 239 in 2014 ^[30][66]. The prevalence of E. faecalis strains resistant to linezolid was found to be 2.8% in Asia, whereas, in the Americas, the prevalence of linezolid-resistant E. faecium was observed to be 3.4% ^[31][67]. The primary causes of LRE involve changes in the genetic material of the bacteria. These changes occur through mutations in specific genes responsible for producing 23S ribosomal RNA and regulatory genes that encode ribosomal proteins, namely rplC, D, and lV. These mutations result in the replacement of certain amino acids in various ribosomal proteins, including L3, L4, and L22 ^[32][33][34][68,69,70]. Daptomycin-resistant Enterococcus (DRE) has also been reported. Based on a meta-analysis in 2021 by Dadash et al., the prevalence of DRE (9%) is higher than that of LRE (2.2%). Multiple DRE mechanisms of resistance have been reported. However, two major categories of genes are found in DRE. These consist of regulatory genes for cell-envelope homeostasis and stress response, as well as genes that code for enzymes involved in the phospholipids of the cell membrane. Genes found in DRE faecalis are cls, liaFSR, and gdpD, whereas genes found in DRE faecium are cls, liaSR, and yycFG ^[35][71]. Tigecycline has been marked as a potential treatment option for complex soft tissue and intra-abdominal infections. However, it cannot be used for bloodstream infections due to inadequate antibiotic concentration in the bloodstream ^[36][72]. Dadash et al. also reported tigecycline-resistant Enterococcus (TRE) prevalence rates in Europe. TRE faecium was 3.9% and TRE faecalis was 0.4 ^[31][67]. Major genes that cause TRE are tet (M) and tet (L). Tet (M) is a ribosomal protection protein that alters the binding site of tigecycline, whereas tet (L) is an MFS-type efflux pump ^[35][36][71,72]. These tetracycline resistance genes also confer resistance to tigecycline ^[37][73]. Regarding the antibiotic-resistant risk of probiotic enterococci, no published research that sequences probiotic enterococci for the detection of these LRE, DRE, or DRE-related genes has been conducted.

2.4. Concern of Transfer of Virulence and Antibiotic Resistance

Enterococcal strains currently used in probiotics are not pathogenic, nor confer resistance to antibiotics. However, there is growing concern regarding the potential transfer of virulence and antibiotic-resistance genes between different enterococcal strains. Enterococci have a notable characteristic of possessing mobile genetic elements such as plasmids and transposons, which facilitate the efficient transfer of genes. This feature drives the evolution of certain strains, enabling them to adapt to different antibiotics found in clinical settings ^[38][74]. For instance, enterococcal strains can transfer genetic material like antibiotic resistance or virulence factors to each other or to other strains through the transfer of conjugative plasmids [5]. An example of such a transfer occurred in a study by de Niederhäusern et al., where they successfully transferred the vanA gene (associated with vancomycin resistance) from VRE to Staphylococcus aureus through the horizontal transfer of the Tn1546 transposon containing vanA. This discovery raised concerns about the horizontal transfer of vancomycin resistance, highlighting that VRE strains are capable of transferring their resistance to other pathogenic strains ^[39][75]. Another instance involved a clade B-classified E. faecium strain without vancomycin resistance. This clade B strain was found to possess the vanN gene and exhibited inducible vancomycin resistance. This finding suggests the potential transfer of vancomycin resistance from clade A to clade B strains, indicating that the extent of vancomycin resistance may be underestimated, especially in enterococcal strains previously described as lacking antibiotic resistance ^[28][64]. The highly efficient mechanism of gene transfer implies that a harmless enterococcal strain can acquire virulence or antibiotic resistance through conjugation with a pathogenic strain. In the case of probiotics, where enterococci are consumed in significant quantities, a large population of recipient bacteria is available for the transfer of virulence or antibiotic-resistance genes. This transfer can occur, for example, from pathogenic strains present in the human gastrointestinal tract to harmless probiotic strains ^[40][76]. Such transfer events can lead to the evolution of pathogenic or antibiotic-resistant probiotic strains, which can potentially cause problematic infections. Olanrewaju et al. showed that conjugal transfer of resistance genes could result in an effect of biofiltration in the guts of zooplankton Daphnia magna and D. pulex. PCR and DNA sequencing was used to confirm that filter feeding in aquatic environments could lead to in vivo conjugative transfer of vanA resistance genes in Daphnia. These results showed that host enterococcal strains in Daphnia can acquire vanA simply through the consumption of vanA-containing bacteria in the aquatic environment. Such conclusions raise the possibility of humans being the end host of resistant enterococcal strains through the food chain ^[41][77]. In addition, Moubareck et al. demonstrated in vitro and in vivo conjugative transfer of the vanA resistance gene from vancomycin-resistant enterococcal strains isolated from pigs to vancomycin-susceptible human fecal isolates in gnotobiotic mice. The transfer event occurred in human isolates only 5 h after inoculation with the donor strain, suggesting that human bacteria may be able to acquire vancomycin resistance from enterococci of animal origin in a short time frame ^[42][78]. These findings suggest that the transfer of vancomycin resistance to human hosts is possible. Therefore, it is crucial to carefully monitor enterococcal strains currently deemed “safe” for any potential emergence of virulence or antibiotic resistance. Genetic changes can render these strains pathogenic at any time point in the future ^[38][74].