Abstract

Urogenital infections are a major reason that women visit their family physician and are referred to gastroenterology, gynecology, urology, and infectious disease specialists. The association between abnormal vaginal microbiota and increased risk for sexually transmitted infections, bladder and vaginal infections per se, and a higher rate of preterm labor indicate the need to better understand and manage urogenital health. The concept of probiotics arose from the realization that humans are inhabited with microbes from birth and that these organisms play a role in preventing disease. Defined as live microorganisms, which when administered in adequate amounts confer a health benefit on the host, probiotic strains have already been shown to effectively prevent diarrhea and to hold potential in preventing and treating tonsillitis, caries, renal calculi, and respiratory infections. This review provides a rationale for the use of probiotics in maintaining female vaginal and bladder health and as a treatment option for recurrent bacterial vaginosis, urinary tract infection, yeast vaginitis, and sexually transmitted infections. We consider only probiotic strains that fulfill the United Nations/World Health Organization Guidelines for Probiotics in being fully characterized and clinically documented through scientific investigations describing known or presumed mechanisms of action. Although medical practitioners as yet are unable to access these probiotic strains, an awareness of recent and ongoing research for probiotics is important, as results are encouraging. The concept of probiotic therapy is familiar to many consumers and although it has historically lacked credibility in the medical community, perceptions are changing.

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Scope of the Problem

It seems astonishing that urogenital tract infections (UTIs) in women are viewed with relative apathy by government health and funding agencies, industry, and many investigative clinicians. Globally, an estimated 1 billion women have bladder or vaginal infections each year,[1] with more recent increases noted in candidiasis in Brazil,[2] bacterial vaginosis (BV) in the United States and Indonesia,[3,4] and HIV in Africa.[5] These acute and chronic urogenital infections can significantly affect quality of life in women, although only a single study has investigated (and confirmed) this important facet.[6] Furthermore, secondary complications, such as increased risk for preterm delivery, arise from persistent infections, such as BV.[7] Yet, for all the adverse effects on well-being and the massive expenditure

burden,[8] therapeutic approaches to treatment and prevention of urogenital infections have remained essentially unchanged for many years. Antibiotics and antifungals remain the mainstay of therapy, despite their well-documented side effects ranging from diarrhea, depression, and headaches to renal failure and superinfections. Moreover, these therapies are becoming less effective as a result of antimicrobial resistance[9,10]; in the case of pregnancy, antibiotics may[11] or may not[12] prevent preterm birth even once BV has been cured. Most cases of BV, UTI, and yeast vaginitis arise from the host's gastrointestinal tract, as microbes ascend 4 to 5 cm from the anus, thereby showing that the intestine and urogenital tracts are linked and that intestinal health can influence the vagina and bladder. Every 15 seconds, a child somewhere in the world dies of diarrheal disease, and up to 60 billion cases of gastrointestinal illness occur every year.[13] The approach of boosting the commensal microbial defenses of the host using probiotic organisms thus has consequences for the gut as well as the vagina.

Bacterial Vaginosis Prevalent, Often Misdiagnosed, and Invariably Recurrent

The prevalence of BV varies in different parts of the worldfor example, 15% in rural Brazil[14]; 25% in a group of healthy, white, Canadian women[15]; 27.4% in Chicago[16]; and 29.9% in Indonesia[17] to 85% in some prostitute populations.[18] Such factors as vaginal douching[19]; black ethnicity; and low socioeconomic status increase the risk for BV.[20] In addition, the higher rate of BV among black women[21] raises the question of whether host-cell receptor density, diet, or other factors play a role in altering the vaginal microbiota and thereby increasing risk for infection. Most women are not aware that they have BV. Self-use diagnostic kits are available but are not yet very sensitive or specific.[22] A longitudinal study of women in the United Kingdom showed that at any given time during the menstrual cycle, the vaginal microbiota may be abnormal.[23] When symptoms of pain, discharge, and itching occur, many women diagnose these symptoms as yeast infections and selftreat with over-the-counter antifungals, when in fact they have BV.[24-26] This misdiagnosis and mistreatment can result in adverse consequences.[25] Antimicrobial treatment for BV is suboptimal, with some cure rates as low as 60% 1 month after treatment, and subsequent overgrowth of pathogenic bacteria in the vagina often occurs.[27-29] The common asymptomatic nature of BV has raised the question of whether the condition is actually detrimental to the host. Although specific studies have not been

designed to answer this question, the major causes of BVnamely anaerobic or aerobic organisms such as Gardnerella, Prevotella, and Escherichia colihave all been found to produce virulence factors.[30-32]

