Draft Genome Sequences of Two Strains of Enterococcus lactis Showing High Potential as Cattle Probiotic Supplements

ABSTRACT Probiotic supplements are currently widely used in cattle feeding practices. However, knowledge regarding the genomic landscape of cattle probiotic microorganisms is relatively scarce and is based on analogies with human probiotics. Here, we report on the draft genome sequences of two Enterococcus lactis strains, VKPM B-4989 and VKPM B-4992, which were isolated from the rumen of a healthy calf and utilized as a probiotic additive.

P robiotic additives have been shown to improve productivity and fitness characteristics of cattle and to reduce methane emissions from farms and agricultural companies (1). Lactic acid bacteria, primarily belonging to the genera Bifidobacterium, Enterococcus, and Lactococcus, represent the predominant class of microbial additives, reported to improve weight gain and feed efficiency (2,3). Widespread representatives of the genus Enterococcus are currently widely utilized as a basis for human food supplements (4) and were recently shown to demonstrate significant potential as cattle probiotic additives, having a positive effect on the diversity of rumen microbiota (5). Here, we report the draft genome sequences of two Enterococcus lactis strains, VKPM B-4989 and VKPM B-4992, which were isolated from the rumen of a healthy calf in 1989 in STC Lekbiotech (Moscow, USSR). Later the strains were used as a probiotic additive (6,7).
Before DNA isolation, the strains were cultured in MRS medium for 36 h at 28°C to 30°C under aerobic conditions. The genomic DNA was extracted as described previously (8). Fragment genomic libraries were prepared with a Nextera XT reagent kit (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations. Sequencing was performed on an Illumina MiSeq system using a 2 Â 250-bp MiSeq reagent kit.
Genome-based taxonomic classification of the sequenced strains was performed with the Type Strain Genome Server (TYGS) (14). TYGS analysis identified strains B-4989 and B-4992 as E. lactis with high confidence (digital DNA-DNA hybridization [dDDH] values of .90%) ( Table 1).
Antibiotic resistance analysis performed with the Comprehensive Antibiotic Resistance Database (CARD) Resistance Gene Identifier (RGI) web server v5.2.0 (15), using Perfect and Strict hits only search criteria, discovered two antibiotic resistance genes. The identified genes were present in both strains; AAC(69)-Ib homologs are involved in aminoglycoside antibiotic inactivation, and ABC-F ribosomal protection protein homologs are involved in antibiotic resistance via ribosomal protection.
A search of secondary metabolite gene clusters was carried out with antiSMASH (16). A type III polyketide synthase (PKS) gene cluster was found in both genomes. The genome of strain B-4989 includes two ribosomally synthesized and posttranslationally modified peptide product (RiPP)-like gene clusters, both of which encode class II bacteriocin and related biosynthetic enzymes and transporters. The genome of strain B-4992 includes two nonribosomal peptide synthetase clusters.
An NCBI search showed a lack of sequenced genomes of cattle probiotic microorganisms. E. lactis strains VKPM B-4989 and VPKM B-4992 possess antibiotic resistance genes and secondary metabolite gene clusters; however, in-depth analysis is needed to characterize cattle-specific genetic determinants of their probiotic properties.
Data availability. The genomes have been deposited in NCBI GenBank and are available under accession numbers JAGPYO000000000.1 (E. lactis strain B-4989) and JAGPYP000000000.1 (E. lactis strain B-4992). Raw sequencing reads are available in the NCBI SRA under accession numbers SRX10881548 (E. lactis strain B-4989) and SRX10881549 (E. lactis strain B-4992).

ACKNOWLEDGMENTS
Sequencing was supported by a grant from the Ministry of Science and Higher Education of the Russian Federation, allocated to the Kurchatov Center for Genome Research (agreement 075-15-2019-1659). Genome analysis was supported by the NRC Kurchatov Institute (order 1056 from 2 July 2020). Cultivation and DNA extraction were supported by Russian State assignment AAAA-A20-120093090016-9 and were carried out using the equipment of the Unique Scientific Facility of the Biological Resource Center (BRC), Russian National Collection of Industrial Microorganisms (VKPM).