Normal as well as abnormal microbiotas play a critical role in the progression and outcome of complex microbial diseases (e.g., see references 19
, and 36
). In order to better understand the disease process, to gain clearer understanding of the influence of the microbiota that are present, and to better evaluate methods for diagnosing and treating these complex diseases, it is important to understand the impact that variability in collection protocols and sampling techniques may have on diagnosis and data interpretation.
Here, we report considerable variation between the microbiotas among samples collected from three vaginal sites (cervix, fornix, outer vaginal canal) and by three different sampling methods (lavaging, swabbing, scraping). Previous culture-based studies have reported differences in site-specific vaginal microbial profiles (2
), but the scope of these studies was limited due to cultivation biases. This is the first study examining niche variation in the vaginal tract by using molecular methods, which allowed for greater resolution in the detection of site-specific differences. In addition, this is the first study to address potential sampling bias introduced by use of swabs only, which may not adequately collect adherent bacteria. Finally, results for culture-independent analysis of the influence of sampling methods on the detection of vaginal microbes have also not been previously reported.
The lavage sample detected the least diversity in the vaginal microbiota and may not be representative of the true diversity of the vaginal bacteria. Though the lavage sample was the most dilute of the six samples collected, dilution would not be expected to alter the overall microbial composition of the samples, merely the total quantity of microbes within that sample. An interesting observation was the complete lack of Proteobacteria
sequences from the lavage samples of all the subjects, even though Proteobacteria
constituted the second largest representation in the overall vaginal clone library. Little or no Fusobacteria
, or Bacteroidetes
were found in the three swab or lavage clone libraries for six of the eight subjects (groups 1 and 2), even though Fusobacteria
, and, to a lesser extent, Bacteroidetes
were present in the scrape samples, which presumably would facilitate the collection of more adherent organisms. Prior studies have shown that the vaginal microbiota includes organisms that form loosely and tightly tissue-adherent biofilms (9
), possibly accounting for the differences in the bacteria detected among the three sampling methods utilized. G. vaginalis
, a member of the phylum Actinobacteria
, forms adherent biofilms (29
), and this may explain the greater prevalence of Actinobacteria
in the scrape samples of some subjects. These results suggest that a single sample from an individual might not be sufficient to reflect the complexity of the vaginal microbiota within a subject, and these results provide a framework for microbial ecologists and population biologists who seek to identify and characterize factors that underlie the establishment and maintenance of barriers that separate and define microenvironments.
In this study, we were particularly concerned about causing perturbations in the microbial community composition at sites that were swabbed or scraped and, thus, collected a single sample from each site by any particular method. Nonetheless, we collected both a swab sample and a scrape sample from the lower one-third region of the vaginal canal of each individual. For six of the eight subjects, the microbial community compositions of the swab and scrape samples were considerably different. It is not currently clear whether these differences were strictly due to differences in the sampling methods or due to perturbations at the sampling site during collection, but this work underscores the importance of considering the potential effects of sample collection methodology on microbial community structure.
The composition of the vaginal microbiota varied substantially among the subjects in this study, in agreement with observations from other culture-independent studies of the vaginal microbiota (14
). Individuals from group 1 and group 2 all had Nugent scores of 0, consistent with what is generally considered a “healthy” vaginal microbiota (22
). The vaginal microbiotas of group 1 subjects exhibited the least microbial diversity, and all the sample types were dominated by Lactobacillus
spp. and, thus, most closely resembled what is conventionally considered to be a “healthy” microbial composition (1
species dominated all the samples from group 2, but the relative abundances of each varied widely among the sample types within and between individuals. Given the abundance of Pseudomonas
in most of the subjects in this study and in those from a recent study by Hyman et al. (17
spp. may be previously unrecognized common members of the healthy vaginal ecosystem.
was present in all the samples from subject 403 (group 3) and in most samples from subject 409 (group 4), but the relative abundances varied among the sample types. The presence of G. vaginalis
sequences in the clone libraries of these two subjects is consistent with the Gram staining results for these individuals. While all of the subjects in this study were asymptomatic and did not exhibit any clinical signs of BV, the Nugent scores and the greater microbial diversities observed for subjects 403 and 409 suggest that subject 403 may have been transitioning into or out of disease and subject 409 may have had asymptomatic BV at the time of specimen collection. Alternatively, it is possible that our understanding of BV is incomplete, and a high Nugent score does not necessarily reflect a diseased state in all cases. Moreover, subject 409, who exhibited the greatest heterogeneity in bacterial composition among all of the sample types, reported having been infected with chlamydia in the 6 months prior to the study, indicating that the recently perturbed microbiota may have not yet recovered from the infection. However, this subject tested negative for chlamydia at the time of sample collection, and Chlamydia
16S rRNA gene sequences were not detected in the clone libraries from this individual. Interestingly, subjects 403 and 409 were also the only two subjects reporting new sexual partners within the 6 months prior to sample collection. History of multiple sexual partners is a known risk factor for BV (4
), but given the small sample size, it is not possible to draw any conclusions regarding sexual history and vaginal microbiota in these study participants.
These results are highly significant for medical microbiologists who seek to correlate microbial community composition to the pathophysiology of vaginal diseases, and especially for clinical practitioners interested in predictive measures for vaginal health and disease, susceptibility to sexually transmitted disease, reproductive fecundity, and pregnancy outcome. Notably, standard clinical practice today mandates a single swab of the vaginal area, but protocols are poorly defined. The data presented here indicate that the bacterial diversity detected by combining swab samples only from three different locations in the vaginal canal is lower than that detected using both swab and scrape samples from multiple locations within an individual. The broader implication of this demonstration of the differences between samples within individuals is that it highlights the importance of the sampling site and sampling method in determining the composition of vaginal microbial communities within individuals.
The methods utilized in this study are applicable to both clinical and laboratory settings. Our clinical partners have been implementing the specimen collection methods described here with relative ease. Clinical laboratory techniques are changing rapidly and exploring the use of PCR and DNA gene-chip technologies for diagnostics. Consequently, it is very likely that culture-independent bacterial detection techniques such as 16S sequencing could be utilized in a clinical laboratory setting in the near future.
These findings could significantly impact specimen collection protocols and sampling techniques for experimental studies and for clinical diagnostic evaluation and treatment of vaginal disease, as microbes indicative of an unhealthy vaginal state may be absent or underrepresented in one sampling site or by one sampling method but may be predominant in another. This niche variation is particularly important with regard to obstetric and neonatal care in considering the appropriate collection technique for the determination of group B streptococcal colonization in pregnant women, a critical risk factor for adverse postpartum outcome (15
). Thus, based on the results of this study, an overall scoop of the entire vaginal tract that includes collection of adherent bacteria might provide a more representative view of the vaginal microbial composition and may be important for accurate clinical evaluation of individuals, especially those with highly heterogeneous microbiota or those who may be transitioning into disease.