Abstract
Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four “clades” identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome.
Highlights
Gardnerella vaginalis is recognized as a ubiquitous element of the complex of vaginal organisms in healthy women, but is more abundant in women diagnosed with bacterial vaginosis
Despite well-known phenotypic diversity [3], the possibility that G. vaginalis may consist of several species with distinct roles in bacterial vaginosis (BV) pathogenesis has only recently been investigated [5, 7, 8]
Studies based on 16S rRNA variable-region targeted sequencing have known biases against detecting Gardnerella at all [24, 25], much less resolving subgroups, one early deep sequencing study did distinguish four G. vaginalis subgroups based on a single nucleotide difference in variable region 6 of the 16S rRNA gene [26]
Summary
The objectives of the current study were: 1) to reconcile cpn UT-based molecular subgroups A-D with previously published clades 1–4, 2) to define cpn UT subgroups of 112 Gardnerella isolates and compare to classification based on ARDRA and clade-specific realtime PCR assays and 3) to determine sialidase gene presence and activity in all 112 isolates, in order to clarify distribution of this virulence factor across G. vaginalis subgroups
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