The Vaginal Microbiota and Influence of Select Probiotic Lactobacilli Strains


by Anthony Thomas, PhD

 

 

Specific, highly complex microbial communities (microbiota), including their collective genetic material (microbiome), differ between anatomic sites of an individual (e.g., intestinal, vaginal, oral, skin) as well as between people.(1) An increasing body of scientific evidence has demonstrated these microbial communities markedly influence human health. Furthermore, clinical research is demonstrating the targeted manipulation of these microbial communities with specific probiotic strains offers a promising strategy to improve and maintain health.
     
The vagina is a dynamic environment colonized by various microorganisms (microbes), thus, collectively referred to as the vaginal microbiota. The composition and function of the vaginal microbiota has been linked to women's health status. Microbes inhabiting the vagina are thought to provide the first line of defense in the urogenital tract. Although there is not a definitive 'normal" vaginal microbiota, current scientific knowledge has revealed that lactobacilli predominance is generally the hallmark of a healthy vaginal microbiota as high lactobacilli abundance is associated with the promotion and maintenance of vaginal microbial ecosystem balance. In 1892, Albert Döderlein, a German obstetrician and gynecologist considered one of the founders of gynecological bacteriology, first described a Gram-positive vaginal bacillus (Döderlein's bacillus) occurring in normal vaginal secretions of asymptomatic pregnant women, which was later renamed Lactobacillus. He argued that in normal vaginal secretions, Döderlein's bacilli and lactic acid produced by the bacilli were essential to keep the vagina free of pathogenic bacteria. Low numbers or absence of vaginal lactobacilli is more often associated with increased risk of bacterial vaginosis (BV), yeast overgrowth ('yeast infection"), aerobic vaginitis (AV), urinary tract infections (UTIs), and adverse obstetric outcomes (e.g., miscarriage, premature rupture of membranes (PROM), preterm birth, ventilation/respiratory distress at birth, neonatal sepsis, neonatal intensive care unit admission).(2-6) Clinical research has established the therapeutic value of administering specific probiotic lactobacilli strains for the restoration and maintenance of a healthy vaginal microbiota.

Vaginal Acidification: Lactobacilli Production of D- and L-Lactic Acid

Lactobacilli are facultative anaerobic (aerotolerant) bacteria that produce lactic acid via the fermentation of glucose.(7) The vaginal lactic acid concentration is inversely associated with vaginal pH in women with a lactobacilli-dominated vaginal microbiota, indicating lactic acid is predominantly responsible for vaginal acidification.(8) Production of lactic acid and vaginal acidification plays a prominent role in imparting the broad protection against other microbes associated with a lactobacilli-dominated vaginal microbiota.(9) Furthermore, lactic acid reinforces lactobacilli predominance and maintenance of an acidic vaginal pH in support of a balanced microbial ecosystem with limited diversity.
     
Vaginal lactobacilli are the primary source of lactic acid in the vagina(10,11) and only source of the D-isomer as human cells can only produce L-lactic acid, with < 15% of L-lactic acid produced by vaginal mucosal epithelial cells.11 The production of the D-lactic acid isomer by some Lactobacillus strains enhances protection against microbial invasion of the upper genital tract by supporting the integrity of the cervical external orifice of the uterus.(12) The majority of preterm births result from infections caused by bacteria from the vagina that have traversed the cervix. In addition to vaginal acidification, lactic acid has been shown to directly inactivate various reproductive and urinary tract pathogens.(13-15) The active microbicidal/viricidal form of lactic acid is the protonated (LAH) rather than the un-protonated lactate anion (LA-),(16,17) with the latter form a function of both the concentration of total lactate (LAH + LA-) and hydrogen ions (H+)/pH.

Vagmic1.jpg

Lactic acid may also regulate host immune responses to evoke protection against potentially pathogenic microbes within the vaginal microbiota. In the presence of a synthetic analogue of double-stranded viral RNA, lactic acid potentiates the production of protective pro-inflammatory cytokines (IL-8 and IL-1b) by vaginal epithelial cells.(19) Lactic acid was shown to potentiate interleukin-23 production from innate immune cells in response to lipopolysaccharide (endotoxin), which may promote activation of T-helper type 17 subclass of T-lymphocytes in response to Gram-negative bacteria.(20) Each of these activities exemplifies enhanced activation of host anti-microbial innate and acquired immunity.

Other Potential Protective Mechanisms of Vaginal Lactobacilli

In addition to lactic acid production and vaginal acidification, Lactobacilli are thought to utilize several mechanisms to inhibit pathogen colonization of the vaginal tract: co-aggregation of non-pathogens and pathogens to interfere with the infectious capacity of pathogenic species, production of biosurfactants to disrupt adhesion to the vaginal mucosa by pathogenic species, production of antimicrobial bacteriocins and hydrogen peroxide, competitive exclusion of pathogenic species via competing for nutrients and host surfaces, reinforcing the integrity of the vaginal mucosal epithelial barrier (up-regulation of tight junction proteins to limit damage caused to vaginal epithelium by inflammatory processes or pathogens), and regulation of host immune responses (production of antimicrobial peptides/proteins such as defensins, lactoferrin and lysozyme, and alkaline phosphatases, which can bind to lipopolysaccharide/endotoxin to neutralize toxicity).(21)

