The Antibacterial Efficacy of a Compound Extracted from Marine Sediment Bacterium Enterococcus Lactis (S-2): A Comparative Analysis Through In-Vitro and In-Silico Assessments.

BACKGROUND: Marine sediment bacteria have been generating considerable attention lately due to their potential as valuable reservoirs of novel antimicrobial agents. AIM: In vitro and in silico antibacterial activities of antibacterial compounds isolated from the marine sediment bacterium Enterococcus Lactis (S-2). METHODS: Coastal sediment samples were collected from Rameswaram, Ramnathapuram District, Tamil Nadu, India. Bacteria were isolated using the crowded plate method, and their phenotypic and genotypic characteristics were studied. Purified bacteria were cultured in large volumes, secondary metabolites were extracted, and novel antibacterial agents were isolated from the aqueous extract. Novel compound antibacterial activity was studied through in-silico and invitro. The mechanism activity of antibacterial activity was confirmed by a high-resolution transmission electron microscope. RESULTS: Genotypic analysis confirmed that the isolated S-2 bacteria were Enterococcus lactis, and the aqueous extract showed antibacterial activity against Staphylococcus aureus (17 mm zone of inhibition) and Proteus mirabilis (12 mm zone of inhibition). A bioactive molecule, 13- hydroxy-9-(1-hydroxyethyl)-11-methoxy-2,4dioxapentacyclo[10.7.1.0³,4.05,²¹.0¹³,¹6]icosa- 1(20),5,7,12,14(19), 16-hexane-18-one, was isolated from aqueous extracts of the S-2 bacterium. Chromatography and spectroscopic analysis confirmed the identity of the isolated compound. Novel compound potential antibacterial activity showing against S. aureus (18 mm zone of inhibition) and MIC 250 µg/mL, which was confirmed by tetrazolium staining. The antibacterial activity mechanism was confirmed by transmission electron microscopy. Molecular docking studies show good binding (-9.9 kcal/mol) of the compound with 3U2D, while molecular dynamic simulation studies confirm the conformationally stable structure of the complex between 3U2D and 13-hydroxy-9-(1-hydroxyethyl)-11-methoxy-2,4-dioxapentacyclo [10.7.1.0³,4.05,²¹.0¹³,¹6]icosa-1(20),5,7,12,14(19), 16-hexane-18-one. It has been observed from the docking study of 3U2D with standard drug ciprofloxacin that the lower affinity is compared to the test ligand, which has a docking score of 7.3 kcal/mol. Out of interacting residues of protein 3U2D residue, Thr173 and Ile86 formed conventional hydrogen bonds. CONCLUSION: Marine bacterium E. lactis produces a novel antibacterial compound (13-hydroxy- 9-(1-hydroxyethyl)-11-methoxy-2,4-dioxapentacyclo[10.7.1.0³,4.05,²¹.0¹³,¹6]icosa- 1(20),5,7,12,14(19),16-hexane-18-one), which shows antibacterial activity against clinical S. aureus, confirmed by in vitro and in silico analysis. This molecule can used as a lead molecule for antibacterial activity.

Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium.

Enterococcus faecium is a gut commensal bacterium which is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition-dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,ß-hydrolases, SGNH-hydrolases, phospholipases, and amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.

Assessment of the feed additive consisting of Enterococcus lactisNCIMB 10415 (Cylactin®) for cats and dogs for the renewal of its authorisation (DSM Nutritional Products Ltd.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of the authorisation of Cylactin® as a zootechnical feed additive for cats and dogs. The active agent of the additive is Enterococcus lactis NCIMB 10415 and the micro-encapsulated formulation, Cylactin® LBC ME5 PET, was assessed. The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. The Panel concluded that the additive remains safe for cats and dogs. Regarding user safety, the additive was not shown to be skin and eye irritant, but it should be considered a respiratory sensitiser. No conclusions can be drawn on the skin sensitisation. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Genome-based taxonomic identification and safety assessment of an Enterococcus strain isolated from a homemade dairy product.

The aim of this study was to explore the taxonomic identification and evaluate the safety of a bacterium, Enterococcus lactis IDCC 2105, isolated from homemade cheese in Korea, using whole genome sequence (WGS) analysis. It sought to identify the species level of this Enterococcus spp., assess its antibiotic resistance, and evaluate its virulence potential. WGS analysis confirmed the bacterial strain IDCC 2105 as E. lactis and identified genes responsible for resistance to erythromycin and clindamycin, specifically msrC, and eatAv, which are chromosomally located, indicating a minimal risk for horizontal gene transfer. The absence of plasmids in E. lactis IDCC 2105 further diminishes the likelihood of resistance gene dissemination. Additionally, our investigation into seven virulence factors, including hemolysis, platelet aggregation, biofilm formation, hyaluronidase, gelatinase, ammonia production, and ß-glucuronidase activity, revealed no detectable virulence traits. Although bioinformatic analysis suggested the presence of collagen adhesion genes acm and scm, these were not corroborated by phenotypic virulence assays. Based on these findings, E. lactis IDCC 2105 presents as a safe strain for potential applications, contributing valuable information on its taxonomy, antibiotic resistance profile, and lack of virulence factors, supporting its use in food products.

