Sexually Transmitted Diseases:
Lactobacillus Vaginal Microbiota of Women Attending a Reproductive Health Care Service in Benin City, Nigeria
Anukam, Kingsley C. PhD*†; Osazuwa, Emmanuel O. PhD†; Ahonkhai, Ijeoma PhD†; Reid, Gregor PhD*‡
From the *Canadian Research & Development Centre for Probiotic, Lawson Health Research Institute, London, Ontario, Canada; the † Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Nigeria; and the ‡ Departments of Microbiology and Immunology, and Surgery, University of Western Ontario, London, Ontario, Canada
The support of NSERC of Canada is appreciated and the technical assistance of Ms. Christine Heineman, Ms. Dominique Lam, and Ms. Sheri Saunders of LHRI are appreciated. The authors also thank Dr. M. Ngwu, a gynecologist, Shepherd Clinic and Maternity, and Dr. E. C. Ohanaka, Dr. Onakewhor, Dr. G. I. Osemene, and Mr. Martin Duru of the University of Benin Teaching Hospital (UBTH), for their assistance in the collection of the vaginal samples.
Correspondence: Kingsley C. Anukam, PhD, Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Nigeria. E-mail: firstname.lastname@example.org.
Received for publication March 22, 2005, and accepted May 31, 2005.
Objective: The objective of this study was to determine whether Lactobacillus species found in African women differ substantially to those of white decent, described in previous studies. The vaginal microbiota play an important role in female health, and when the naturally dominant lactobacilli are displaced resulting in bacterial vaginosis (BV), the host is more at risk of acquiring sexually transmitted diseases, including HIV.
Methods: Vaginal samples were collected from 241 healthy, premenopausal Nigerian women, which were then Gram-stained for Nugent scoring. Microbial DNA was extracted, amplified using polymerase chain reaction (PCR) and Lactobacillus primers, and processed by denaturing gradient gel electrophoresis (DGGE). Lactobacillus species were identified by DNA sequencing and BLAST algorithm.
Results: Of the samples, 207 (85.8%) had PCR products for lactobacilli, whereas 34 (14.2%) showed absence of lactobacilli, which correlated to the BV Nugent scores. On sequencing of amplicons, 149 subjects (72%) had sequence homologies to lactobacilli. Most women (64%) were colonized by L. iners as the predominant strain, similar to previous findings in Canadian and Swedish women. L. gasseri was found in 7.3% samples, followed by L. plantarum, L. suntoryeus, L. crispatus, L. rhamnosus, and other species.
Conclusion: The findings indicate that even with geographic, racial, and other differences, the predominant vaginal Lactobacillus species is similar to species in women from Northern countries.
THE VAGINAL MICROBIOTA PLAY an important role in female health. When the naturally dominant lactobacilli are displaced resulting in bacterial vaginosis (BV), the host is at major risk for sexually transmitted diseases, including the acquisition of HIV/AIDS.1 Lactobacillus presence is severely depleted or absent in BV, replaced by potentially pathogenic bacteria that form dense biofilms in the vaginal vault. Symptoms are not often present,2 but BV results in elevated pH and inflammatory IL-1 and IL-8.3 This inflammatory condition is of most concern in countries such as Nigeria, where HIV infection in women is skyrocketing. Studies have shown that black women are at higher risk of BV,4 and one might wonder if this is because of their vaginal Lactobacillus species differing from those of whites.
The increased risk of HIV among some black women,5 albeit associated with various factors such as douching practices,6 has raised the possibility that the Lactobacillus microbiota is depleted or comprised of species different from those in white women.
In the last century, culture-based studies showed that L. acidophilus was the most predominant species in the microbiota of the healthy vagina,7 with other species such as L. fermentum, L. plantarum, L. brevis, L. jensenii, L. casei, L. delbrueckii, L. vaginalis, and L. salivarius being secondary.8 The use of culture followed by whole-chromosomal DNA probes indicated that colonization was primarily by L. crispatus or L. jensenii.9,10 Very recently, nonculture polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) and clone library methods have identified L. iners as the most common species detected in the human vagina of white women.11–14
One method of displacing BV and restoring a healthy vaginal tract has been to administer probiotic lactobacilli.15,16 If the vaginal microbiota of African women is similar to those of northern populations where probiotic studies have been undertaken, a case could be made for use of such therapy in Africa. In the current study, the Lactobacillus composition of healthy Nigerian women was investigated using a Nugent scoring Gram stain system17 and PCR-DGGE.
Materials and Methods
Two hundred forty-one healthy (as defined by having no symptoms or signs of major disease including HIV) premenopausal women attending a reproductive healthcare service in Benin City provided a vaginal swab collected by a physician with sterile speculum. The age of the subjects ranged between 16 and 48 years (32 ± 16). The swabs were smeared onto glass slides in Nigeria, for Nugent scoring, then placed in ice packs and transported by courier to the Lawson Health Research Institute, London, Canada, for DNA extraction and sequencing. The swabs were received within 4 days.
