Abstract: The search for Lactobacilli of African origin with probiotic properties formed the basis of the study. Two hundred and forty one premenopausal healthy (as defined by having no symptoms of vaginal infections and are HIV negative) Nigerian women provided vaginal swabs. The swabs were cultured on de Man Rogosa Sharpe (MRS) agar (pH 5.5) and incubated anaerobically at 37°C for 48 h. Microbial DNA was extracted from the colonies and directly from the swabs, amplified using Polymerase Chain Reaction (PCR) with Lactobacillus primers and processed by Denaturing Gradient Gel Electrophoresis (DGGE). DGGE bands were excised, re-amplified, purified, V2-V3 region of 16S rRNA gene sequenced with 3730xl ABI prism BigDye Terminator and sequence identification was by BLAST. Lactobacillus strains were tested for probiotic properties (H2O2, Pathogen inhibition and Biosurfactant production). Out of 241 swab samples cultured on MRS agar, only 24 (10%) samples had growth of Lactobacillus species at pH 4.5. Lactobacillus gasseri was isolated from 6 (3.3%)samples, followed by L. plantarum (2.4%), L. vaginalis (1.6%), L. fermentum (0.8%), L. crispatus (0.8%) and L. rhamnosus (0.8%). Two species, each of Lactobacillus plantarum and L. fermentum produced huge amount of biosurfactants. In addition they produced H2O2, inhibited the growth of intestinal and urogenital pathogens. The study presents a new understanding by culture method showing lactobacilli composition of the vagina of some Nigerian women. Two Lactobacillus species exhibited probiotic characteristics and clinical studies with these strains are now planned following investigation of the genomic regions encoding probiotic functionality.
INTRODUCTION
The human vagina is a complex ecosystem containing an abundance of microorganisms (Tannock, 1995). It has been reported that more than 60 different bacterial species including aerobes, facultative anaerobes and obligate anaerobes colonize the healthy vagina (Hooton and Stamm, 1996; Redondo-Lopez et al., 1990). Studies have shown that urogenital cells are covered by dense bacterial biofilms (Reid et al., 1990), whose composition changes constantly, but in which members of the genus Lactobacillus predominate in 50 to 90% of women, at least until menopause (Reid et al., 1996).
The dominant presence of lactobacilli in the urogenital microflora of healthy women and the obliteration of lactobacilli in patients who develop Urinary Tract Infections (UTI) (Stamey, 1973), Bacterial Vaginosis (BV) and many other genital infections have led to a focus on these bacteria.
Eastern Europeans, some of whom were apparently long-lived, consumed lactobacillus fermented dairy products as part of their daily diet. This was taken as proof of efficacy and milk fermented with the Bulgarian bacillus of Metchnikoff subsequently enjoyed some vogue in Western Europe till today (Tannock, 2004). Metchnikoff’s theory thus gave birth to the concept of probiotics. However, in 2001, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) collaborated and defined probiotics as Live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/WHO, 2001). Europe, has a highly developed, growing market; with probiotic yogurt in at least nine European countries (UK, France, Germany, Spain, Belgium, Netherlands, Denmark, Finland and Sweden) totaled 250 million kg in 1997, with France representing the largest with sales of 90 million kg, valued at US$219 million (Hilliam, 1998).
Probiotics are rarely found in sub-Saharan Africa. In Nigeria, no probiotic products are available but conventional yogurts fermented with starter cultures of Lactobacillus delbruekii var bulgaricus and Streptococcus thermophilus abound in rural and urban cities. These organisms are designed to ferment milk and few if any pass into the gastrointestinal tract or confer health benefits beyond those of nutrition.
The concept of probiotic biotherapy is gaining an increasing momentum in developed countries, while the idea is yet to be grasped by the people of sub-saharan Africa, that are in dire need of probiotic benefits. The search for Lactobacilli of African origin with probiotic properties formed the basis of the study.
MATERIALS AND METHODS
Two hundred and forty one premenopausal healthy (as defined by having no symptoms of vaginal infections and are HIV negative) Nigerian women provided vaginal swabs. The swabs were collected by clinicians, after approval by the ethical review board of the University of Benin. The women were attending reproductive health care centers in Benin City in June 2004 and the samples were taken after informed consent. The age of the women ranged between 18 and 48 years. The swabs were packaged in ice-packs and subsequently transported via courier to the laboratory at the Lawson Health Research Institute, London, Ontario, Canada, for DNA extraction, sequencing and testing of probiotic characteristics.
Isolation of Lactobacilli from Vaginal Swabs
The vaginal swabs were vigorously agitated in sterile 1 mL of Phosphate
Buffered Saline (PBS), pH 7.1) to dislodge cells. Aliquots of each sample were
plated in duplicates on MRS (de Man Rogosa Sharpe) agar plates which were prepared
according to manufacturer’s instructions. The pH of the MRS agar was adjusted
to pH 4.5 with hydrochloric acid and the value determined with digital pH-meter
(Ö240 pH/Temp Meter, Beckman, USA). The inoculated plates were incubated
anaerobically by using the BBL™ GasPack system (Becton Dickinson,
NY, USA) at 37°C for 48 h. Pale straw coloured colonies were identified
as presumptive lactobacillus genus by Gram stain morphology and catalase negative
test.