Preparation and Mechanistic Exploration of Fermented Shrimp Surimi Gel Utilizing Enterococcus lactis S-15.

This study aimed to utilize Enterococcus lactis S-15 for the preparation of fermented shrimp gels. The gel properties and the gelation mechanism of proteins were investigated under acid-induced denaturation and protein degradation, and the quality of the gel was evaluated. Results showed that the pH of the shrimp surimi decreased from 7.35 to 4.74. The optimal gel strength observed at 24 h of fermentation was 326.41 g × cm, and disulfide bonds played a crucial role in the fermented gel. The fermented gel exhibited higher cooking loss rates and freeze-thaw loss rates compared to the heat-induced gel (control). However, fermented gels exhibited high overall acceptability both before and after cooking. The volatile basic nitrogen content in the fermented gel remained below 28.00 mg/100 g, within the safe range, and no histamine was detected. The results provide valuable data for the development and reprocessing of fermented shrimp surimi gel.

Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Safety and efficacy of a feed additive consisting of Enterococcus lactis NCIMB 11181 (Lactiferm®) for chickens for fattening or reared for laying, other poultry species for fattening or reared for laying, and ornamental birds (Chr. Hansen A/S).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of Enterococcus lactis NCIMB 11181 (Lactiferm®) as a zootechnical additive (gut flora stabiliser) for chickens for fattening, chickens reared for laying, other poultry species for fattening or reared for laying, and ornamental birds. The additive is available in two formulations: Lactiferm WS200 and Lactiferm Basic 50. The FEEDAP Panel concluded that the use of the additive is safe for chickens for fattening or reared for laying, other poultry species for fattening or reared for laying, and ornamental birds. The Panel also concluded that the use of the feed additive is safe for consumers, and the environment. Lactiferm WS200 is not irritant to skin or eyes. Owing to the proteinaceous nature of the active agent, both formulations of the additive are considered respiratory sensitisers. It was not possible, however, to conclude on the irritancy potential for skin and eyes of the Lactiferm Basic 50 formulation or on the potential of both formulations of the additive to cause skin sensitisation. The efficacy studies submitted did not allow to draw a conclusion on the efficacy of the additive for the target species. Lactiferm® is considered compatible with the coccidiostats monensin sodium and decoquinate.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

Comparative pangenome analysis of Enterococcus faecium and Enterococcus lactis provides new insights into the adaptive evolution by horizontal gene acquisitions.

BACKGROUND: Enterococcus faecium and E. lactis are phylogenetically closely related lactic acid bacteria that are ubiquitous in nature and are known to be beneficial or pathogenic. Despite their considerable industrial and clinical importance, comprehensive studies on their evolutionary relationships and genomic, metabolic, and pathogenic traits are still lacking. Therefore, we conducted comparative pangenome analyses using all available dereplicated genomes of these species. RESULTS: E. faecium was divided into two subclades: subclade I, comprising strains derived from humans, animals, and food, and the more recent phylogenetic subclade II, consisting exclusively of human-derived strains. In contrast, E. lactis strains, isolated from diverse sources including foods, humans, animals, and the environment, did not display distinct clustering based on their isolation sources. Despite having similar metabolic features, noticeable genomic differences were observed between E. faecium subclades I and II, as well as E. lactis. Notably, E. faecium subclade II strains exhibited significantly larger genome sizes and higher gene counts compared to both E. faecium subclade I and E. lactis strains. Furthermore, they carried a higher abundance of antibiotic resistance, virulence, bacteriocin, and mobile element genes. Phylogenetic analysis of antibiotic resistance and virulence genes suggests that E. faecium subclade II strains likely acquired these genes through horizontal gene transfer, facilitating their effective adaptation in response to antibiotic use in humans. CONCLUSIONS: Our study offers valuable insights into the adaptive evolution of E. faecium strains, enabling their survival as pathogens in the human environment through horizontal gene acquisitions.