Researched NUTRITIONALSCoreBioticAD_0118.jpg

Lactobacillus Species Predominance of Vaginal Microbiota

Unlike any other anatomical site of the human body, most vaginal microbial communities (>70%) are dominated by one or more species of Lactobacillus that constitute >50% of all genetic sequences obtained. Recent studies using high-throughput 16S rRNA gene sequencing studies have shown that composition and relative abundance of vaginal microbial communities in reproductive-aged women cluster into at least five core vaginal microbiota, termed community state types (CSTs).(22-24) Four of these CSTs, representing the majority (>70%) of women, are dominated by a different Lactobacillus species: L. crispatus (CSTI), L. gasseri (CSTII), L. jensenii (CSTV), and L. iners (CSTIII), whereas CSTIV is characterized by low proportions of lactobacilli and is composed of a diverse mixture of primarily strict anaerobic bacteria including species of the genera Gardnerella, Atopobium, Mobiluncus, Prevotella and other taxa in the order Clostridiales, as seen with states of BV.
     
Of note, L. iners is present in most women, including both healthy and those with dysbiosis/BV,4 whereas L. crispatus is typically only observed in healthy women. Ravel et al.(22) observed that L. crispatus-dominated vaginal microbiota have lower vaginal pH compared to communities dominated by other species. Vaginal microbiotas dominated by L. iners have lower vaginal concentrations of D-lactic acid as L. iners lacks the gene coding for D-lactate dehydrogenase, so cannot produce this lactic acid isomer,(12) which may in part mediate the higher observed frequency of BV and preterm delivery in these women.(25) D-lactic acid levels were significantly higher when L. crispatus was the dominant vaginal bacterial species than when L. iners or Gardnerella dominated the vaginal microbiota.(12) It has been suggested that the increased proportion of L. iners in women with BV may be due to increased tolerance of this Lactobacillus species to an elevated pH, characteristic of BV, more than other Lactobacillus species.(26) In contrast, L. crispatus, L. gasseri, and L. jensenii are all producers of the D-lactic acid isomer as well as hydrogen peroxide. L. iners also does not produce antimicrobial hydrogen peroxide.
     
Hydrogen peroxide-producing lacto-bacilli are more likely to sustain long-term vaginal colonization, and women colonized by hydrogen peroxide-producing lactobacilli have decreased acquisition of human immunodeficiency virus (HIV) infection,(27) gonorrhea,(27) and BV.(28) Evidence supports a vaginal microbiota dominated by Lactobacillus species other than L. iners is optimal to support vaginal health (i.e., strains of L. iners have not been considered candidates as probiotics to support women's urogenital health).(29,30)

Ethnic Differences

Differences in the composition of these vaginal microbial communities have been observed between women of different ethnic backgrounds. Lactobacilli-dominated vaginal microbial communities (CST I, II, III, V) were observed in ~80% and ~90% of Asian and white women, respectively, but only ~60% and ~62% of Hispanic and black women, respectively.(22) Over-representation of CSTIV in Hispanic and black women was associated with a higher median pH in these ethnic groups as well. Such differences in vaginal microbial communities between women of different ethnic backgrounds have been observed in other studies as well.(24,31)
     
Although CSTIV can be observed in otherwise healthy women, asymptomatic for BV, it is associated with higher Nugent scores (a Gram stain  scoring system from 0 – 10 for vaginal swabs reflecting abundance of Gram-positive rods [lactobacilli] and Gram-negative variable rods and cocci [G. vaginalis, Prevotella, etc.] to diagnose BV with 0 – 3 considered normal, 4 – 6 indicative of intermediate bacterial counts, and 7 – 10 diagnostic of BV) and may be a risk factor for adverse gynecologic and obstetric outcomes.(3,32,33)

Pregnancy and Estrogen Promotion of Vaginal Lactobacilli

Composition of the vaginal microbiota can be dynamic and capable of rapid shifts within a short period of time (e.g., < 24 hours), although more stable in many women and during different physiologic states such as pregnancy. Vaginal microbial communities of pregnant women are more stable and have higher relative abundance of lactobacilli than non-pregnant women.(34,35) The most common lactobacillus species of the vaginal microbiota observed in healthy pregnant women in the late first trimester were L. crispatus and L. gasseri (>50%), followed by L. jensenii (~20%) and L. rhamnosus (~10%), as well as combinations thereof (~10%).(36)
     
Estrogen is thought to play an important role in promoting a lactobacilli-dominated vaginal microbiota by stimulating accumulation of glycogen in the vaginal epithelial mucosa,(37,38) which is thought to contribute to the increased lactobacilli predominance and stability of the vaginal microbiota observed in healthy pregnant women. Nutrient-containing vaginal secretions and glycogen-containing (as a source of glucose) vaginal epithelial cells that are sloughed and subsequently lyse are thought to be primary nutrient sources for the vaginal microbiota. Estrogen increases the volume of vaginal secretions and induces thickening of the vaginal epithelium along with glycogen accumulation, thought to support growth of glucose-fermenting lactobacilli.(39) Changes in relative abundance of vaginal lactobacilli are associated with both estrogen levels and glycogen content across the various life-stages of women (e.g., pre-pubertal, pubertal/reproductive age, postmenopausal).(40) Additionally, use of oral hormonal contraceptives (i.e., estrogen) and a decreased prevalence of BV has been consistently observed in epidemiological studies.(41-44)