DNA Preparation, PCR, DGGE and Sequencing of Isolated Lactobacilli
Briefly, an isolated presumptive lactobacillus colony was picked from the
MRS agar plate and resuspended in 1 mL of autoclaved water in a microfuge tube.
The cells were pelleted by centrifugation (Eppendorf, Digital Centrifuge 5417C)
at 10,000 g for 5 min and later washed by re-suspending cells in PBS, centrifuged
at 13,000 g for 3 min. The pellets were re-suspended in 200 μL Instagene
Matrix, incubated for 20-30 min in a water bath (Isotemp®, Fisher Scientific,
USA) at 55°C. The sample was vortexed for 10 sec and boiled at 100°C
(Tekstir® Hot plate) for 8 min. The sample was vortexed for 10 seconds and
centrifuged at 13000g for 3 min. The supernatant containing the DNA was stored
at-20°C. PCR amplification, Denaturing Gradient Gel Electrophoresis (DGGE),
sequencing of the re-amplified fragments were determined by dideoxy chain termination
(Sequencing facility, John P. Roberts Research Institute, London, Ontario).
Analysis of the partial 16S rRNA sequences was conducted using the GenBank DNA
database and the BLAST algorithm (Altschul et al., 1990).
Testing for Hydrogen Peroxide Production (H2O2)
H2O2 production by Lactobacillus strains was tested
for by the method previously described by Eschenbach et al. (1989).
Inhibition Test Against Some Selected Pathogens
Six isolated hydrogen peroxide producing lactobacillus strains were screened
for their inhibition against selected pathogens such as E. coli o157:
H7 and Salmonella typhi, Staphylococcus aureus, Klebsiella
aerogenes and Neisseria gonorrhea, using a modified version of the
agar overlay technique as previously described (McGroaty and Reid, 1988).
Biosurfactant Production by Dialysis and Lyophylisation
Biosurfactant production was tested for six candidates; Lactobacillus
plantarum, Lactobacillus fermentum, L. gasseri, L. vaginalis,
L. crispatus and L. rhamnosus, in large quantities using 300 mL
of overnight (18 h) cultures. Briefly, a colony of cells was picked from a precultured
MRS plate and inoculated into 15 mL MRS broth. The broth was incubated at 37°C
for 24 h. Thereafter, the incubated 15 mL broth containing the cells was added
to 300 mL of MRS broth in flask and incubated at 37°C for 18 h. The cells
were harvested by centrifugation (10,000 x g) for 5 min, at 10°C (cold room)
washed twice in demineralized water by centrifugation (10,000 x g) for 10 min.
The cells were re-suspended in 150 mL of Phosphate buffered saline (PBS), (pH
7.1). The bacteria were kept at room temperature for biosurfactant release with
gentle stirring (Magnetic stirrer) for 2 h. Following biosurfactant release,
the cells were pelleted by centrifugation (10,000 x g) in falcon tubes, for
15 min, at 10°C (cold room). The biosurfactant containing supernatant was
filtered through a series of pore size filters (1.2, 0.8 μ, 0.4 μ
and finally 0.2 μm) (Millipore). The biosurfactant was dialyzed against
demineralized water at 4°C in a Spectrapor membrane tube (molecular weight
cut off 6,000-8,000, Spectrum Medical Industries Inc., California, USA). The
dialysate was lyophilized (freeze dried) and stored at -20°C.
RESULTS
Out of the 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%) (Table 1).
| Table 1: | Lactobacillus species isolated with culture dependent method on MRS agar plate and identified by DNA sequencing |
| Fig. 1: | Growth inhibition of intestinal and urogenital pathogens: zone of inhibition (mm) for L. fermentum (L.f), L. plantarum (L.p), L. crispatus (L.c), L. rhamnosus (L.r), L. vaginalis (L.v), L. gasseri (L.g). GR-1 (Lactobacillus probiotic) |
| Table 2: | Summary of Biosurfactants and H2O2 production by isolated Lactobacillus species |
| *** Lactobacillus plantarum and Lactobacillus fermentum are the potential candidates. Lactobacillus GR-1 is a positive control for biosurfactant and hydrogen peroxide producer | |
Generally all the six Lactobacillus species tested exhibited various degrees of inhibition against the tested pathogens as shown in Fig. 1. Lactobacillus fermentum (Lf) and Lactobacillus plantarum (Lp) had more inhibitory effect on Neisseria gonorrhea with zones of inhibition above 40 mm. The inhibitory effects of all the Lactobacillus strains against Escherichia coli and Klebsiella pneumoniae did not go beyond 30 mm zone. Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus rhamnosus and Probiotic GR-1 had growth inhibition zones against Salmonella typhi above 30 mm.
Among the six Lactobacillus strains, tested for hydrogen peroxide production (Table 2), only Lactobacillus vaginalis did not produce hydrogen peroxide. Out of 6 strains of Lactobacillus plantarum, 4 were strongly positive and 2 weakly positive for H2O2 production. All the two strains of Lactobacillus fermentum were strongly positive for H2O2 production. Of the 8 strains of Lactobacillus gasseri, 4 were strongly positive for H2O2 production, 1 weakly positive and 3 were negative for H2O2 production.
