ANALISIS E INTERPRETACIÓN DE RESULTADOS

Species of Lactobacillus have long been associated with the human vaginal microbiota, though they are found in smaller proportions in the gastrointestinal tract and oral cavity (158). The genus is a member of the lactic acid bacteria group: a clade of Gram-positive, low-GC bacteria that produce lactic acid as a major fermentation product. This characteristic is thought to have a major functional role in the vagina where abundant lactic acid maintains a low pH unfavorable to pathogenic organisms (159, 160). This represents a potential mutualistic relationship where the vaginal epithelial cells produce glycogen that is converted by the bacteria to lactic acid (13). Lactobacillus species are also known to produce small antimicrobial molecules called bacteriocins, and hydrogen peroxide - both considered defense mechanisms against other organisms. Although recent studies have suggested H2O2 is not produced in sufficient quantities in the microaerobic

vaginal environment to have an antagonistic effect against other organisms (159, 160). Other than production of toxic metabolic byproducts, competitive exclusion by adherence to the mucosal surface and auto-aggregation has been proposed as an important protective strategy (161).

Revealed by comparative genomics, the genus Lactobacillus is highly diverse with evidence for extensive gene loss and gain via horizontal gene transfer (162). Species found in the vagina (including L. crispatus, L. iners, L. gasseri, L. jensenii, L. johnsonii, L. vaginalis, L. reuteri) are phylogenetically more similar than other Lactobacillus

species (163, 164).

Notably, although BV is in general a low-Lactobacillus state, several studies have indicated L. iners can remain in relatively high abundance during BV (18, 48, 165) and post-antimicrobial treatment (166). This species was overlooked due to non-standard cultivation requirements until it was cultured on blood agar and described by Falsen et al.

(17). Molecular sequencing techniques have uncovered L. iners as the most common constituent of the vaginal microbiota (24, 29, 48). Unlike other Lactobacillus species, L. iners seems to exclusively inhabit the human vagina and isn’t detectable in saliva or fecal samples (167), nor has it been reported in non-human primate vaginas (25). Although notably, the methods used in these studies may be limited in detection sensitivity.

Genome sequencing and in vitro assays have uncovered functions of particular strains that could explain their role in the vagina during BV. Gardnerella vaginalis is abundant in many cases of BV, but is a common constituent of the normal vaginal biota (45). This observation has lead to studies that suggest different strains of G. vaginalis could have different functions and result in varied risks for the host (168). Genome studies of G. vaginalis, once thought to be a single organism, reveal a diverse complement of genes between different strains with as much as 31% divergence in gene content and order (169). Two studies have used comparative genomic analysis on strains isolated from healthy or asymptomatic women compared to BV-isolated strains. Harwich et al. (170) found a point mutation in the protein encoding vaginolysin, a cytolysin active against human cells (171), that rendered it less cytotoxic than the same protein from the health-

associated strain. The same BV-associated strain had better adherence, aggregation, and biofilm formation. Biofilm formation is thought to be one of the major strategies G. vaginalis uses to persist (172) and resist antimicrobial compounds (173) and lactic acid (174). In addition, adherence of G. vaginalis biofilms to vaginal epithelial cells is responsible for the “clue cells” observed during BV and used as part of the Amsel diagnostic (175). The second comparative genomic study of four strains by Yeoman et al.

(176) found a number of conserved genes with virulent potential including exopolysaccharide (EPS) biosynthesis for biofilm formation, pili, hemolytic/cytolytic potential, and several bactericidal toxins. Of the four strains, one (409-05) associated with a healthy asymptomatic woman was lacking genes encoding enzymes for mucin degradation – a trait associated with BV (177). In comparison to other BV isolates from different genera, G. vaginalis strains were determined to have a higher virulent potential by Patterson et al. (178) by assaying adherence, cytotoxicity, and biofilm activity. One study has implicated G. vaginalis in a symbiotic relationship with another BV-associated organism Prevotella bivia whereby amino acids produced by G. vaginalis supports the growth of P. bivia that in turn produces ammonia utilized by G. vaginalis (179). A similar symbiosis has been described between P. bivia and Peptostreptococcus anaerobius (180). Prevotella and Gardnerella species, and in particular P. bivia, have been shown to have sialidase activity (181) which is associated with risk of pre-term labour (182).

Other bacteria associated with the vaginal microbiota are less characterized due to the effort required to culture them for biochemical assays and sequencing (183). Atopobium vaginae has only been described relatively recently (184), but has a high specificity with BV where it is detectible by sequencing methods (183, 185). Most strains are resistant to metronidazole (186), and at least one affects local immunity by inducing release of IL-6, IL-8, and β-defensin 4 from epithelial cells in vitro (187). Other recently detected organisms include the BV-associated bacteria (BVAB) of the Clostridiales order which have high specificity for BV (18). Many of these low abundance, difficult-to-culture species, could be forming co-occurrence networks with other species in the vagina, as suggested by Srinivasan et al. (38). The collective effort of the individual strains could benefit the community as a whole if, for example, several mucinases produced by

different members of the community allowed access to the vaginal epithelium where attachment and lysis can occur (46). These yet to be characterized organisms reflect the complexity of the vaginal environment and the potential interactions that can occur between multiple taxa contributing to the etiology of BV and other conditions.

1.8 High-throughput sequencing techniques for