Loss of Vaginal Lactobacilli: Common Factors

Various common factors are thought to influence the vaginal microbiota and have been associated with dysbiosis/BV: hygiene/intravaginal practices (douching), sexual activity (e.g., increased frequency and number of partners, lack of male circumcision/condom use), stress, smoking, and use of antibiotics/anti-fungals.(3,45) Antimicrobials have been the primary therapeutic intervention utilized for the treatment of urogenital 'infections" (overgrowth of potentially pathogenic microbes) for more than four decades. Unfortunately, antimicrobial treatment of urogenital infections is often ineffective, particularly for BV; and there is a high rate of recurrent infections without preventative continuation of antimicrobial therapies. Efficacy is also diminishing with increasing development of antimicrobial resistance. The antimicrobial, metronidazole, is the most commonly used treatment for BV; however, cure rates associated with this treatment are low (as low as 61% one month post-therapy(46) with a high incidence of overgrowth of potentially pathogenic bacteria following treatment.(47)

Antibiotics

Antibiotics affect not only pathogenic microorganisms, but many human residential symbiotic and administered probiotic bacterial strains too. Many strains of the most prevalent vaginal lactobacilli species (L. crispatus, L. iners, L. jensenii, and L. gasseri) were all demonstrated to be susceptible to commonly used systemic antibiotics including ampicillin, cefazolin, cefotaxime, and vancomycin, but insensitive to metronidazole and trimethoprim/sulfamethoxazole along with differential sensitivity to others (gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline). For example, treatment with clindamycin was shown to suppress/eradicate L. crispatus and induce the selective accumulation of L. iners and L. gasseri.

Bacterial Vaginosis and Urinary Tract Infections

Many women experience a transient, often recurrent, loss of a lactobacilli-dominated vaginal microbiota and reduced vaginal acidity, which is associated with increased risk of urogenital infections as the reduction in lactobacilli makes for a more conducive vaginal environment for the proliferation of many anaerobic bacteria such as Gardnerella vaginalis (G. vaginalis) and Atopobium vaginae (A. vaginae).
     
BV and UTIs are common infections, afflicting hundreds of millions of women annually,(48,49) with BV the most common cause of vaginal symptoms among women. In the United States, the prevalence of BV (determined by a Nugent score of 7-10) was estimated to be 21.2 million (29.2%) among women aged 14 – 49 years, based on a nationally representative sample of women who participated in the National Health and Nutrition Examination Survey (NHANES) 2001 – 2004.(42) BV is a risk factor for acquisition of both bacterial (gonorrhea, chlamydia, and Trichomonas vaginalis infection) and viral (HIV, HSV, and HPV) sexually transmitted diseases as well as adverse obstetric outcomes (e.g., miscarriage, fetal distress syndrome, PROM, preterm birth).(3,6,50)
   
G. vaginalis and A. vaginae are commonly associated with BV51,(52) whereas the majority (>80%) of UTIs are caused by uropathogenic E. coli (UPEC) and often associated with AV.(53) These pathogenic bacteria colonize the vagina via the formation of biofilms, which results in increased tolerance to adverse conditions for better persistence in hostile environments (i.e., protection from the immune system and decreased susceptibility to antibiotics).(54,55)
     
Adherent biofilm comprised of mostly G. vaginalis and A. vaginae was observed to persist for three weeks following one-week treatment with orally administered metronidazole in women with BV.(52) In the UK, BV is frequently treated with topical clindamycin. The proportion of group B streptococci isolated from neonatal blood cultures that are resistant to clindamycin or erythromycin has risen substantially over recent years in the UK (Health Protection Report 2013, 7:46), most likely as a result of exposure to these antibiotics.

Lactobacillus Strains to Support Women's Urogenital Health

Bacterial migration from the colon to the vagina across the perineum occurs naturally, thus is a source of both potential pathogens as well as certain lactobacilli. Given that lactobacilli are generally recognized as the hallmark of a healthy vaginal microbiota, the rationale for probiotic use in support of women's urogenital tract health is strong.(56) Certain lactobacilli strains can safely colonize the vagina after oral as well as vaginal administration, displace and kill pathogens, and modulate host immune responses. Maintenance of a healthy vaginal microbiota could reduce the incidence of urogenital infections, the spread of sexually transmitted infections, and adverse pregnancy outcomes, thus decrease the need for conventional treatments.(57)
     
However, it is important to keep in mind that these inhibitory mechanisms and activities are generally strain specific; therefore, not all strains of a given lactobacillus species have the same probiotic potential. Furthermore, strains contained in a multi-strain combination need to be compatible (i.e., not antagonistic) and preferably synergistic for a targeted health condition beyond general gastrointestinal health (e.g., women's urogenital tract health), which must be demonstrated by clinical research. Although administration of vaginal suppositories is the most common way of delivering lactobacilli to the vagina, oral administration represents a more user-friendly alternative that may be more effective as a preventative strategy in the long run, given the recognition of the gastrointestinal tract as a reservoir for vaginal colonization by lactobacilli for the maintenance of a normal vaginal microbiota. It should be noted that any vaginally inserted capsule should not be enteric-coated, common for oral probiotics to protect the live bacteria from stomach acid, thus appropriate for orally administered probiotics to support women's urogenital tract health.
     