Association Between an Abnormal Vaginal Microbiota and the Spread of HIV in Women
Women are the fastest-growing HIV-infected population; worldwide, approximately 50% of adults with AIDS are women. According to 2001 data, the proportion of women between 15 and 24 years of age living with HIV/AIDS is 62% worldwide (and 67% in sub-Saharan Africa).[33] Factors contributing to greater transmission among women and girls include lack of access to health information, lack of negotiation power over sexual encounters, rape, dependence on men for housing and income, diminished educational opportunities, low male use of condoms, and young age at first intercourse (12 to 14 years). Studies have also shown that the absence or depletion of lactobacilli in the vagina associated with overgrowth of anaerobic pathogens causing BV results in significantly increased risk for HIV (as well as gonorrhea, chlamydia, and herpes simplex virus infections).[34-38] By mechanisms not yet understood, BV displaces lactobacilli, elevating vaginal pH[39] and creating an environment within which the pathogens survive and can infect the host.

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The Protective Role of the Commensal Vaginal Microbiota

Culture-based methods followed by molecular typing have shown Lactobacillus crispatus and L jensenii to be the dominant organisms in most healthy, premenopausal women.[40] However, use of denaturing gradient gel electrophoresis without culture has recently identified the strict anaerobe L iners as the most common habitant, at least in 1 primarily white population.[41] After menopause, some 25% to 30% of women still have lactobacilli present, and this number rises to between 60% and 100% with use of vaginal or oral estrogen-replacement therapy.[42-45] Correspondingly, rates of UTI and vaginal infections increase with age and decrease with estrogen-replacement therapy. Although oral and vaginal sex can lead to vaginal infections, many cases of vaginitis and UTI arise from ascension of the person's own microorganisms. The net effect of ascension of these abnormal microorganisms is creation of a vaginal environment in which lactobacilli occur in low concentrations or are absent and are replaced by pathogenic Gram-positive cocci, Gram-negative rods, or yeast. Clinical

implications of the distinction between normal and abnormal microbiota were noted more than 30 years ago in studies of the role of the normal microflora in protecting the host.[46,47] The fluctuations between a normal and an abnormal (and potentially unhealthy) vaginal environment have more recently become a focus of investigation by other groups.[48,49] An analysis of the vaginal microflora over the course of the menstrual cycle has shown that only 22% women maintain a lactobacilli-dominated flora,[50] and although epithelial cell receptivity to lactobacilli adhesion increases at peak estrogen levels midcycle,[51] it is still not clear what factors cause such a dramatic alteration in the flora. The concept of a protective role for the microflora normally found in a healthy vagina is the basis for the research on application of beneficial bacteria to the vagina, and the positive results obtained in recent studies[15, 52, 53] point to a future in which probiotics will soon be an option for vaginal healthcare.

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What Are Probiotics?

The concept of probiotics dates back more than 100 years. Most of the research has focused on intestinal benefits of lactic acid bacteria, resulting in narrow definitions for the term probiotics. Recently, a much broader definition has been developed: Live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.[54] This recognizes the use of probiotics for urogenital diseases. An argument has been made that patients should consume probiotics as part of their daily diet.[55] The essence of this argument is that the modern diet is designed to inhibit pathogens and is bereft of beneficial organisms. Increased exposure to antimicrobials and preservatives through the food chain that deplete the commensal flora, impaired or suboptimal immunity that reduces the ability to fight infection, and recognition of the important role that certain bacteria (lactobacilli, bifidobacteria, and others) play in health have resulted in this global trend in the interest in probiotic foods. In this light, the new guidelines developed to define precisely what is expected of an organism or product designated to be probiotic are of great importance.[56] These guidelines were developed after requests from member nations of the Food and Agriculture Organization of the United Nations. The guidelines are critical because many so-called probiotic products currently on the market have never been proven, using acceptable methods, to have health benefits; indeed, many have been found to

either contain dead organisms or species that are different from those stated on the label.[57-60] Furthermore, some less credible marketing companies make outlandish or subtly incorrect claims in their publicity material, including content placed on Web sites.[61] As a result, the concept of probiotics has lacked credibility in the medical community and many consumers have purchased and used unreliable products. But a broadening of education about probiotics spurred on by good science and rigorous clinical studies are changing perceptions in the medical community about probiotics. (For example, a recent Medscape Medical News CME program examined the safety and tolerability of probiotics added to infant formula.[62]) Following are probiotic strains that are both relatively easy to purchase in the United States and Europe and could be useful additions to the diet:

