ABSTRACT
The identity of Lactobacilli colonizing the vagina of premenopausal Nigerian women has not been studied with the most recent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and gene sequencing techniques. The resultant paucity of information on Lactobacilli leaves a very large gap in the knowledge of the species that may be propagated and used as biotherapeutic agents. This study was designed to (a) determine the Lactobacilli colonizing the vagina of women of childbearing age, (b) test if the Lactobacilli isolated exhibit probiotic properties, (c) compare culture-independent and dependent methods in Lactobacillus identification, (d) determine the safety of known probiotics in an animal model and (e) determine the receptivity of probiotics among premenopausal women for the maintenance of urogenital and gastrointestinal health.
Two hundred and forty one premenopausal Nigerian women (18-48 years) provided vaginal swabs which were Gram stained for Nugent scoring, cultured on MRS agar, microbial DNA was extracted, amplified using PCR, with Lactobacillus primers, and processed by DGGE. DGGE bands were excised, re-amplified, purified, V2-V3 region of 16S rRNA gene sequenced with ABI prism (3730xl) BigDye Terminator. Lactobacillus strains were tested for probiotic properties (H2O2 and biosurfactant production; acid tolerance and pathogen inhibition). Safety of Western probiotics was determined in an animal model and a non-parametric survey was employed for probiotic receptivity.
Of the two hundred and forty one vaginal samples that were Gram Stained for Nugent rating, 84 (34.8%) had Normal rating (0-3), 123 (51%) had Intermediate score (4-6), while 34 (14.2%) had bacterial vaginosis (BV), with 7-10 score. There was a positive association between absence of lactobacilli PCR product and Nugent scores interpreted as BV (85.8% vs. 14.2%. χ2 [2]= 4.12, P=0.05). Sequence results revealed that most women (64%) were colonized by Lactobacillus iners as the most predominant strain. L. gasseri was found in 7.3% samples, followed by L. plantarum (6.0%), L suntoryeus (6.0%), L. crispatus (3.0%), L. rhamnosus, and other species. Out of 241 swab samples cultured on MRS agar, only 24 (10%) samples had growth of Lactobacillus at pH 4.5. Lactobacillus gasseri (3.3%) was isolated from 8 samples, followed by L. plantarum (2.4%), L. vaginalis (1.6%), L. fermentum (0.8%), L. crispatus (0.8%), and L. rhamnosus (0.8%). There was a significant difference between culture independent and dependent methods [85% vs.10%, χ2 [2] = 148.72, P<0.001]. Lactobacillus plantarum and L. fermentum produced H2O2, were acid tolerant (pH 2 - 5.5), inhibited pathogen growth and produced biosurfactants. The safety profile of Probiotic GR-1 and RC-14 showed no significant alteration on the hematological parameters of Sprague Dawley albino rats. There was a significant increase in the birth weight of rat pups whose mothers were fed with probiotic GR-1 and RC-14 (6.5g vs.4.5g; P=0.01). The survival rate of rat pups from mothers fed with Probiotics, and controls, up to 21 days post weaning showed a significant difference in the rate of mortality (18% vs. 2.7%, χ2 [2]= 4.84, P=0.05). Receptivity for probiotic products among female university students indicated that 82% would welcome oral or vaginal probiotics to improve their health.The study presents a new understanding of the nature of the vaginal Lactobacillus microbiota of some women in Nigeria and some strains of the lactobacilli possess probiotic properties for biotherapeutic applications.