It is worth noting the current internationally endorsed definition of probiotics established by an expert panel commissioned in 2001 by the Food and Agriculture Organization (FAO) of the United Nations and supported by the World Health Organization (WHO), which states, 'Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." A health benefit to the host, humans in our case, must be realized and demonstrated to be superior to that of placebo/control (i.e., clinical research), but the majority of fermented foods and products labeled as or as containing probiotics on the market have not been appropriately tested and verified as such. How 'probiotic" containing products are regulated, marketed, and sold often has nothing to do with the definition. Many consumers/patients appear to be influenced by the live cell count (quantity) and number of strains offered in products labeled as containing probiotics, naively believing the more of each the better. Companies indulge this naivety and even actively propagate this messaging to consumers, often masquerading as educational, and eluding the lack of clinical validation for these strains (when strains are even identified on the label). Yet, the definition of probiotics militates against excessively high counts (dosing) of strains lacking clinical validation as touted by many commercially available products in favor of efficacious strains and dosing, substantiated by clinical research.
     
Orally and vaginally administered probiotic lactobacilli strains have been investigated as both an adjuvant and alternative therapy for the treatment and prevention, including recurrence post-treatment, of BV and UTIs. For example, orally administered (twice daily) probiotic lactobacilli strains L. rhamnosus GR-1 (1 billion) and L. reuteri RC-14 (1 billion) for 30 days in conjunction with oral metronidazole (500 mg) treatment during the first week was shown to be significantly more effective at curing BV than metronidazole treatment alone in premenopausal women, with significantly higher abundance of vaginal lactobacilli at day 30 in women receiving oral probiotic supplementation.(58) Additionally, oral supplementation with probiotic lactobacilli strains was as effective as daily antibiotics for UTIs.(59) Used in conjunction with and following antibiotic treatment helps restore a healthy vaginal microbiota to increase cure rate and reduce relapse.(56,58)
     
'Kitchen sink" products marketed to support women's vaginal health often tout a laundry list of different bacterial species and claim as many as 100 billion per capsule, albeit generic, clinically unsubstantiated strains for this indication. These levels have no rationale in light of the fact that the successful clinical studies utilized dosing in the 1 - 10 billion live organisms per day range, the difference being clinically validated strains with probiotic attributes to serve this function. Clinically validated strains are expensive, thus a product containing 100 billion (live organisms guaranteed through the listed 'best used before date" and not at time of manufacture) clinically validated strains per capsule would be prohibitively expensive and excessively dosed. Again, and most importantly, the specific probiotic strain(s) indicated to support women's urogenital tract health must be provided in sufficient quantity as validated by clinical research; otherwise, it does not constitute a true probiotic for this condition.

Not All Strains Are Equal

Again, it should be noted that not all lactobacilli strains, even those recognized more generally as a probiotic to support gastrointestinal health, are effective for supporting women's urogenital tract health. For example, daily oral administration of the widely recognized probiotic strain to support gastrointestinal health, L. rhamnosus GG, for 28 days failed to influence vaginal health despite the administration of 10 billion, whereas oral administration of only 1.6 billion of the combination of L. rhamnosus GR-1 and L. reuteri RC-14 supported a healthy vaginal microbiota.(60) Furthermore, daily oral administration of a commercially available dietary probiotic containing L. rhamnosus GG (40 billion) for six months failed to demonstrate vaginal colonization by this specific L. rhamnosus strain or reduce the recurrence of UTIs.(61)
     
The majority of probiotic products marketed for support of women's vaginal health, generally multi-strain combinations of lactobacilli with or without strains from additional genera such as Bifidobacteria, are not supported by clinical research for this indication. Additionally, the strain designation for each bacterium within the combination is often not disclosed (only genus and species). It is well recognized scientifically that probiotics are strain, dose, and condition specific. Strains of the same bacterial species can be different as exemplified by aforementioned examples comparing L. rhamnosus GR-1 vs. L. rhamnosus GG (unique strains of the same bacterial genus and species) to support women's urogenital tract health (condition), a functional distinction that was not overcome with administration of markedly greater abundance (dose) of L. rhamnosus GG.
     
Strain functionality and associated health claims beyond general support of gastrointestinal health in humans require substantiation of efficacy with clinical trials. Strain designation links unique bacterial strains to the scientific research supporting probiotic characteristics and efficacy for a specific condition in target host organisms (e.g., humans).
     
Guidelines established by an expert working group, convened jointly by the FAO of the United Nations and the WHO, state, 'Proper identification to the level of strain of all probiotics in the product," and Dr. Mary Ellen Sanders of the International Scientific Association for Probiotics and Prebiotics, an internationally recognized consultant in the area of probiotic microbiology, has stated, 'Manufacturers should designate the strains in their products so that consumers know what they're getting. It's pretty much a consensus among probiotic scientists that this is the responsible thing to do."

