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Appl Environ Microbiol. Jan 2011; 77(1): 378–381.
Published online Nov 12, 2010. doi:  10.1128/AEM.00973-10
PMCID: PMC3019699

Pyrosequencing of the Genital Microbiotas of HIV-Seropositive and -Seronegative Women Reveals Lactobacillus iners as the Predominant Lactobacillus Species[down-pointing small open triangle]

Abstract

The species of vaginal lactobacilli in HIV-seropositive and -seronegative women were determined by 16S gene pyrosequencing. Lactobacillus iners sequences were the predominant lactobacillus sequences in 66% of HIV+ women and 90% of HIV women. This has implications for resistance of HIV+ and HIV women to genital colonization by pathogenic organisms.

Lactobacillus is the predominant bacterial genus in the lower genital tract of many women. Common species include L. crispatus, L. jensenii, L. gasseri, and L. iners (2, 13, 26, 32). Predominant vaginal colonization by lactobacilli is believed to be beneficial in inhibiting development of bacterial vaginosis (BV), which causes conditions such as preterm delivery and pelvic inflammatory disease (16, 20, 32). Colonization by lactobacilli is also associated with lower rates of infection with several sexually transmitted diseases (1, 6, 7, 19, 24, 28).

Different species of lactobacilli have been associated with differing protective effects from colonization by pathogenic microorganisms. For example, the presence of H2O2-producing lactobacilli was associated with a lower occurrence of BV, and L. crispatus and L. jensenii frequently produce hydrogen peroxide while L. iners and L. gasseri do not (2, 9, 14, 15, 21, 30). Thus, identification of the types of lactobacilli that colonize the genital tract can potentially help in understanding susceptibility to pathogens.

Many studies that identify the lactobacilli in genital tract samples first culture lactobacilli and then use molecular methods to determine the species. Using this approach, several studies showed that L. crispatus and L. jensenii are most frequently the predominant species (2, 23, 29). However, some culture methods can select certain types of lactobacilli over others (10, 11, 32), possibly introducing a bias into identification. To avoid culture bias, recent studies characterized vaginal microbiota by cloning and sequencing the bacterial 16S rRNA gene. These studies also showed that L. crispatus was frequently predominant (12, 17).

Since it is of interest to identify the species of lactobacilli present in women because of their protective ability, and since no studies have done this using culture-independent methods in HIV-seropositive women, a group that is already at increased risk of many types of infections due to compromised T-cell immunity, we sequenced a region of the 16S gene to determine the species of vaginal lactobacilli present in HIV+ and HIV women.

The subjects were a subset of those enrolled in the Women's Interagency HIV Study (WIHS) (4). Informed consent was obtained from all subjects. The HIV+ women were selected randomly from 406 HIV+ subjects studied previously (25), and the HIV women were described previously (27). A total of 30 of the 36 HIV+ women and 9 out of 10 of the HIV women were African-American. The median age was 39 years (range, 32 to 49) for the HIV+ women and 37 years (range, 25 to 45) for the HIV women. None of the women were undergoing current antibiotic treatment, and none had current infection/colonization with Trichomonas or yeast (determined by wet mount and KOH). None of the women were on highly active antiretroviral therapy (HAART).

Methods for genital tract sample collection by cervical-vaginal lavage, DNA isolation, multitag pyrosequencing of a portion of the 16S rRNA gene spanning the V1 and V2 regions, CD4 counts, and Nugent Gram stain were previously described (4, 22, 27). Lactobacillus 16S rRNA gene sequences were identified using the Ribosomal Database II Project (RDP 10) classifier (8) and assembled with reference sequences into phylogenetic trees with a 96% overlap identity and 80% confidence threshold using Geneious Pro 4.6.1 software (Auckland, New Zealand). Reference sequences were AF257097 (L. crispatus), M58820 (L. gasseri), AF243176 (L. jensenii), AF243177 (L. vaginalis), and AY526083 (L. iners).

A total of 48,000 16S gene sequences from 46 women were analyzed. For the 36 HIV+ women, (Table (Table1)1) a median of 24% (range, 0 to 99%) of the sequences corresponded to lactobacilli (3 had no lactobacilli). For the 10 HIV women, a median of 44% of the sequences corresponded to lactobacilli (range, 1 to 98%).

TABLE 1.
Proportions of species of Lactobacillus in HIV+ and HIV women

In the HIV+ women, L. iners, L. crispatus, L. gasseri, and L. jensenii were the predominant lactobacillus sequences in 66%, 18%, 9%, and 3% of subjects, respectively. In the 10 HIV women, L. iners was predominant in 90%. L. iners sequences were also present at substantial levels in several of the women who did not have L. iners as the predominant type. The percentage of L. iners was significantly higher in the HIV group (mean, 85%) than in the HIV+ women (mean, 60%; P = 0.004, two-tailed Mann-Whitney test), although this difference may have been due to the relatively small number of subjects (n = 10) in the HIV group.

