Genomic sequence analysis and characterization of Sneathia amnii sp. nov

Abstract

Background: Bacteria of the genus Sneathia are emerging as potential pathogens of the female reproductive tract. Species of Sneathia, which were formerly grouped with Leptotrichia, can be part of the normal microbiota of the genitourinary tracts of men and women, but they are also associated with a variety of clinical conditions including bacterial vaginosis, preeclampsia, preterm labor, spontaneous abortion, post-partum bacteremia and other invasive infections. Sneathia species also exhibit a significant correlation with sexually transmitted diseases and cervical cancer. Because Sneathia species are fastidious and rarely cultured successfully in vitro; and the genomes of members of the genus had until now not been characterized, very little is known about the physiology or the virulence of these organisms.

Results: Here, we describe a novel species, Sneathia amnii sp. nov, which closely resembles bacteria previously designated "Leptotrichia amnionii". As part of the Vaginal Human Microbiome Project at VCU, a vaginal isolate of S. amnii sp. nov. was identified, successfully cultured and bacteriologically cloned. The biochemical characteristics and virulence properties of the organism were examined in vitro, and the genome of the organism was sequenced, annotated and analyzed. The analysis revealed a reduced circular genome of ~1.34 Mbp, containing ~1,282 protein-coding genes. Metabolic reconstruction of the bacterium reflected its biochemical phenotype, and several genes potentially associated with pathogenicity were identified.

Conclusions: Bacteria with complex growth requirements frequently remain poorly characterized and, as a consequence, their roles in health and disease are unclear. Elucidation of the physiology and identification of genes putatively involved in the metabolism and virulence of S. amnii may lead to a better understanding of the role of this potential pathogen in bacterial vaginosis, preterm birth, and other issues associated with vaginal and reproductive health.

Figure 1

Prevalence of Sneathia species in mid-vaginal samples. (A) Relative abundance of Sneathia species in mid-vaginal samples. Sneathia was identified by 16S rDNA analysis in 319 of 736 mid-vaginal samples collected from volunteers recruited from outpatient clinics using a 0.1% abundance threshold; i.e., Sneathia was considered present if at least 0.1% of total 16S rRNA reads classified to Sneathia. (B) Abundance of different species of Sneathia (S. sanguinegens, S. amnii, or non-speciated Sneathia) in mid-vaginal samples. The majority of Sneathia reads (76.3%) identified in the mid-vaginal microbiome profiles classify to Sneathia amnii.

Figure 2

Maximum likelihood phylogenetic tree of S. amnii and related organisms within the family Fusobacteriaceae. The tree was inferred from 1,271 aligned characters of the 16S rRNA gene sequence (Additional file 1) and rooted in accordance with the current taxonomy. Numbers at nodes correspond to the support values from 1,000 bootstrap replicates.

Figure 3

Genomic atlas of S. amnii Sn35. Coordinates are indicated starting from the predicted origin of replication at base 1. Circles represent the following (from the outer circle inward): 1) Coding regions are marked on the first two rings: orange color if encoded on the positive strand and teal color if encoded on the negative strand. 2) location of the tRNA. 3) GC content using a 10-kbp sliding window, green, positive GC skew; gray, negative GC skew. The innermost graph shows the GC skew, with sharp changes in skew occurring at the putative origin and terminus of replication. The atlas was constructed using DNAPlotter [58].

Figure 4

Comparison of the distribution of predicted proteins of S. amnii and related organisms. Predicted ORFs from S. amnii, S. moniliformis, L. buccalis and S. termiditis were classified into COG functional categories. The percent of genes belonging to each COG is represented for each species.

Figure 5

Electron Micrographs of S. amnii. S. amnii were fixed to either glass cover slips, or copper grids, and visualized by SEM (a) or TEM (b), respectively.

Figure 6

S. amnii was cytotoxic and adhered to Me180 cells. To assess cytotoxicity, bacteria were added to Me180 monolayers and monitored by light microscopy during the incubation for phenotypic alterations. In stark contrast to monolayers treated with PBS alone as a control (a), monolayers exposed to S. amnii rapidly induced cell rounding and loss of adherence (b). To determine whether or not S. amnii adhered, bacteria were added to Me180 monolayers grown on glass coverslips. Following incubation, the glass coverslips were washed extensively in PBS, stained, and analyzed by SEM (c).