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

Department of Pediatrics, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, United States of America.
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e40425. DOI: 10.1371/journal.pone.0040425
Source: PubMed


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.
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).
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.
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.

Download full-text


Available from: Ana Cecilia Rodriguez, Oct 09, 2015
1 Follower
36 Reads
  • Source
    • "The Type IV community type is most closely associated with BV, as defined by a Nugent score of 7–10. A study examining the cervical microbiome has largely recapitulated the vaginal findings (Smith et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.
    Frontiers in Cellular and Infection Microbiology 06/2014; 4:72. DOI:10.3389/fcimb.2014.00072 · 3.72 Impact Factor
  • Source
    • "Different second-generation sequencing platforms can also yield different results on community composition (e.g. [25,26]), requiring that each technology be examined separately. Illumina is increasingly being used for evaluating bacterial community composition but soil preservation techniques have yet to be explored on this platform. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.
    PLoS ONE 07/2013; 8(7):e70460. DOI:10.1371/journal.pone.0070460 · 3.23 Impact Factor
  • Source
    • "Although PCR-DGGE has some shortcomings, including the fact that it needs costly equipment, the “GC clamp” is very expensive, and the formamide is poisonous, it is a powerful tool for microbiota studies. But the next generation sequencing [26] might will replace the current molecular biotechnology in the near future. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: The female genital tract is an important bacterial habitat of the human body, and vaginal microbiota plays a crucial role in vaginal health. The alteration of vaginal microbiota affects millions of women annually, and is associated with numerous adverse health outcomes, including human papillomavirus (HPV) infection. However, previous studies have primarily focused on the association between bacterial vaginosis and HPV infection. Little is known about the composition of vaginal microbial communities involved in HPV acquisition. The present study was performed to investigate whether HPV infection was associated with the diversity and composition of vaginal microbiota. Methods: A total of 70 healthy women (32 HPV-negative and 38 HPV-positive) with normal cervical cytology were enrolled in this study. Culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis was used to measure the diversity and composition of vaginal microbiota of all subjects. Results: We found significantly greater biological diversity in the vaginal microbiota of HPV-positive women (p < 0.001). Lactobacillus, including L. gallinarum, L. iners and L. gasseri, was the predominant genus and was detected in all women. No significant difference between HPV-positive and HPV-negative women was found for the frequency of detection of L. gallinarum (p = 0.775) or L. iners (p = 0.717), but L. gasseri was found at a significantly higher frequency in HPV-positive women (p = 0.005). Gardnerella vaginalis was also found at a significantly higher frequency in HPV-positive women (p = 0.031). Dendrograms revealed that vaginal microbiota from the two groups had different profiles. Conclusions: Our study is the first systematic evaluation of an association between vaginal microbiota and HPV infection, and we have demonstrated that compared with HPV-negative women, the bacterial diversity of HPV-positive women is more complex and the composition of vaginal microbiota is different.
    BMC Infectious Diseases 06/2013; 13(1):271. DOI:10.1186/1471-2334-13-271 · 2.61 Impact Factor
Show more