Four Unique Vaginal Probiotic Lactobacilli Strains

Domig et al.(62) demonstrated an extensive, multi-step scientific process to identify candidate probiotic strains, representing the predominant Lactobacillus species colonizing the vagina of healthy pregnant women (L. crispatus, L. jensenii, L. gasseri, L. rhamnosus), for oral administration to support women's urogenital tract health. From 68 isolates belonging to these species, which were derived from 99 isolates from the genus Lactobacillus out of a total of 127 isolates from healthy pregnant women in their late-first trimester, four final candidate strains were selected for targeted formulation based on a battery of criteria such as ability to grow under both aerobic and anaerobic conditions, acidification capacity, glycogen utilization, extracellular hydrogen peroxide production, stability under acidic conditions and resistance to bile salts (important for survival during gastrointestinal transit post-oral administration), anti-microbial activity against multiple strains of common vaginal pathogens (i.e., Candida albicans, Candida krusei, Candida glabrata, E. coli, and G. vaginalis), compatibility, safety (e.g., lack of virulence factors, antibiotic susceptibility/lack of antibiotic resistance), and encapsulated stability of the multi-strain formulation (forecasting shelf-life stability for use in commercial dietary supplements).
     
The strains of this probiotic formulation representing the predominant Lactobacillus species of the vaginal microbiota of healthy pregnant women have been designated L. crispatus LbV 88, L. jensenii LbV 116, L. gasseri LbV 150N, and L. rhamnosus LbV 96. These strains, as part of multi-strain probiotic formulation, were subsequently demonstrated in multiple clinical studies to increase vaginal lactobacilli abundance and acidification in support of urogenital tract health.

Klaire1017SMALL.jpg

The Neovagina: A Challenging Microbial Environment

The neovaginal microbiota of male-to-female transsexual women is a diverse community of both aerobic and anaerobic species with very limited colonization by lactobacilli, more reflective of the abnormal vaginal microbiota characteristic of BV.63 The neovaginal environment may not adequately support the growth of lactobacilli despite transsexual women having comparable estrogen levels to those of postmenopausal women receiving hormone replacement therapy,(63) with one study observing a neovaginal lactobacilli colonization rate of only 4%.(64)
     
In a prospective, randomized, placebo-controlled study, twice daily oral supple-mentation with the multi-strain probiotic formulation (2 X 2.5 billion live cells/dose) for only one week significantly enriched lactobacilli and resulted in a lower Nugent score in the neovagina of male-to-female transsexual women compared to placebo.(65) Neovaginal lactobacilli abundance was five to six times higher in the intervention group compared to the placebo group. In contrast to previous observations reported in the scientific literature, an unexpectedly high proportion (30%) of the participants in this study had a normal Nugent score of < 3. When those participants with a baseline Nugent score < 3 were excluded from the analysis or only those participants with BV (Nugent score > 7) were included, an improvement in the Nugent score was observed in the intervention group, but not in the placebo group.

Fortified vs. Conventional Yogurt as Adjuvant Therapy for Bacterial Vaginosis

In another randomized, double-blind, placebo-controlled clinical trial, oral supplementation with the selected probiotic strains and yogurt significantly improved cure rate and symptoms of BV compared to control.(66) Women with newly diagnosed BV (based on Amsel criteria, diagnostic criteria for BV of which 3 of 4 criteria must be met: pH > 4.5, positive whiff test, presence of discharge, and presence of clue cells in the wet smear) were administered metronidazole (2 x 500 mg/day) for one week and the multi-strain probiotic formulation twice daily in 125 g yogurt (intervention group; n = 17) or acidified yogurt (control group; n = 17), which naturally contained live fermentation starter cultures Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus.
     
After a four-week intervention period, 0/17 women had BV in the intervention group vs. 6/17 (35%) in the control group, a statistically significant and clinically relevant difference in cure rate. Amsel score was significantly decreased in the intervention group by a median value of 4 compared to a median value of only 2 in the control group. Odor and discharge (Amsel 2 and 3) was significantly decreased in the intervention group vs. control group, 2 vs. 1, respectively.

Immunosuppressed Pregnant Women and Obstetric Outcomes

The mother is the main source of microbes, both non-pathogenic and pathogenic, for newborn colonization. Dysbiotic vaginal microbiota characteristic of BV with marked reductions of lactobacilli increases risk of obstetric complications such as placental insufficiency, premature birth, fetal growth restriction, and postpartum endometritis,(6,50,67,68) which is particularly relevant for women with immunosuppression and herpes virus infection (HVI).
     
The selected probiotic strains were evaluated for efficacy in the complex therapeutic and preventative intervention for pregnant women with HVI.(69) Sixty pregnant women with HVI either received a patented food supplement to restore and support the vaginal microbiota twice daily for one week containing the selected probiotic strains and the prebiotic carbohydrate, fructooligosaccharides (intervention group; n = 30), or only prenatal care (comparator group; n = 30). Fifty healthy pregnant women without HVI were included as a control group. Intestinal lactobacilli and bifidobacteria were significantly increased in conjunction with significant decreases in pathogenic microbes (hemolytic E. coli, Klebsiella pneumoniae, Staphylococcus aureus, candida yeast species) in the intervention group vs. the comparison group, post-intervention levels which were similar to levels in healthy pregnant women of the control group. Prior to the intervention, 40% of women with HVI complained of symptoms associated with dysbiosis of the intestinal microbiota, namely bloating/abdominal discomfort, constipation, and mucus in the feces, but these complaints were reduced to only 12% of participants in the intervention group.
     