HIV+ women with sequences that were >50% lactobacilli had a significantly (P = 0.006, two-tailed Mann-Whitney) higher proportion of L. crispatus sequences (median, 4%) than women with <50% lactobacilli (median, 0%). There was a trend (P = 0.07) toward higher levels of L. jensenii in HIV+ women with >50% lactobacillus sequences. Conversely, the proportion of L. iners sequences was significantly lower (P = 0.05) in HIV+ women with microbiota comprised of >50% lactobacilli (median, 30%; mean, 44%) than in women with <50% lactobacilli (median, 81%; mean, 73%).

In HIV women, L. crispatus levels were not significantly higher in the five women with lactobacilli comprising >50% of sequences than in those with <50%. However, L. iners levels were significantly lower in the five with >50% than in those with <50% (P = 0.05; median, 89% versus 100%).

L. iners, compared to L. crispatus or L. jensenii, produces less H2O2 and relatively lower resistance to colonization by pathogens (2, 9, 14, 15, 21, 30). Our observations are in contrast to those that showed L. crispatus as the predominant lactobacillus species using either culture-dependent or -independent methods. A study that used culture-independent methods (12) found that in eight women with predominant lactobacilli, L. crispatus sequences were predominant in five and comprised 40 to 49% of sequences in three. Antonio et al. (2), using culture, reported that the largest numbers of women were colonized by L. crispatus (32%) and L. jensenii (23%) while other lactobacilli colonized at much lower frequencies. Other culture-based studies also showed that L. crispatus was the prevalent lactobacillus species in a majority of women (23, 29). One study using molecular methods, however, found that women were frequently colonized with L. iners (5).

Interestingly, we found that L. jensenii was absent or present at relatively low levels in all women except one. In contrast, culture-based studies found substantial levels of L. jensenii (2, 3). While L. jensenii and L. crispatus are easily cultured on medium commonly used for isolation, strains of L. iners are not (11). Thus, culture bias could alter the proportions of species reported in some studies, possibly providing an explanation for why L. iners was found less frequently in some previous studies.

The CD4 cell count for the HIV+ women ranged from 0 to 726 cells/mm3 (median, 245) (Table (Table1).1). Thirteen of the women had CD4 counts of ≤200 cells/mm3, and the percentage of L. crispatus in these women was significantly lower (P = 0.04, Mann-Whitney) than in the 17 women with CD4 counts of >200 cells/mm3 (median of 0% L. crispatus for both groups). In contrast, the amount of L. iners in the 17 women with CD4 counts of ≤200 cells/mm3 was significantly higher in women with low CD4 counts (median, 83%; P = 0.05) than in women with high CD4 counts (median, 32%). L. jensenii levels and total lactobacillus levels were not significantly different between the CD4 groups. No previous studies compared lactobacillus types with CD4 levels, although a prospective study over time (18) found that vaginosis was more prevalent and persistent among HIV+ women and even more pronounced in those with CD4 counts of ≤200. We speculate that this could have been due to lower levels of H2O2-producing L. crispatus and L. jensenii (2, 9, 14, 15, 21). Studies of the WIHS have not demonstrated an inverse relationship between BV and HIV status, but this may reflect behavioral factors such as condom use rather than biologic susceptibility (31). It should be noted that the HIV group of women analyzed here had higher levels of L. iners than the HIV+ group even though they had no known immunodeficiency. The explanation for why L. iners levels were lower in HIV+ women with high CD4 numbers but higher in women with no decrease in CD4 (HIV) may have been the relatively small sample size of the HIV group.

In conclusion, we showed that L. iners was the predominant lactobacillus in these HIV+ and HIV women. Since previous studies indicate that L. iners is less effective at preventing vaginal colonization by pathogens than other lactobacilli (L. crispatus and L. jensenii), this study has important implications for the resistance of women to colonization by pathogens and the effects of HIV disease on this resistance.

Acknowledgments

This work was supported by NIH grant P01 AI082971, the Chicago Women's Interagency HIV Study (WIHS), and P30 AI082151. WIHS is funded by the National Institute of Allergy and Infectious Diseases (grant UO1 34993), with supplemental funding from the National Cancer Institute and the National Institute on Drug Abuse (grants UO1 AI35004, UO1 AI31834, UO1 AI34994, UO1 AI34989, UO1 AI34993, and UO1 AI42590). Funding is also provided by the National Institute of Child Health and Human Development (grant UO1 HD 23632) and the National Center for Research Resources (grants MO1-RR-00071, MO1-RR-00079, and MO1-RR-00083).

Footnotes

[down-pointing small open triangle]Published ahead of print on 12 November 2010.

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