Assessment of a feed additive containing Enterococcus lactis NCIMB 11181 (Lactiferm®) for weaned piglets, calves for fattening and calves for rearing for the renewal of its authorisation (Chr. Hansen A/S).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of the authorisation of Enterococcus lactis NCIMB 11181 (Lactiferm®) as a zootechnical additive for weaned piglets, calves for fattening and calves for rearing. The product under assessment is based on a strain originally identified as Enterococcus faecium. During the current assessment, the active agent has been reclassified as Enterococcus lactis. The additive currently authorised is marketed in two formulations: Lactiferm Basic 50 (a solid formulation to be used in feed), and Lactiferm WS200 (a solid 'water-soluble' formulation to be used in water for drinking). The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. The Panel concludes that the use of Lactiferm® under the authorised conditions of use remains safe for the target species (calves up to 6 months and weaned piglets up to 35 kg), consumers and the environment. The Lactiferm WS200 formulation of the additive is not irritant to skin or eyes. Owing to the proteinaceous nature of the active agent, both formulations of the additive are considered respiratory sensitisers. It is not possible to conclude on the irritating potential for skin and eyes of the Lactiferm Basic 50 formulation or on the potential of both forms of the additive to cause skin sensitisation. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Safety and efficacy of the feed additive consisting of Enterococcus lactisNCIMB 10415 (Cylactin®) for all poultry for fattening or reared for laying/breeding and ornamental birds, all Suidae, calves and other ruminant species for fattening or rearing (DSM Nutritional products Ltd).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of Cylactin® as zootechnical additive. The active agent of the additive is Enterococcus lactis NCIMB 10415, and three additive formulations currently authorised: Cylactin® LBC G35, Cylactin® LBC ME10 and Cylactin® LBC ME20 plus. The additive is currently authorised in the EU for use in poultry (chickens and minor poultry species for fattening, chickens and minor species reared for laying), calves and kids for rearing and for fattening, sows, suckling and weaned piglets and pigs for fattening. The applicant is now seeking the renewal of its authorisation and the extension of use for chickens and minor poultry species reared for breeding, turkeys for fattening and reared for breeding, ornamental birds, lambs for rearing and for fattening, minor or other ruminants' species for rearing and fattening, minor suckling and weaned Suidae species, pigs and minor Suidae species for fattening, rearing or reproduction. In addition, the applicant is seeking authorisation for use in water for drinking for all above-mentioned target species and categories. The applicant has provided evidence that the additive currently on the market complies with the conditions of authorisation. The FEEDAP Panel concludes that the additive is safe for the target animals, consumers and the environment under the authorised/new proposed conditions of use. The Cylactin® LBC ME10 and LBC ME20 plus are not skin and eye irritants, but no conclusion could be drawn on the potential of Cylactin® LBC G35 to be skin and eye irritant. Moreover, no conclusions could be drawn on the additive skin sensitisation potential. The additive is considered a potential respiratory sensitiser. The efficacy for the new target species/categories as well its use in water was extrapolated from the previous efficacy studies.

Assessment of the feed additive consisting of Enterococcus lactis DSM 7134 and Lacticaseibacillus rhamnosus DSM 7133 (Provita LE) for calves for rearing for the renewal of its authorisation (Lactosan GmbH & Co.KG).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of the authorisation of the additive Provita LE for calves for rearing, consisting of Enterococcus lactis DSM 7134 (formerly identified as Enterococcus faecium) and Lacticaseibacillus rhamnosus DSM 7133 (formerly identified as Lactobacillus rhamnosus) as a zootechnical feed additive. The applicant has provided evidence that the additive currently on the market complies with the existing terms of the authorisation. The FEEDAP Panel concluded that the use of the feed additive in animal nutrition remains safe for calves for rearing, consumers and the environment under the authorised conditions of use. The additive is not irritant to skin or eyes but should be considered a respiratory sensitiser. It was not possible to draw conclusions on the skin sensitisation potential of the additive under assessment. There was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Assessment of the feed additive consisting of Enterococcus lactisDSM 7134 (Bonvital®) for chickens reared for laying and minor poultry species other than those used for laying for the renewal of its authorisation (Lactosan GmbH & Co.KG).

Bonvital® is the trade name for a feed additive currently authorised for use in piglets, pigs for fattening, sows, chickens for fattening, laying hens, chickens reared for laying and minor poultry species (other than those used for laying). This opinion concerns the renewal of the authorisation of Bonvital® as a zootechnical additive for chickens reared for laying and minor poultry species other than those used for laying. The active agent of Bonvital® was originally identified as Enterococcus faecium. During the current assessment, the strain was reassigned to Enterococcus lactis. The applicant provided evidence that the additive currently on the market complies with the existing terms of authorisation. There is no new evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concluded that the additive remains safe for the target species, consumers and the environment under the authorised conditions of use. Bonvital® was found to be non-irritant to skin and eyes, but a potential respiratory sensitiser. No conclusions could be drawn on the skin sensitisation potential of the additive. There is no need to assess the efficacy of Bonvital® in the context of the renewal of the authorisation.