At the start of the study, vaginal lactobacilli were only detected in 13.3% and 16.7% of participants in the intervention and comparison groups, respectively, which was significantly increased in the intervention group to 46.7% after the one-week intervention, but not significantly different in the comparison group (20%). The percentage of women in the intervention group with a vaginal pH > 4.5, complaining of profuse vaginal discharge, swelling (hyperemia), and itching (pruritus), and with a positive amine test of vaginal discharge, were significantly decreased in the intervention group and no longer different than healthy pregnant women of the control group, whereas these parameters did not change in the comparison group.
     
The incidence of placental insufficiency and fetal distress were significantly reduced about two-fold in women of the intervention vs. comparison group. The percentage of aggravated pregnancy was significantly lower in women of the intervention group (33.3%) vs. the comparison group (53.3%). Furthermore, the percentage of women with other pregnancy complications (i.e., threatened miscarriage, threat of premature birth, pre-eclampsia, and pathology of amniotic fluid) was 25-50% fewer in the intervention vs. comparison group, which may be clinically relevant, but was not statistically significantly different in this study. This result was likely due to the small study population and variability and worth investigating in future, larger clinical trials.

Postmenopausal Women

Given the positive influence of estrogen on vaginal lactobacilli abundance, postmenopausal women experience a decrease in vaginal lactobacilli and increase in vaginal pH with increased incidence of UTIs due to increased colonization by enterobacteria. Clinical symptoms include vaginal dryness, burning, itching, dyspareunia, dysuria, urinary frequency, and recurrent UTIs.(70,71)
     
The genitourinary symptoms of meno-pause are particularly common in women with breast cancer due to chemotherapy and estrogen deprivation therapy. Vaginal estrogen therapy increases vaginal lactobacilli and decreases colonization by UPEC to reduce UTI recurrence,(72) but estrogen treatment for women with breast cancer is used with restraint due to frequent estrogen sensitivity of the tumor.(73) Thus, an appropriate probiotic intervention would be an alternative therapeutic option to enhance vaginal lactobacilli to discourage vaginal dysbiosis and recurrent UTIs.
     
Indeed, in a proof-of-principle pilot study,(74) twice daily oral supplementation with the multi-strain probiotic formulation (2 X 2.5 billion live cells/dose) for two weeks in postmenopausal women with breast cancer receiving chemotherapy, with vaginal atrophy and an intermediate vaginal microbiota (Nugent score 4 - 6) (n = 11/group), improved the Nugent score (-1.3) towards a normal microbiota (< 3), whereas there was a deterioration in the control group receiving placebo (+0.45). One week post-discontinuation, the Nugent score regressed in those women previously receiving the probiotic intervention (to only -0.57 from baseline) and further deteriorated in the control group (+2.5 from baseline), suggesting the protective effect of the probiotic intervention is not sustained with discontinuation in this at-risk group.

True Probiotics

The application of true probiotics to support women's vaginal health (i.e., clinically validated dosing of probiotic formulations such as L. crispatus LbV 88 + L. jensenii LbV 116 + L. gasseri LbV 150N + L. rhamnosus LbV 96) in clinical practice has gained increasing recognition as a therapy and for prophylactic prevention. However, in a society that focuses on disease and drug therapy more so than natural preventative measures, significant efforts will be needed to get such probiotics into mainstream practice. The failure of most medical education programs to teach future physicians about the human microbiota, its relationship to health, and appropriate applications of specific, validated probiotic strains and multi-strain formulations, ultimately diminishes care for patients. It is critical that healthcare practitioners acknowledge the human microbiota and consider its role in health maintenance. Clinical research, dissemination of research results, and education will be key, as confusion about what constitutes a true probiotic-based intervention and misinforming marketing campaigns are widespread.

 

Anthony Thomas, PhD, earned his bachelor's degree in nutrition, food science, and dietetics from California State University Northridge, his doctorate in nutritional biology from the University of California at Davis, and conducted postdoctoral research at the University of California at Los Angeles Larry Hillblom Islet Research Center. His primary research interests (via both pre-clinical and clinical studies) have focused on the influence of dietary and lifestyle factors (i.e., physical activity, circadian disruption) on the pathogenesis of chronic cardiovascular/metabolic diseases including obesity, insulin resistance syndrome, and type 2 diabetes. He has authored/co-authored multiple peer-reviewed scientific manuscripts and has served as a referee with relevant expertise in the fields of nutrition, obesity, and diabetes for multiple scientific journals.

 

References

 

1. Human Microbiome Project C. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486:207-214.

2. Green KA, et al. Gynecologic health and disease in relation to the microbiome of the female reproductive tract. Fertil Steril 2015, 104:1351-1357.

3. Lewis FM, et al. Vaginal Microbiome and Its Relationship to Behavior, Sexual Health, and Sexually Transmitted Diseases. Obstet Gynecol 2017, 129:643-654.

