Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Ohkusu K

PROVIDER: S-EPMC10315194 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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

Ohkusu Kiyofumi K

Bioscience of microbiota, food and health 20230126 3

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 ...[more]

PMID: 37404571

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Project description: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.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

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Project description:Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

Dataset Information

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

Publications

<i>Enterococcus faecium</i> 129 BIO 3B is classified as <i>Enterococcus lactis</i> 129 BIO 3B.

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