Six of the strains that were tested for hydrogen peroxide production were equally tested for biosurfactants production. Interestingly, two strains Lactobacillus plantarum and Lactobacillus fermentum were positve, while the rest of the tested strains were negative for biosurfactants (Table 2).
DISCUSSION
The isolation of lactobacilli by cultural method has some limitations and identification has been uncertain because of unreliability of phenotypic classifications, which employ sugar fermentations. Our study revealed six lactobacillus species, with Lactobacillus gasseri predominating, contrary to previous studies, based on culture methods, indicating the predominance of Lactobacillus acidophilus (Redondo-Lopez et al., 1990). However our recent published study showed that Lactobacillus iners is the predominant species colonizing the vagina of premenopausal Nigerian women using non-culture method involving 16S rRNA gene sequencing (Anukam et al., 2006a). Lactobacillus iners, is a viable but non-culturable species when cultured in media used for the cultivation of Lactobacilli.
There is the hypothesis that commensal lactobacilli in the lower genital tract reduce the risk of gonococcal infection in women through the production of hydrogen peroxide (St.Amant et al., 2002). The tested lactobacilli exhibited growth inhibition against all the tested pathogens. Although the specific mechanism of action involved in the inhibition of growth of the pathogens was not investigated. Other studies have suggested that the production of lactic acid may contribute to the inhibition. Besides, the approach of competitive exclusion seems to have potential in this regard. Zhao et al. (1998), tested a probiotic culture consisting of several non-pathogenic strains of E. coli isolated from the intestinal tracts of cattle for the prevention of E. coli O157: H7 colonization in young dairy calves. Their study revealed that probiotic cultures were able to reduce E. coli counts in the rumen and the colon.
The inhibition of pathogen growth demonstrated in vitro with some Lactobacillus strains in this study is important for the reduction of the risk of infection and improve recovery from diarrhea caused by E. coli O157: H7 and Salmonella typhi as well as urogenital infections. This finding supports previous studies on the ability of Lactobacilli to stimulate host defenses and reduce translocation and infectivity of Salmonella typhimurium (Reid et al., 2002; Raza et al., 1995).
The fact that not all lactobacilli possess properties that were required to colonize the vagina and inhibit urogenital pathogens needs to be emphasized. Only twelve Lactobacillus strains (Table 2) emerged with excellent production of hydrogen peroxide, thus supporting other studies that not all strains of lactobacilli produce hydrogen peroxide (Reid and Bruce, 2001). It should be noted that hydrogen peroxide production has been proposed as an explanation for the success of lactobacilli as vaginal colonizers and as a further mechanism by which lactobacilli may inhibit the growth of other genital pathogens (Eschenbach et al., 1989). In one cross-sectional study (limited period of time) pregnant women colonized by hydrogen peroxide lactobacilli were less likely than women with hydrogen peroxide negative lactobacilli to have bacterial vaginosis, vulvo-vaginal candidiasis, vaginal trichomoniasis or vaginal colonization by other pathogens (Hillier et al., 1992). Also the clinical relevance of hydrogen peroxide producing isolates of lactobacilli was suggested by Hawes et al. (1996). In a longitudinal study (long period of time) of non-pregnant women. After adjustment was made for douching and having multiple sex partners, it was shown that nonpregnant women lacking hydrogen peroxide producing vaginal lactobacilli were twice as likely to develop BV than were women colonized by hydrogen peroxide producing lactobacilli. Klebanoff et al. (1991), demonstrated in vitro that hydrogen peroxide produced by vaginal lactobacilli was the source of the bactericidal activity against Gardneralla vaginalis and Prevotella bivia.
Two strains Lactobacillus plantarum and Lactobacillus fermentum produced huge quantities of biosurfactant (Table 2) and were positive for hydrogen peroxide production. An important component of any lactobacillus strain that can serve as a probiotic is the production of biosurfactant, which inhibits adhesion of a range of uropathogens (Conway et al., 1987; Reid and Bruce, 2001). This is based on the fact that strains of L. plantarum are known to be able to bind to vaginal cells (Bonetti et al., 2003), produce bacteriocins (Nes et al., 1996) and be good candidates for anti-infective therapy (Grangette et al., 2004).
In a study on the potential of lactobacilli isolated from local ogi slurry in Nigeria, results indicated that L. Plantarum is a possible candidate as a probiotic (Oyetayo and Osho, 2004).
CONCLUSIONS
The present study has shown two potential probiotic organisms of African origin that may be propagated, although further studies are needed to determine the safety and efficacy of the species towards conferring health benefits on the human host. As knowledge of probiotics is very poor among health care providers in Nigeria (Anukam et al., 2006b), concerted research efforts are needed in Nigeria to support and embrace the probiotic concept for general health maintenance.
ACKNOWLEDGMENTS
This study was supported by Chidak Medical Laboratories, Benin City, Nigeria and Canadian R and D Centre for probiotics, Lawson Health Research Institute, London, Ontario, Canada.