4. Petrova MI, et al. Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health. Front Physiol 2015, 6:81.

5. Kovachev SM. Obstetric and gynecological diseases and complications resulting from vaginal dysbacteriosis. Microb Ecol 2014, 68:173-184.

6. Dingens AS, et al. Bacterial vaginosis and adverse outcomes among full-term infants: a cohort study. BMC Pregnancy Childbirth 2016, 16:278.

7. Wilson WA, et al. Regulation of glycogen metabolism in yeast and bacteria. FEMS Microbiol Rev 2010, 34:952-985.

8. O’Hanlon DE, et al. Vaginal pH and microbicidal lactic acid when lactobacilli dominate the microbiota. PLoS One 2013, 8:e80074.

9. Tachedjian G, et al. The role of lactic acid production by probiotic Lactobacillus species in vaginal health. Res Microbiol 2017.

10. Boskey ER, et al. Acid production by vaginal flora in vitro is consistent with the rate and extent of vaginal acidification. Infect Immun 1999, 67:5170-5175.

11. Boskey ER, et al. Origins of vaginal acidity: high D/L lactate ratio is consistent with bacteria being the primary source. Hum Reprod 2001, 16:1809-1813.

12. Witkin SS, et al. Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections. MBio 2013, 4.

13. Conti C, et al. Inhibition of herpes simplex virus type 2 by vaginal lactobacilli. J Physiol Pharmacol 2009, 60 Suppl 6:19-26.

14. Graver MA, Wade JJ. The role of acidification in the inhibition of Neisseria gonorrhoeae by vaginal lactobacilli during anaerobic growth. Ann Clin Microbiol Antimicrob 2011, 10:8.

15. Juarez Tomas MS, et al. Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli. J Med Microbiol 2003, 52:1117-1124.

16. Aldunate M, et al. Vaginal concentrations of lactic acid potently inactivate HIV. J Antimicrob Chemother 2013, 68:2015-2025.

17. Owen DH, Katz DF. A vaginal fluid simulant. Contraception 1999, 59:91-95.

18. Aldunate M, et al. Antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis. Front Physiol 2015, 6:164.

19. Mossop H, et al. Influence of lactic acid on endogenous and viral RNA-induced immune mediator production by vaginal epithelial cells. Obstet Gynecol 2011, 118:840-846.

20. Haas R, et al. Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol 2015, 13:e1002202.

21. Reid G, et al. Microbiota restoration: natural and supplemented recovery of human microbial communities. Nat Rev Microbiol 2011, 9:27-38.

22. Ravel J, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 2011, 108 Suppl 1:4680-4687.

23. Gajer P, et al. Temporal dynamics of the human vaginal microbiota. Sci Transl Med 2012, 4:132ra152.

24. Zhou X, et al. Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J 2007, 1:121-133.

25. Petricevic L, et al. Characterisation of the vaginal Lactobacillus microbiota associated with preterm delivery. Sci Rep 2014, 4:5136.

26. Lamont RF, et al. The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 2011, 118:533-549.

27. Martin HL, et al. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 1999, 180:1863-1868.

Researched Nutritionals RxAD_0118.jpg

28. Hawes SE, et al. Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J Infect Dis 1996, 174:1058-1063.

29. van de Wijgert JH, et al. The vaginal microbiota: what have we learned after a decade of molecular characterization? PLoS One 2014, 9:e105998.

30. Bradshaw CS, Brotman RM. Making inroads into improving treatment of bacterial vaginosis - striving for long-term cure. BMC Infect Dis 2015, 15:292.

31. Fettweis JM, et al. Differences in vaginal microbiome in African American women versus women of European ancestry. Microbiology 2014, 160:2272-2282.

32. Fredricks DN, et al. Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005, 353:1899-1911.

33. Fox C, Eichelberger K. Maternal microbiome and pregnancy outcomes. Fertil Steril 2015, 104:1358-1363.

34. MacIntyre DA, et al. The vaginal microbiome during pregnancy and the postpartum period in a European population. Sci Rep 2015, 5:8988.

35. Romero R, et al. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome 2014, 2:4.

36. Kiss H, et al. Vaginal Lactobacillus microbiota of healthy women in the late first trimester of pregnancy. BJOG 2007, 114:1402-1407.

37. Brotman RM, et al. Microbiome, sex hormones, and immune responses in the reproductive tract: challenges for vaccine development against sexually transmitted infections. Vaccine 2014, 32:1543-1552.

38. Mirmonsef P, et al. Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH. PLoS One 2014, 9:e102467.

39. Farage M, Maibach H. Lifetime changes in the vulva and vagina. Arch Gynecol Obstet 2006, 273:195-202.

40. Nunn KL, Forney LJ. Unraveling the Dynamics of the Human Vaginal Microbiome. Yale J Biol Med 2016, 89:331-337.

41. Achilles SL, Hillier SL. The complexity of contraceptives: understanding their impact on genital immune cells and vaginal microbiota. AIDS 2013, 27 Suppl 1:S5-15.

42. Koumans EH, et al. The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis 2007, 34:864-869.

43. Riggs M, et al. Longitudinal association between hormonal contraceptives and bacterial vaginosis in women of reproductive age. Sex Transm Dis 2007, 34:954-959.

