Lactobacillus Vaginal Microbiota of Women Attending a
Reproductive Health Care Service in Benin City, Nigeria
KINGSLEY C. ANUKAM, PHD,*† EMMANUEL O. OSAZUWA, PHD,† IJEOMA AHONKHAI, PHD,†
AND GREGOR REID, PHD*‡
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 trans-
mitted diseases, including HIV.
Methods: Vaginal samples were collected from 241 healthy, premeno-
pausal Nigerian women, which were then Gram-stained for Nugent scor-
ing. Microbial DNA was extracted, amplified using polymerase chain
reaction (PCR) and Lactobacillus primers, and processed by denatur-
ing 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 lacto-
bacilli, 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, ra-
cial, and other differences, the predominant vaginal Lactobacillus
species is similar to species in women from Northern countries.
The objective of this study was to determine whether
THE VAGINAL MICROBIOTA PLAY an important role in fe-
male health. When the naturally dominant lactobacilli are dis-
placed resulting in bacterial vaginosis (BV), the host is at major
risk for sexually transmitted diseases, including the acquisition of
HIV/AIDS.1Lactobacillus 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,2but
BV results in elevated pH and inflammatory IL-1 and IL-8.3This
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,4and 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,5albeit
associated with various factors such as douching practices,6has
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. aci-
dophilus was the most predominant species in the microbiota of
the healthy vagina,7with other species such as L. fermentum, L.
plantarum, L. brevis, L. jensenii, L. casei, L. delbrueckii, L. vagi-
nalis, and L. salivarius being secondary.8The use of culture
followed by whole-chromosomal DNA probes indicated that col-
onization was primarily by L. crispatus or L. jensenii.9,10Very
recently, nonculture polymerase chain reaction–denaturing gradi-
ent 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.15,16If the vag-
inal 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 system17and
Materials and Methods
Two hundred forty-one healthy (as defined by having no symp-
toms 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,
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 Pharmaceu-
tical Microbiology, Faculty of Pharmacy, University of Benin, Nigeria.
Received for publication March 22, 2005, and accepted May 31, 2005.
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
Sexually Transmitted Diseases, January 2006, Vol. 33, No. 1, p.59–62
Copyright © 2005, American Sexually Transmitted Diseases Association
All rights reserved.
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.17A 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
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
publications11,12,18and is only summarized here. DNA was ex-
tracted 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 am-
plification 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 ?L PCR product) were analyzed by elec-
trophoresis (Bio-Rad) in agarose gels (1.5%) at 100 V for 45
minutes, followed by staining with ethidium bromide and destain-
ing with 1 ? TAE for 10 minutes. Gels were visualized by
ultraviolet transillumination and recorded with Polaroid 667 in-
stant 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 for-
mamide. 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 Labora-
tories). 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 over-
night at 4°C.
PCR reamplification was conducted using the same PCR Master
Mix to a total volume of 50 ?L 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, Can-
ada). For sequencing, 10 ?L of the purified DNA was mixed with
3 ?L of either LGC-1 or LGC-2 primers and 2 ?L 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 Gen-
Bank DNA databases (www.ncbi.nih.gov) and the standard nucle-
otide–nucleotide BLAST algorithm.20The identities of isolates
were determined on the basis of the highest similarity.12
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 ab-
sence of lactobacilli PCR product and Nugent scores interpreted as
BV (85.8% vs 14.2%, ?2? 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
Reaction (PCR) Product and Nugent Scores of Normal,
Intermediate, and Bacterial Vaginosis
Showing Presence of Lactobacilli Polymerase Chain
N ? 241
Presence of lactobacilli
Absence of lactobacilli
Fig. 1. Showing a representative denaturing gradient gel electro-
phoresis bands. Lane 1 to 10 represents 10 Subjects. Lane 9 with 5
bands, whereas lane 2, 3, and 8 have bands, respectively.
Sexually Transmitted Diseases ● January 2006
ANUKAM ET AL
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. fermen-
tum (1.3%), L. helveticus (1.3%), L. johnsonii (1.3%), and L.
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.12–14Indeed, 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 Lacto-
bacillus return. Based on the data here, there is no reason to think
that “Western” strains, L. rhamnosus GR-1 and L. reuteri (for-
merly 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.21,22There is
certainly local receptivity for this approach in Nigeria,23and
animal safety studies have been successfully performed in preg-
nant and nonpregnant rats.24,25
L. gasseri and L. plantarum were next most commonly recov-
ered, 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.27However, strains of L. gasseri also produce
high levels of H2O2,28whereas strains of L. plantarum are known
to be able to bind to vaginal cells,29,30produce bacteriocins,31and
be good candidates for antiinfective therapy.32This 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 H2O2levels 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,11no 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.33In 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.34Therefore, the absence of lactobacilli in BV sub-
jects here could have a number of explanations. Further studies
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.23The 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.21The
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 applica-
tion of probiotic lactobacilli would be worth assessing to deter-
mine if a change to more women having a “normal” flora is
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 pat-
terns 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.35Rather, the de-
creased risk of HIV and viral shedding requires the restoration of
a lactobacilli-dominated vaginal microbiota.36This provides a
strong argument for testing whether lactobacilli probiotics, known
to increase the vaginal lactobacilli levels after oral and vaginal
administration,16,22can reduce the occurrences of infections
caused by sexually transmitted pathogens, including HIV and
herpes simplex virus.
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Results of Lactobacillus Species Sequence Analysis
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16S rRNA Gene
L. iners clone FX177–4
and FX181–4, FX181–1
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