The Halotolerant Probiotic Bacterium Enterococcus lactis ASF-2 from Al-Asfar Lake, Saudi Arabia, Reduces Inflammation in Carrageenan-Induced Paw Edema.

Inflammation-related diseases are major causes of mortality and disability worldwide. This study aimed to identify and investigate probiotic bacteria that could be present in Al-Asfar Lake in Al-Ahsa City, Saudi Arabia to prevent the inflammatory responses of carrageenan-induced paw edema. In total, seven active strains were isolated, and three isolates (ASF-1, ASF-2, and ASF-3) exhibited a positive Gram stain and viable growth at 20% NaCl salinity; they also lacked catalase and hemolytic activities and had high levels of cell surface hydrophobicity (CSH). They also demonstrated potent antibacterial activity against Salmonella typhi and Staphylococcus aureus. These results revealed that ASF-2 had probiotic qualities, and it was selected for further research. ASF-2 demonstrated significant anti-inflammatory effects in an experimental model of carrageenan-induced paw edema; the experimental model showed decreased levels of pro-inflammatory markers, such as interleukin 6 (IL-6), interleukin 17 (IL-17), and transforming growth factor-ß (TGF-ß), and an increased level of an anti-inflammatory marker (interferon gamma (IFN-γ)). Animals in the control group saw a 45% decrease in edema when compared to mice in the carrageenan group. When comparing tissue damage and infiltration in the ASF-2-treated and non-treated mice, the histological examination of the sub-planar tissues of the hind leg revealed that the inflamed tissues had healed. The 16S rRNA sequencing method was utilized to establish that ASF-2 is, in fact, Enterococcus lactis with a 99.2% sequence similarity. These findings shed further light on ASF-2's potential as a biocompatible anti-inflammatory medication.

Enterococcus faecium 129 BIO 3B is classified as Enterococcus lactis 129 BIO 3B.

Enterococcus faecium 129 BIO 3B is a lactic acid bacterium that has been safely used as a probiotic product for over 100 years. Recently, concerns about its safety have arisen because some species of E. faecium belong to the vancomycin-resistant enterococci. The groups of E. faecium with less pathogenic potential have been split into a separate species (Enterococcus lactis). In this study, I investigated the phylogenetic classification and safety of E. faecium 129 BIO 3B as well as E. faecium 129 BIO 3B-R, which is naturally resistant to ampicillin. Mass spectrometry and basic local alignment search tool analysis using specific gene regions failed to differentiate 3B and 3B-R into E. faecium or E. lactis. However, multilocus sequence typing successfully identified 3B and 3B-R as the same sequence types as E. lactis. Overall genome relatedness indices showed that 3B and 3B-R have high degrees of homology with E. lactis. Gene amplification was confirmed for 3B and 3B-R with E. lactis species-specific primers. The minimum inhibitory concentration of ampicillin was confirmed to be 2 µg/mL for 3B, which is within the safety standard for E. faecium set by the European Food Safety Authority. Based on the above results, E. faecium 129 BIO 3B and E. faecium 129 BIO 3B-R were classified as E. lactis. The absence of pathogenic genes except for fms21 in this study demonstrates that these bacteria are safe for use as probiotics.

Safety assessment of Enterococcus lactis based on comparative genomics and phenotypic analysis.

Enterococcus faecium is sometimes used in food production; however, its acquisition of antibiotic resistance has become an alarming health concern. The E. lactis species is closely related to E. faecium and has good probiotic potential. This study aimed to investigate the antibiotic resistance of E. lactis. We analyzed the antibiotic resistance phenotype and whole-genome sequences of 60 E. lactis isolates (23, 29, and 8 isolates from dairy products, Rice wine Koji, and human feces, respectively). These isolates showed varying degree of resistance to 13 antibiotics, and were sensitive to ampicillin and linezolid. The E. lactis genomes carried only a subset of commonly reported antibiotic resistance genes (ARGs) in E. faecium. Five ARGs were detected across the investigated E. lactis, including two universally present genes (msrC and AAC(6')-Ii) and three rarely detected ARGs (tet(L), tetM, and efmA). To identify other undescribed antibiotic resistance-encoding genes, a genome-wide association study was performed, returning 160 potential resistance genes that were associated with six antibiotics, namely chloramphenicol, vancomycin, clindamycin, erythromycin, quinupristin-dalfopristin, and rifampicin. Only around one-third of these genes encode known biological functions, including cellular metabolism, membrane transport, and DNA synthesis. This work identified interesting targets for future study of antibiotic resistance in E. lactis. The fact that the lower number of ARGs present in E. lactis supports that it may be an alternative to E. faecalis for use in the food industry. Data generated in this work is of interest to the dairy industry.