Smears were made on microscope slides from vaginal swabs collected from each subject. The slides were Gram-stained and scored by Nugent criteria.17 A score of 0 to 10 was assigned considering the relative proportions of large Gram-positive rods (indicative of lactobacilli), small Gram-negative or gram-variable rods (Bacteroides, Prevotella, or Gardnerella species), and curved Gram-variable rods (Mobiluncus species). A score of 0 to 3 was interpreted as consistent with normal microbiota, a score of 4 to 6 as intermediate, and a score of 7 to 10 was considered consistent with BV.
Extraction of DNA From Vaginal Swabs, Polymerase Chain Reaction Amplification of the DNA Template/Sample, and Denaturing Gradient Gel Electrophoresis
This methodology has been described in detail in previous publications11,12,18 and is only summarized here. DNA was extracted from the vaginal swabs using Instagene Matrix (Bio-Rad Laboratories) according to the manufacturer’s instructions. The supernatant containing the DNA was stored at −20°C. The amplification reactions of the DNA template/sample were carried out in 0.2 mL PCR single tube-RNase/DNase/pyrogen free (Diamed, Lab Supplies, Mississauga, ON, Canada) with a hinged flat cap in a Thermocycler (Eppendorf Mastercycler). The primers used were those of Walter et al19: Lac-1, 5′-AGC AGT AGG GAA TCT TCC A-3′ and Lac2-GC with the sequence; 5′-CGC CCG GGG CGC GCC CCG GGC GGC CCG GGG GCA CCG GGG GAT TYC ACC GCT ACA C-3′ (Invitrogen; Life Technologies), which have been previously been used with DNA from other human clinical samples. The PCR amplification program consisted of an initial DNA denaturation step at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 1 minute, and elongation at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. To confirm amplicon production, samples (5 μL PCR product) were analyzed by electrophoresis (Bio-Rad) in agarose gels (1.5%) at 100 V for 45 minutes, followed by staining with ethidium bromide and destaining with 1 × TAE for 10 minutes. Gels were visualized by ultraviolet transillumination and recorded with Polaroid 667 instant film. For DGGE, the denaturing environment was created by a combination of uniform temperatures, typically between 50° and 65°C and a linear denaturant gradient formed with urea and formamide. Preparation of gel gradients and electrophoresis was carried out according to the manufacturer’s instructions for the D-Code Universal Mutation Detection System (Bio-Rad Laboratories). After electrophoresis, gels were removed, allowed to cool, stained, and recorded, as described previously. DGGE fragments were excised from the gels washed in buffer and incubated overnight at 4°C.
PCR reamplification was conducted using the same PCR Master Mix to a total volume of 50 μL and Lactobacillus primers Lac-1 and Lac-2 without the GC clamp. The amplification, annealing, and extension conditions were the same as described before. The DNA fragments from PCR reactions were purified using QIAquick purification Kit protocol (QIAGEN Inc., Mississauga, ON, Canada). For sequencing, 10 μL of the purified DNA was mixed with 3 μL of either LGC-1 or LGC-2 primers and 2 μL of Milli-QH2O. Sequences of the fragments were determined by the automatic Big Dye (dideoxy chain terminator) sequencer method ABI PRISM 3,730 (Sequencing Facility, John P. Robarts Research Institute, London, ON, Canada). Sequences were edited to exclude the PCR primer binding sites and manually corrected with Chromas 2.3 (Chromas version 2.3; www.technelysium.com.au.chromas.html). For identification, sequences were compared with those available in the V2–V3 region of the 16S rRNA sequences using the GenBank DNA databases (www.ncbi.nih.gov) and the standard nucleotide–nucleotide BLAST algorithm.20 The identities of isolates were determined on the basis of the highest similarity.12
Nonparametric statistical methods such as chi-squared were used to test the association between presence of lactobacilli PCR product and Nugent scores indicating normal, intermediate, and BV.
Of 241 vaginal samples that were Gram-stained for Nugent rating, 84 (34.8%) had a normal rating (0–3), 123 (51%) had intermediate score (4–6), whereas 34 (14.2%) had BV (Table 1). Chi-squared analysis showed a positive association between absence of lactobacilli PCR product and Nugent scores interpreted as BV (85.8% vs 14.2%, χ2 = 4.12, P = 0.05).
DGGE analysis of 207 PCR products (Fig. 1) showed that 73 (35%) typically yielded one band, 60 (29%) had 2 bands, followed by 42 (20%) with 3 bands, 26 (12.5%) with 4 bands, and 6 (2.8%) yielding 5 DGGE bands.
