Description of Microbacterium luticellae sp. nov., Microbacterium alcoholitolerans sp. nov. and Microbacterium daqui sp. nov. isolated from baijiu pit mud and daqu

Four Gram-stain-positive bacterial strains, designated as YY-01^T, YY-02^T, YY-03^T and YY-04, were isolated from pit mud (YY-01^T, YY-02^T and YY-04) and daqu (YY-03^T) samples collected at a winery in Shandong Province, China. Phylogenetic and genomic analyses, along with bioassays and antibiotic susceptibility testing, revealed that the four strains, YY-01^T, YY-02^T, YY-03^T and YY-04, represent three novel species within the genus <i>Microbacterium</i>. Phylogenetic reconstruction based on 16S rRNA gene sequences revealed the closest relatives as follows: strain YY-01^T shares closest affinity with <i>Microbacterium pseudoresistens</i> CC-005209^T (97.1% similarity), strains YY-02^T and YY-04 cluster with <i>Microbacterium esteraromaticum</i> DSM 8609^T (98.3-98.4% similarity) and strain YY-03^T aligns with <i>Microbacterium mitrae</i> M4-8^T (97.6% similarity). Genome phylogeny further resolved strain YY-01^T as a sister lineage to <i>Microbacterium laevaniformans</i> DSM 20140^T, while strains YY-02^T, YY-03^T and YY-04 exhibited phylogenetic relationships congruent with the 16S rRNA topology. Genomic relatedness analysis demonstrated that strains YY-02^T and YY-04 share an OrthoANIu value of 99.9% and a digital DNA-DNA hybridization (dDDH) value of 100%, confirming their classification as conspecific. In contrast, OrthoANIu and dDDH values between strains YY-01^T, YY-02^T and YY-03^T and other <i>Microbacterium</i> species were ≤82.1 and 28.3%, respectively, supporting their distinct species status. Chemotaxonomic analysis revealed distinct profiles for each strain. Strain YY-01^T was characterized by MK-13 menaquinones; major fatty acids anteiso-C_{15 : 0}, anteiso-C_{17 : 0} and iso-C_{16 : 0}; and the polar lipids diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). Strain YY-02^T contained predominant menaquinones MK-11 and MK-13 and major fatty acids anteiso-C_{17 : 0}, anteiso-C_{15 : 0} and iso-C_{16 : 0}, along with DPG and PG. Strain YY-03^T possessed major menaquinones MK-11 and MK-13; major fatty acids anteiso-C_{15 : 0}, iso-C_{15 : 0} and iso-C_{16 : 0}; and polar lipids DPG and PG. Integrative genomic, phenotypic and phylogenetic evidence supports the proposal of three novel species: <i>Microbacterium luticellae</i> sp. nov. (type strain YY-01^T=GDMCC 1.5102^T=KCTC 59478^T), <i>Microbacterium alcoholitolerans</i> sp. nov. (type strain YY-02^T=GDMCC 1.5103^T=KCTC 59479^T) and <i>Microbacterium daqui</i> sp. nov. (type strain YY-03^T=GDMCC 1.5104^T=KCTC 59480^T).