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Comparative genome analyses of Lactobacillus crispatus isolated from different ecological niches reveal an environmental adaptation of this species to the human vaginal environment.

ABSTRACT: Vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the vaginal microbiota is dominated by members of the Lactobacillus genus, whose relative abundance and microbial taxa composition are dependent on the healthy status of this human body site. Particularly, among members of this genus, the high prevalence of Lactobacillus crispatus is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 healthy vaginal microbiome samples through shotgun metagenomics analyses. Based on our results we observed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Therefore, we isolated fifteen L. crispatus strains from different environments in which this species is abounding, ranging from vaginal swabs of healthy women to chicken fecal samples. The genomes of these strains were decoded and their genetic content was analyzed and correlated with their physiological features. An extensive comparative genomic analysis encompassing all publicly available genome sequences of L. crispatus and combined with those decoded in this study, revealed a genetic adaptation of strains to their ecological niche. In addition, in vitro growth experiments involving all isolated L. crispatus strains together with a synthetic vaginal microbiota reveal how this species is able to modulate the composition of the vaginal microbial consortia at strain level. Overall, our findings suggest that L. crispatus plays an important ecological role in reducing the complexity of the vaginal microbiota by depleting pathogenic bacteria.Importance The vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the high prevalence of Lactobacillus crispatus species is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 public healthy vaginal samples through shotgun metagenomics analyses. Results showed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Moreover, we isolated and sequenced the genome of new L. crispatus strains from different environments and the comparative genomics analysis revealed a genetic adaptation of strains to their ecological niche. In addition, in-vitro growth experiments display the capability of this species to modulate the composition of the vaginal microbial consortia. Overall, our findings suggest an ecological role exploited by L. crispatus in reducing the complexity of the vaginal microbiota toward a depletion of pathogenic bacteria.

SUBMITTER: Mancabelli L

PROVIDER: S-EPMC8091109 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. Post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.

Project description:BackgroundLactobacillus crispatus is the dominant species in the vaginal microbiota associated with health and considered a homeostasis biomarker. Interestingly, some strains are even used as probiotics. However, the genetic mechanisms of L. crispatus involved in the control of the vaginal microbiome and protection against bacterial vaginosis (BV) are not entirely known. To further investigate these mechanisms, we sequenced and characterized the first four L. crispatus genomes from vaginal samples from Brazilian women and used genome-wide association study (GWAS) and comparative analyses to identify genetic mechanisms involved in healthy or BV conditions and selective pressures acting in the vaginal microbiome.MethodsThe four genomes were sequenced, assembled using ten different strategies and automatically annotated. The functional characterization was performed by bioinformatics tools comparing with known probiotic strains. Moreover, it was selected one representative strain (L. crispatus CRI4) for in vitro detection of phages by electron microscopy. Evolutionary analysis, including phylogeny, GWAS and positive selection were performed using 46 public genomes strains representing health and BV conditions.ResultsGenes involved in probiotic effects such as lactic acid production, hydrogen peroxide, bacteriocins, and adhesin were identified. Three hemolysins and putrescine production were predicted, although these features are also present in other probiotic strains. The four genomes presented no plasmids, but 14 known families insertion sequences and several prophages were detected. However, none of the mobile genetic elements contained antimicrobial resistance genes. The genomes harbor a CRISPR-Cas subtype II-A system that is probably inactivated due to fragmentation of the genes csn2 and cas9. No genomic feature was associated with a health condition, perhaps due to its multifactorial characteristic. Five genes were identified as under positive selection, but the selective pressure remains to be discovered. In conclusion, the Brazilian strains investigated in this study present potential protective properties, although in vitro and in vivo studies are required to confirm their efficacy and safety to be considered for human use.

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Comparative genome analyses of Lactobacillus crispatus isolated from different ecological niches reveal an environmental adaptation of this species to the human vaginal environment.

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