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Evaluating the Safety of Potential Probiotic Enterococcus durans KLDS6.0930 Using Whole Genome Sequencing and Oral Toxicity Study.

ABSTRACT: Enterococcus durans KLDS6.0930 has previously been shown to have probiotic potential. However, being a potential clinical pathogen, it becomes necessary to evaluate its safety status for novel potential probiotic use. The purpose of this study is to systematically evaluate the safety of E. durans KLDS6.0930 based on its genomics, phenotypic characteristics and oral toxicity. The complete genome of E. durans KLDS6.0930 was sequenced and analyzed for safety-related genes. Antibiotic susceptibility and the production of harmful metabolites were tested. A 28-day repeated oral dose toxicity test was implemented in rats. In vitro, E. durans KLDS6.0930 was resistant to five antibiotics, with intrinsic resistances to four antibiotics and no identified genes for the last. E. durans KLDS6.0930 was not hemolytic and virulence factors were non-functional in its genome. E. durans KLDS6.0930 produced a small amount of tyramine and phenethylamine; genes encoding tyramine decarboxylase were identified. In addition, genotype and phenotype analyses showed that the strain did not have the ability to generate D-lactic acid, indole, or nitroreductase. In vivo, E. durans KLDS6.0930 did not induce adverse effects on the organs, hematological and serum biochemical parameters, or cecal bacterial populations in the oral toxicity test. These results indicate that E. durans KLDS6.0930 can be safely used as a potential probiotic for human consumption and animal feed.

SUBMITTER: Li B

PROVIDER: S-EPMC6110905 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To evaluate the probiotic characteristics and safety of Enterococcus durans isolate A8-1 from a fecal sample of a healthy Chinese infant, we determined the tolerance to low pH, survival in bile salts and NaCl, adhesion ability, biofilm formation, antimicrobial activity, toxin gene distribution, hemolysis, gelatinase activity, antibiotic resistance, and virulence to Galleria mellonella and interpreted the characters by genome resequencing. Phenotypically, E. durans A8-1 survived at pH 5.0 in 7.0% NaCl and 3% bile salt under aerobic and anaerobic condition. The bacterium had higher adhesion ability toward mucin, collagen, and Bovine Serum Albumin (BSA) in vitro and showed high hydrophobicity (79.2% in chloroform, 49.2% in xylene), auto-aggregation activity (51.7%), and could co-aggregate (66.2%) with Salmonella typhimurium. It had adhesion capability to intestinal epithelial Caco-2 cells (38.74%) with moderate biofilm production and antimicrobial activity against several Gram-positive pathogenic bacteria. A8-1 can antagonize the adhesion of S. typhimurium ATCC14028 on Caco-2 cells to protect the integrity of the cell membrane by detection of lactate dehydrogenase (LDH) and AKP activities. A8-1 also helps the cell relieve the inflammation induced by lipopolysaccharide by reducing the expression of cytokine IL-8 (P = 0.002) and TNF-α (P > 0.05), and increasing the IL-10 (P < 0.001). For the safety evaluation, A8-1 showed no hemolytic activity, no gelatinase activity, and had only asa1 positive in the seven detected virulence genes in polymerase chain reaction (PCR), whereas it was not predicted in the genome sequence. It was susceptible to benzylpenicillin, ampicillin, ciprofloxacin, levofloxacin, moxifloxacin, tigecycline, nitrofurantoin, linezolid, vancomycin, erythromycin, and quinupristin/dalofopine except clindamycin, which was verified by the predicted lasA, lmrB, lmrC, and lmrD genes contributing to the clindamycin resistance. The virulence test of G. mellonella showed that it had toxicity lower than 10% at 1 × 107 CFU. According to the results of these evaluated attributes, E. durans strain A8-1 could be a promising probiotic candidate for applications.

Project description:The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3')-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.

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Evaluating the Safety of Potential Probiotic Enterococcus durans KLDS6.0930 Using Whole Genome Sequencing and Oral Toxicity Study.

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