44. Vodstrcil LA, et al. Hormonal contraception is associated with a reduced risk of bacterial vaginosis: a systematic review and meta-analysis. PLoS One 2013, 8:e73055.

45. Mayer BT, et al. Rapid and Profound Shifts in the Vaginal Microbiota Following Antibiotic Treatment for Bacterial Vaginosis. J Infect Dis 2015, 212:793-802.

46. Schmitt C, et al. Bacterial vaginosis: treatment with clindamycin cream versus oral metronidazole. Obstet Gynecol 1992, 79:1020-1023.

47. Reid G, Bruce AW. Urogenital infections in women: can probiotics help? Postgrad Med J 2003, 79:428-432.

48. Allsworth JE, Peipert JF. Prevalence of bacterial vaginosis: 2001-2004 National Health and Nutrition Examination Survey data. Obstet Gynecol 2007, 109:114-120.

49. Stamm WE, Norrby SR. Urinary tract infections: disease panorama and challenges. J Infect Dis 2001, 183 Suppl 1:S1-4.

50. Denney JM, Culhane JF. Bacterial vaginosis: a problematic infection from both a perinatal and neonatal perspective. Semin Fetal Neonatal Med 2009, 14:200-203.

51. Hummelen R, et al. Deep sequencing of the vaginal microbiota of women with HIV. PLoS One 2010, 5:e12078.

52. Swidsinski A, et al. An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after standard therapy with oral metronidazole. Am J Obstet Gynecol 2008, 198:97 e91-96.

53. Donders GG, et al. Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG 2002, 109:34-43.

54. Danese PN, et al. Biofilm formation as a developmental process. Methods Enzymol 2001, 336:19-26. 55. Hardy L, et al. Bacterial biofilms in the vagina. Res Microbiol 2017.

56. MacPhee RA, et al. Probiotic strategies for the treatment and prevention of bacterial vaginosis. Expert Opin Pharmacother 2010, 11:2985-2995.

57. Spurbeck RR, Arvidson CG. Lactobacilli at the front line of defense against vaginally acquired infections. Future Microbiol 2011, 6:567-582.

58. Anukam K, et al. Augmentation of antimicrobial metronidazole therapy of bacterial vaginosis with oral probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14: randomized, double-blind, placebo controlled trial. Microbes Infect 2006, 8:1450-1454.

59. Bruce AW, Reid G. Intravaginal instillation of lactobacilli for prevention of recurrent urinary tract infections. Can J Microbiol 1988, 34:339-343.

60. Reid G, et al. Probiotic Lactobacillus dose required to restore and maintain a normal vaginal flora. FEMS Immunol Med Microbiol 2001, 32:37-41.

61. Kontiokari T, et al. Randomised trial of cranberry-lingonberry juice and Lactobacillus GG drink for the prevention of urinary tract infections in women. BMJ 2001, 322:1571.

62. Domig KJ, et al. Strategies for the evaluation and selection of potential vaginal probiotics from human sources: an exemplary study. Benef Microbes 2014, 5:263-272.

63. Weyers S, et al. Microflora of the penile skin-lined neovagina of transsexual women. BMC Microbiol 2009, 9:102.

64. Weyers S, et al. Cytology of the ‘penile’ neovagina in transsexual women. Cytopathology 2010, 21:111-115.

65. Kaufmann U, et al. Ability of an orally administered lactobacilli preparation to improve the quality of the neovaginal microflora in male to female transsexual women. Eur J Obstet Gynecol Reprod Biol 2014, 172:102-105.

66. Laue C, et al. Effect of a yoghurt drink containing Lactobacillus strains on bacterial vaginosis in women - a double-blind, randomised, controlled clinical pilot trial. Benef Microbes 2017:1-16.

67. Vedmedovska N, et al. Preventable maternal risk factors and association of genital infection with fetal growth restriction. Gynecol Obstet Invest 2010, 70:291-298.

68. Jacobsson B, et al. Bacterial vaginosis in early pregnancy may predispose for preterm birth and postpartum endometritis. Acta Obstet Gynecol Scand 2002, 81:1006-1010.

69. Anoshina TM. Role of microbiota correction in complex treatment of pregnant women with herpesvirus infection. Perinatologiya Pediatriya 2016, 4:22-25.

70. Portman DJ, Gass ML, Vulvovaginal Atrophy Terminology Consensus Conference P. Genitourinary syndrome of menopause: new terminology for vulvovaginal atrophy from the International Society for the Study of Women’s Sexual Health and the North American Menopause Society. Maturitas 2014, 79:349-354.

71. Pandit L, Ouslander JG. Postmenopausal vaginal atrophy and atrophic vaginitis. Am J Med Sci 1997, 314:228-231.

72. Raz R, Stamm WE. A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infections. N Engl J Med 1993, 329:753-756.

73. Krychman ML, Katz A. Breast cancer and sexuality: multi-modal treatment options. J Sex Med 2012, 9:5-13; quiz 14-15.

74. Marschalek J, et al. Influence of orally administered probiotic Lactobacillus strains on vaginal microbiota in women with breast cancer during chemotherapy: A randomized placebo-controlled double-blind pilot study. Breast Care 2017, 12:335-339.