DNA sequencing was conducted on excised DGGE fragments, which were reamplified with PCR. Sequences as revealed by the BLAST algorithm (Table 2) shows that 95 (64%) of the samples were colonized by L. iners: 3 clones FX177–4, FX181–4, and FX181–1 dominated. The L. salivarius identity level was only 94% and although this is lower than the 97% to 100% preferred, it was the closest species found in the search. After L. iners, the most commonly found organisms were L. gasseri (7.3%), L. plantarum (6.0%), L. suntoryeus (6.0%), L. crispatus (3.0%), L. rhamnosus (2.7%), L. vaginalis (2.7%), Lactobacillus sp. (2.7%), L. fermentum (1.3%), L. helveticus (1.3%), L. johnsonii (1.3%), and L. salivarius (1.3%).
This is the first study using Nugent rating and PCR-DGGE 16S rRNA gene sequencing to investigate the vaginal lactobacilli of apparently “healthy” African women. The subjects were most commonly colonized by the same Lactobacillus species, L. iners, similar to studies of white women in Canada, the United States, and Sweden.12–14 Indeed, this organism, which does not grow on Rogosa or MRS media traditionally used for lactobacilli recovery, was present in 64% of the samples. This similarity in species among women in different locales is quite remarkable given the different races, diets, and lifestyles.
A principal rationale for administering probiotic lactobacilli to women with a history of urinary tract infection and BV is to displace the pathogens and reestablish a lactobacilli population, which in turn creates an environment in which indigenous Lactobacillus return. Based on the data here, there is no reason to think that “Western” strains, L. rhamnosus GR-1 and L. reuteri (formerly fermentum) RC-14, selected 20 years ago as probiotics for vaginal health, could not be effective in colonizing African women and reducing the risk of BV and its complications.21,22 There is certainly local receptivity for this approach in Nigeria,23 and animal safety studies have been successfully performed in pregnant and nonpregnant rats.24,25
L. gasseri and L. plantarum were next most commonly recovered, and although both species are known to colonize whites,14,26 they are not generally as prevalent as L. crispatus and L. jensenii. It has been proposed that a low recovery of L. crispatus and L. jensenii could increase the risk of BV, because these species produce high levels of hydrogen peroxide that are detrimental to BV propagation.27 However, strains of L. gasseri also produce high levels of H2O2,28 whereas strains of L. plantarum are known to be able to bind to vaginal cells,29,30 produce bacteriocins,31 and be good candidates for antiinfective therapy.32 This data plus the low prevalence of BV among the women tested suggests that the L. crispatus and L. jensenii species are not critical for defense of the vagina, and although H2O2 levels were not measured, it does not appear that this is the key critical factor in health because relatively few of the normal Nugent scored women had the L. crispatus and L. jensenii species detected.
Of interest, unlike studies on white women with BV in whom PCR-DGGE detected lactobacilli,11 no lactobacilli were detected here. The handling and processing were performed similarly, so it raises the question as to whether an increase in Mycoplasma hominis, the most commonly detected pathogen (35%) in the BV subjects, was associated with BV. A previous study showed an increased presence of this organism in African women with BV compared with those with a normal Nugent.33 In addition, carriage of intron 2 of the IL-2 receptor antagonist allele 2 is associated with elevated vaginal pH and chronic inflammation (like with BV), and increased presence of Mycoplasma and deceased lactobacilli.34 Therefore, the absence of lactobacilli in BV subjects here could have a number of explanations. Further studies are warranted.
The women sampled here and others who responded to a survey at this African site were extremely concerned about acquiring HIV, and indeed 55% believed they may be at risk of becoming infected.23 The finding that only 34.8% of the women sampled here had a normal, lactobacilli-dominated microbiota is consistent with results obtained from white premenopausal women.21 The women sampled here were well educated with good personal care practices. Although the BV rate was only 14.2%, the large number of subjects with intermediate Nugent scores (51%) raises the question as to whether these women will go on to be at risk of infection or revert to a lactobacilli-dominated flora. The application of probiotic lactobacilli would be worth assessing to determine if a change to more women having a “normal” flora is feasible.
On the other hand, it is possible that the intermediate flora is actually somehow protective for the Nigerian subjects, and it represents a “normal” status in that population. The study here was not designed to identify all the organisms in each subject, but a random sampling of intermediate microbiota did not identify patterns different from those found in white women with the same Nugent scores. Furthermore, there has been no evidence from epidemiologic studies to suggest that an intermediate Nugent flora is protective against HIV.
What is clear is that antibiotic treatment of BV does not by itself reduce viral shedding in HIV-positive patients.35 Rather, the decreased risk of HIV and viral shedding requires the restoration of a lactobacilli-dominated vaginal microbiota.36 This provides a strong argument for testing whether lactobacilli probiotics, known to increase the vaginal lactobacilli levels after oral and vaginal administration,16,22 can reduce the occurrences of infections caused by sexually transmitted pathogens, including HIV and herpes simplex virus.
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