reAnalyze #4 - Vaginal Infection

What is reAnalyze?

Recent advancements in sequencing technology and database development have provided new insights into microbial communities. Notably, many microbial genomes have been sequenced from metagenomes without official descriptions. With up-to-date databases, especially those incorporating genomospecies, and improved software, we can revisit existing datasets to uncover previously overlooked organisms.

Our goal with these reAnalyze blog posts is to utilize EzBioCloud’s comprehensive databases and proprietary pipeline to explore public metagenomic datasets and share relevant findings.

A New Perspective on Vaginal Microbiome Alterations in HPV16 (Human Papillomavirus 16) Infection

Let's reAnalyze: The Alterations of Vaginal Microbiome in HPV16 Infection as Identified by Shotgun Metagenomic Sequencing by Yang et al., 2020. This study examined the differences between HPV16-positive (without lesion) and HPV-negative vaginal microbiomes. Vaginal samples were collected from 27 HPV16-positive women and 25 age-matched HPV-negative controls. Swab samples were taken from near the vaginal fornix and cervix. The sequencing data are publicly available under the BioProject ID: PRJNA576566.

For microbial profiling, the authors used shotgun metagenomic sequencing, a comprehensive method that identifies bacterial taxa down to the species level and provides functional analysis of the microbiome. While this approach was advanced for its time, revisiting these datasets with modern tools and updated databases could yield additional insights.

The Overlooked Frontier: Vaginal Microbiome

When we think of the microbiome, our minds often turn to the gut. However, the vaginal microbiome plays a crucial role in female reproductive health, acting as a multifaceted defense system against infections and diseases.

The vaginal microenvironment is influenced by hormonal cycles, personal hygiene, and sexual activity, supporting diverse microbial populations. These factors create unique microbial signatures specific to each individual.

Six undescribed species prevalent in the vaginal microbiome

Figure 1. Group composition of HPV16 positive[up] and negative[down], each with sampling site (vaginal fornix[left] and cervix[right]).

Our first reAnalyze finding was six undescribed taxa in the 20 most prominent species across groups.

Lactobacillus crispatus dominates in vaginal fornix group, while Lactobacillus iners and Gardnerella group species are dominant in cervix group. While some changes from HPV16 infection could be identified in the figure between positive and negative group, still there could be bias due to relative abundance comparison. Significantly different species will be identified in differential analysis.

Furthermore, comparing the top 20 species with the reAnalyze results with paper result: our result indicates more precise names, with better indication without additional family level.

Table 1. Top 20 species comparing the paper and reAnalyze results

Top	Paper	reAnalyze
1	Lactobacillus crispatus	Lactobacillus crispatus
2	Lactobacillus iners	Lactobacillus iners
3	Gardnerella vaginalis	Gardnerella swidsinskii group
4	Alpha papillomavirus-9	Gardnerella piotii
5	Atopobium vaginae	Gardnerella ADEV_s
6	Lactobacillus sp 7_1_47FAA	Lactobacillus mulieris
7	Gardnerella sp 304	Fannyhessea vaginae
8	Lactobacillus jensenii	Gardnerella vaginalis
9	Gardnerella sp 2612	Megasphaera lornae
10	Lactobacillus gasseri	KQ959578_g MSSCM00739441_s
11	Megasphaera sp UPII 1996	Lactobacillus johnsonii
12	Megasphaera genomosp type 1	Moryella AY995258_s
13	Veillonellaceae bacterium DNF00751	Dialister KQ960846_s
14	Alpha papillomavirus-14	Fannyhessea MSSCM01081469_s
15	Lactobacillus johnsonii	Phocaeicola vulgatus group
16	Veillonellaceae bacterium DNF00626	Megasphaera hutchinsoni
17	Streptomyces turgidiscabies	Staphylococcus epidermidis
18	Mycobacterium leprae	Lactobacillus jensenii
19	Veillonellaceae bacterium KA00182	Lactobacillus gasseri
20	Aerococcus christensenii	Gardnerella KQ956810_s

* Veillonellaceae family contains Megasphaera, Veillonella, Dialister, etc.

More information on the six genomospecies identified in the vaginal samples can be found below:

Gardnerella ADEV_s

KQ959578_g MSSCM00739441_s

Moryella AY995258_s

Dialister KQ960846_s

Fannyhessea MSSCM01081469_s

Gardnerella KQ956810_s

Vaginal microbiomes display different diversity between sampling sites

Figure 2. Principal coordinate analysis plots comparing the vaginal microbiome of fornix and cervix individuals. (Blue: N-cervix, Orange: N-fornix, Red: P-cervix, Green: N-cervix)

The principal coordinate analysis (PCoA) plots significant difference between vaginal fornix group and cervix group, but not on infection status. To understand these observations, it is due to the collection status, which infer that similar health condition, geographical region, ethnicity, and so on.

“There were no differences in demographics between the two groups, including age, BMI, menarche age and days since last menstrual period, nor were there differences in contraceptive methods, age at first sexual intercourse, sexual frequency, number of sexual partners and contraceptive methods. In order to validate the findings of sequencing, a validation cohort was recruited, including 88 HPV16 positive and 81 HPV negative women. Also, there were no significant differences in demographics between the two groups in the validation cohort, except for the number of sexual partners.”

These insights from the PCoA plots can indicate that less discriminative significant taxon would be discovered from the data.

Several microbial species significantly different from vaginal infection

The beta diversity analysis showed overlapping of microbial diversity but with our differential abundance analysis (DAA), we found few significant species level comparing HPV16 positive and negative group, including vaginal fornix and cervix.

Although microbiomes are complex communities comprised of countless individuals responding to each other and their environments, it can take just one species or strain to disrupt that system. Biomarkers can be indicative of such perturbers of the peace or beneficial microbes or, simply, commensals associated with a particular cohort. Below are reAnalyze results using seven differential abundance analysis tools to find consensus over candidate biomarkers.

