The Cervical Microbiome over 7 Years and a Comparison of Methodologies for Its Characterization

Abstract

Background The rapidly expanding field of microbiome studies offers investigators a large choice of methods for each step in the process of determining the microorganisms in a sample. The human cervicovaginal microbiome affects female reproductive health, susceptibility to and natural history of many sexually transmitted infections, including human papillomavirus (HPV). At present, long-term behavior of the cervical microbiome in early sexual life is poorly understood. Methods The V6 and V6–V9 regions of the 16S ribosomal RNA gene were amplified from DNA isolated from exfoliated cervical cells. Specimens from 10 women participating in the Natural History Study of HPV in Guanacaste, Costa Rica were sampled successively over a period of 5–7 years. We sequenced amplicons using 3 different platforms (Sanger, Roche 454, and Illumina HiSeq 2000) and analyzed sequences using pipelines based on 3 different classification algorithms (usearch, RDP Classifier, and pplacer). Results Usearch and pplacer provided consistent microbiome classifications for all sequencing methods, whereas RDP Classifier deviated significantly when characterizing Illumina reads. Comparing across sequencing platforms indicated 7%–41% of the reads were reclassified, while comparing across software pipelines reclassified up to 32% of the reads. Variability in classification was shown not to be due to a difference in read lengths. Six cervical microbiome community types were observed and are characterized by a predominance of either G. vaginalis or Lactobacillus spp. Over the 5–7 year period, subjects displayed fluctuation between community types. A PERMANOVA analysis on pairwise Kantorovich-Rubinstein distances between the microbiota of all samples yielded an F -test ratio of 2.86 (p<0.01), indicating a significant difference comparing within and between subjects’ microbiota. Conclusions Amplification and sequencing methods affected the characterization of the microbiome more than classification algorithms. Pplacer and usearch performed consistently with all sequencing methods. The analyses identified 6 community types consistent with those previously reported. The long-term behavior of the cervical microbiome indicated that fluctuations were subject dependent.

Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women

“…Other limitations of our study include small sample sizes of some of the comparison groups (most notably the limited number of bacterial STI cases despite oversampling of these cases), imprecise timing of certain behaviors around the time of sampling, lack of a control group of women at low risk for HIV and STIs and the fact that we only used cervical samples (including endoand ectocervix). Although small differences between the cervical and vaginal microbiome have been described, the types and relative quantities of the most abundant bacteria are similar between the two sampling sites and bacterial community compositions are also similar (Nikolaitchouk et al, 2008;Kim et al, 2009;Ravel et al, 2011;Smith et al, 2012).…”

“…To date, 10 studies have described clusters on the basis of next generation sequencing data (Forney et al, 2010;Hummelen et al, 2010;Ravel et al, 2011;Schellenberg et al, 2011;Frank et al, 2012;Martin et al, 2012;Smith et al, 2012;Srinivasan et al, 2012;Drell et al, 2013;Lee et al, 2013). Even though these studies included different study populations and employed a variety of molecular and clustering procedures, consistent clustering patterns can be discerned.…”

Reproductive Microbiomes: Using the Microbiome as a Novel Diagnostic Tool for Endometriosis

“…Consistent with other studies characterizing the cervical and vaginal bacterial communities, the cervical bacterial communities of our patients were predominately Lactobacillus sp. [25]. Unfortunately, the patients cannot be grouped into traditional vaginal community types [3] because taxonomic resolution at the species level is not reliable, thus genus level characterizations were used.…”

“…Recent research has demonstrated that there are robust and diverse uterine [5] and cervical [25] bacterial communities. Studies are now beginning to examine how different diseases may cause deviations in these communities.…”

Exploring the dynamics of bacterial community composition in soil: the pan-bacteriome approach

“…Indeed, environmental changes often affect taxonomic composition and abundance in microbial communities (Allison and Martiny 2008), which may have a strong effect on soil health and plant productivity (Chaparro et al 2012). Several works have been performed inspecting bacterial community variation both in cross sectional (different sites at the same time) and longitudinal studies (the same site studied over time) (Bartram et al 2014;Chen et al 2013;Costello et al 2009;Kuang et al 2012;Logares et al 2013;Pini et al 2012;Smith et al 2012).…”