Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract

Abstract

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6^T, KA00182^T and BV3C16-1^T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6^T and KA00182^T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6^T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182^T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1^T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6^T, KA00182^T and BV3C16-1^T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C_{12 : 0}, C_{16 : 0}, C_{16 : 0} dimethyl acetal (DMA) and summed feature 5 (C_{15 : 0} DMA and/or C_{14 : 0} 3-OH) in UPII 199-6^T; C_{16 : 0} and C_{16 : 1} cis 9 in KA00182^T; C_{12 : 0}; C_{14 : 0} 3-OH; and summed feature 5 in BV3C16-1^T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6^T, KA00182^T and BV3C16-1^T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6^T representing the type strain of this species (=DSM 111201^T=ATCC TSD-205^T), Megasphaera hutchinsoni sp. nov. for KA00182^T representing the type strain of this species (=DSM 111202^T=ATCC TSD-206^T) and Megasphaera vaginalis sp. nov. for BV3C16-1^T representing the type strain of this species (=DSM 111203^T=ATCC TSD-207^T).

Keywords: Megasphaera; Negativicutes; Veillonellaceae; bacterial vaginosis; genital tract bacteria; human vagina.

Fig. 1.

Scanning electron micrograph of cells of (a) UPII 199-6^T, (b) KA00182^T, and (c) BV3C16-1^T. Cells were cultured for 2 days in PYG-mod-YG. Bar, 2 µm.

Fig. 2.

Transmission electron microscope image of cells of (a) UPII 199-6^T, (b) KA00182^T, and (c) BV3C16-1^T. Cells were cultured for 2 days in PYG-mod-YG. Bar, 200 nm.

Fig. 3.

Molecular phylogenetic analysis by the maximum-likelihood method based on 16S rRNA gene sequences showing the phylogenetic positions of M. lornae, M. hutchinsoni and M. vaginalis with closely related members of the family Veillonellaceae. The genital tract isolates clustered within the Megasphaera clade. Bootstrap values (based on 1000 replications) greater than or equal to 70 % are shown as percentages at each node. Bar, 0.02 substitutions per nucleotide position. Selenomonas sputigena ATCC 35185 (NR_025115) from the family Selenomonadaceae was added as an outgroup.

Fig. 4.

Genome-based species delineation using the (a) genome blast distance phylogeny approach. Values less than 70 % are indicative of a different species. Strains: 1, UPII 199-6^T; 2, DNF00751; 3, KA00182^T; 4, UPII 135-E; 5, BV3C16-1^T; 7, M. cerevisiae DSM 20462; 8, M. elsdenii DSM 20460; 9, M. hexanoica MH; 11, M. massiliensis DSM 26228; 13, M. paucivorans DSM 16981; 14, M. stantonii AJH120; 15, M. sueciensis DSM 17042. M. vaginalis HL562, M. indica, M. micronuciformis and M. sueciensis did not have genomes available for this analysis. UPII 199-6^T and DNF00751 belong to the same species with a DDH value of 90.7 % (M. lornae). KA00182^T and UPII 135-E belong to the same species with a DDH value of 89.5 % (M. hutchinsoni). (b) Average nucleotide identity analyses. Identities of <95 % indicate a novel species. The genital tract isolates (1–5) are sufficiently different from validly named Megasphaera species (7–15) to warrant designation as novel species. UPII 199-6^T and DNF00751 are the same species (99.1 % ANI) while KA00182^T and UPII 135-E are the same species (99 % ANI).