Distinction between Enterococcus faecium and Enterococcus lactis by a gluP PCR-Based Assay for Accurate Identification and Diagnostics.

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

Safety and efficacy of a feed additive consisting of Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866 (BIO-THREE®) for chickens for fattening, chickens reared for laying, turkeys for fattening, turkeys reared for breeding, all avian species for rearing/fattening to slaughter and all avian species reared for laying or breeding to point of lay (TOA BIOPHARMA Co., Ltd.).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of BIO-THREE® when used as a feed additive for chickens for fattening, chickens reared for laying, turkeys for fattening, turkeys reared for breeding, all avian species for rearing/fattening to slaughter and all avian species reared for laying or breeding to point of lay. The product under assessment is based on viable cells/spores of Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866. Based on the tolerance study provided, the Panel concluded that the additive is safe for the target species under the conditions of use. The additive is safe for the consumers of products derived from animals receiving the additive. The additive is not irritant to skin and eyes. The additive is a respiratory sensitiser. No conclusions could be drawn on its potential to be a skin sensitiser. The use of the product as a feed additive is of no concern for the environment. The FEEDAP Panel was not in the position to conclude on the efficacy of BIO-THREE® for the target species. BIO-THREE® is compatible with diclazuril, decoquinate and halofuginone. No conclusions could be drawn on the compatibility of BIO-THREE® with monensin sodium, salinomycin sodium, narasin, robenidine hydrochloride and maduramicin ammonium.

Differentiating between Enterococcusfaecium and Enterococcuslactis by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.

Unlike Enterococcus faecium strains, some Enterococcus lactis strains are considered potential probiotic strains as they lack particular virulence and antibiotic resistance genes. However, these closely related species are difficult to distinguish via conventional taxonomic methods. Here, for the first time, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with BioTyper and in-house databases to distinguish between E. faecium and E. lactis. A total of 58 reference and isolated strains (89.2%) were correctly identified at the species level using MALDI-TOF MS with in-house databases. However, seven strains (10.8%) were not accurately differentiated as a single colony was identified as a different species with a similar score value. Specific mass peaks were identified by analyzing reference strains, and mass peaks at 10,122 ± 2 m/z, 3650 ± 1 m/z, and 7306 ± 1 m/z were unique to E. faecium and E. lactis reference strains, respectively. Mass peaks verified reproducibility in 60 isolates and showed 100% specificity, whereas 16S rRNA sequencing identified two different candidates for some isolates (E. faecium and E. lactis). Our specific mass peak method helped to differentiate two species, with high accuracy and high throughput, and provided a viable alternative to 16S rRNA sequencing.

Update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA 15: suitability of taxonomic units notified to EFSA until September 2021.

The qualified presumption of safety (QPS) approach was developed to provide a generic pre-evaluation of the safety of biological agents. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. The QPS list was updated in relation to the revised taxonomy of the genus Bacillus, to synonyms of yeast species and for the qualifications 'absence of resistance to antimycotics' and 'only for production purposes'. Lactobacillus cellobiosus has been reclassified as Limosilactobacillus fermentum. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TU)s. Of the 70 microorganisms notified to EFSA, 64 were not evaluated: 11 filamentous fungi, one oomycete, one Clostridium butyricum, one Enterococcus faecium, five Escherichia coli, one Streptomyces sp., one Bacillus nakamurai and 43 TUs that already had a QPS status. Six notifications, corresponding to six TUs were evaluated: Paenibacillus lentus was reassessed because an update was requested for the current mandate. Enterococcus lactis synonym Enterococcus xinjiangensis, Aurantiochytrium mangrovei synonym Schizochytrium mangrovei, Schizochytrium aggregatum, Chlamydomonas reinhardtii synonym Chlamydomonas smithii and Haematococcus lacustris synonym Haematococcus pluvialis were assessed for the first time. The following TUs were not recommended for QPS status: P. lentus due to a limited body of knowledge, E. lactis synonym E. xinjiangensis due to potential safety concerns, A. mangrovei synonym S. mangrovei, S. aggregatum and C. reinhardtii synonym C. smithii, due to lack of a body of knowledge on its occurrence in the food and feed chain. H. lacustris synonym H. pluvialis is recommended for QPS status with the qualification 'for production purposes only'.