Figure 3. Differential abundance analysis finding candidate biomarkers in HPV negative (red) and HPV16 positive (blue) microbiome cohort. Upset Plot: rank: Species, alpha: 0.05, other parameters: default.

There's a lot happening in this plot with many of the DAA tools indicating potential biomarkers, some in agreement and others independently (Figure 3). Three differential abundance tools are in consensus over three biomarkers associated with 'HPV16 positive' microbiome (blue, far left). One differential abundance tool showed one biomarker associated with 'HPV negative' microbiomes (red, right).

Now, here’s which taxa they are.

Table 2a. Candidate biomarker table excerpt displaying top taxa. Negative scores indicating assocations with HPV16 positive microbiome

Table 2b. Candidate biomarker table excerpt displaying top taxa. Positive scores indicating associations with HPV negative microbiome

Even though the result only showed significance on three among seven different tools, still three taxa with a score of 3 (HPV16 positive group) are likely to be reliably associated with an increase in HPV16 positive group microbiome.

All three were not shown in the top 20 taxonomic composition analysis; Enterococcus faecium, g__CAG-568 MSSCM00921844_s (only found in our database; Erysipelotrichia class), and Veillonella parvula group. With the paper's result, these findings could assist the taxonomic biomarker of HPV16 positive group.

Mapping Vaginal Microbiome Enterotypes in HPV16 Infection

Figure 4. Partitioning Around Medoids (PAM) clustering in species level. E1 (blue), E2 (orange), and E3 (red). Upset Plot: minimal prevalence: 0.1, other parameters: default

The visualization of vaginal microbiome enterotype reveals a clear clustering pattern based on dominant bacterial species. Three distinct enterotypes were identified.

1) E1: Dominated by Lactobacillus crispatus, which is predominantly found in the vaginal fornix and the cluster (blue) shows a tight grouping, indicating a stable and protective microbial community that is less prone to disruption, HPV16 infection.

2) E2: Includes various bacterial species various bacterial species such as Gardnerella swidsinskii group and Fannyhessea vaginae. These species are typically found in the vaginal cervix and are associated with a more diverse and potentially dysbiotic environment.

3) E3: Lactobacillus iners dominant, found in the vaginal cervix. Unlike L. crispatus, L. iners is often associated with a less stable microbiome. This would go result to more susceptible to infection.

The enterotype result indicates that L. crispatus maintains as a taxonomic biomarker in vaginal fornix environment. On vaginal cervix environment, rather than HPV16 infection, the clusters are discriminated by L. iners and the other taxons. But with additional information of each taxon in different paper, we can conclude that E2 taxons correlated to dysbiosis in the vaginal cervix, as potential taxonomic biomarkers.

Deciphering Vaginal Microbiome Cooccurrence Patterns in HPV16 Infection

Figure 5. Network plot using SparCC. Each node provides bacterial species, green link for positive, red link for negative cooccurrence. Upset Plot: rank: Species, minimal prevalence: 0.1, threshold: 0.4

The network analysis highlights several key microbial communities. Lactobacillus cluster (yellow, center) does have negative correlation with Gardnerella cluster (yellow, down) and others (red) most of which are related to enterotype E2 cluster. This red cluster also has negative correlation to different cluster (green), which could be possible taxonomic biomarkers that could be identified as healthy vaginal microbiome.

Conclusion: What insights Were Found

Their results revealed significant alterations in the vaginal microbiome of HPV16-positive women compared to controls. Specifically, there was a lower relative abundance of dominant Firmicutes and higher abundances of Actinobacteria, Fusobacteria, and viruses in the HPV16-positive group. Seventy-seven genera, including Gardnerella, Peptostreptococcus, and Prevotella, were more abundant in HPV16-positive women, while twenty genera, including Lactobacillus and Aerococcus, were less abundant. This suggests that microbial dysbiosis may be associated with HPV infection.

The study identified 12 genes, 17 genera, and 7 species biomarkers with excellent predictive power for distinguishing HPV16-positive individuals, with AUC values of 0.861, 0.819, and 0.918, respectively.

Revisiting this dataset through reAnalyze, we identified that common species like Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis showed similar abundant as paper results. Differential abundance analysis highlighted Enterococcus faecium, g__CAG-568 MSSCM00921844_s, and Veillonella parvula group as significantly more abundant in HPV16-positive samples, while Fusimonas MSSCM00678208_s was more abundant in HPV negative group, offering candidate biomarkers for further exploration.

The comparison of metagenomic cohorts using differential abundance analyses provides valuable insights into the microbial dynamics of healthy and HPV16-infected vaginal environments. By revisiting older datasets with updated tools and databases, we can uncover new findings that enhance our understanding of the vaginal microbiome and its role in health and disease. This is our intention with reAnalyze.

---

References

Gajer P, Brotman RM, Bai G, Sakamoto J, Schütte UM, Zhong X, Koenig SS, Fu L, Ma ZS, Zhou X, Abdo Z, Forney LJ, Ravel J. Temporal dynamics of the human vaginal microbiota. Sci Transl Med. 2012 May 2;4(132):132ra52.

Petrova MI, van den Broek M, Balzarini J, Vanderleyden J, Lebeer S. Vaginal microbiota and its role in HIV transmission and infection. FEMS Microbiol Rev. 2013 Sep;37(5):762-92.

Yang Q, Wang Y, Wei X, Zhu J, Wang X, Xie X and Lu W (2020) The Alterations of Vaginal Microbiome in HPV16 Infection as Identified by Shotgun Metagenomic Sequencing. Front. Cell. Infect. Microbiol. 10:286.