L casei DN_114001Danone's Actimel fermented milk drinks; shown to control diarrhea[55] L acidophilus NCFMoccurs in low numbers in many US probiotic yogurts but can alleviate lactose intolerance when administered in adequate amounts[63] L reuteri SD2112available in capsule form in the United States; shown to be useful in treating rotavirus diarrhea[64] L plantarum 299Vsimilar to strain 299; shown to aid recuperation from surgery.[65]

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Probiotics for Vaginal and Bladder Health

After the commercial introduction of L casei Shirota (Yakult) in Japan in the 1930s, the next probiotic strains to be discovered and extensively studied were L acidophilusNCFM (mid-1970s) for the gut[63] and L rhamnosus GR-1 and L fermentum RC-14 for the urogenital tract (1980 through 1985).[66] Since then, several research groups have attempted to identify suitable candidates for vaginal colonization.[67-69] In vitro selection data are useful to learn more about the organisms but are insufficient to predict efficacy in humans. In addition, simply showing an absence of lactobacilli associated with disease does not mean that application of lactobacilli to the vagina will prevent or treat that ailment. A key factor is whether a strain colonizes over a sufficient period to confer health benefits to the host. Adhesion and

colonization of the vaginal epithelium by the strain for days or even weeks may be necessary.[70, 71] However, longer-term colonization for months or years may not be necessary if the person's own lactobacilli recolonize or the exogenous therapy is re-administered. Some researchers have paid little attention to these points and have tested Lactobacillus strains not particularly well suited to the vagina.[72, 73] Perhaps not surprisingly, the strains had no clinical impact on recurrence of infection. Others have taken products to market with minimal clinical verification of efficacy of a given strain.[74] Although these strains may provide some relief of symptoms and ultimately be a useful treatment option, publication of supportive efficacy data would enhance credibility and acceptance by the medical community and consumers. Insertion of lactobacilli into the vagina via a pessary or capsule is an effective means of boosting content of the flora and overcoming some pathogens or reducing their ability to dominate. This seems to be true for treatment of BV and possibly UTI pathogens,[44,75,76] but there is only anecdotal evidence to suggest that lactobacilli can treat yeast vaginitis.[77, 78] (Whether any effect is attributable to the ability of such strains as GR-1 and RC-14 to inhibit candidal growth and adhesion[79, 80] remains to be determined in human trials.) The dried lactobacilli used in vaginal suppositories appear to be capable of hydrating from the capsule and interfering with pathogenic organisms. Use of skim milk-based preparations can also be effective,[81] but compliance may be a problem for some women because of the need to retain the fluid with a tampon. Oral dosage seems to require around 109 viable bacteria once or twice weekly,[52] although a once-per-day vaginal protocol for 3 days might initially be required to displace large pathogen biofilms in the urogenital tract. Most urogenital microflora originate from the gut and ascend via the rectum. Studies have shown that daily oral intake of L rhamnosus GR-1 and L fermentum RC-14 can modify the vaginal flora.[15, 52, 53] In a randomized, double-blind, placebo-controlled study of 64 healthy women, daily intake of L rhamnosus GR-1 and L fermentum RC-14 resulted in significantly less yeast and fewer coliforms in the vagina.[15] Vaginal swabs (blinded) were sent to an independent laboratory and cultured. Cultures revealed significantly more lactobacilli and less yeast and fewer coliforms in the lactobacilli-treated group; whereas the swabs obtained from women treated with placebo showed a significant increase in yeast and coliforms, perhaps explaining why the vaginal flora is so commonly abnormal. Administration of the probiotic organisms even normalized flora in some cases of BV, making it feasible to study this as an approach to long-

term therapy for pregnant women and those susceptible to BV and UTI. The ability of these organisms to displace other organisms was shown previously in vitro.[82] Studies are needed to determine whether healthy people and those prone to recurrent urogenital infections benefit from daily use of dried probiotics, such as L rhamnosus GG, which is the most clinically documented probiotic strain for gut health and is available in the United States. A study using this strain in fermented milk has suggested some reduction in UTI recurrences.[83] The potential for intestinal probiotics to influence bladder and vaginal health through some form of immune modulation has not been fully explored, but a study using L casei Shirota (available from a few stores in California) suggested that it had potential to reduce recurrence of bladder cancer.[84] The interactions among microbes at the vaginal mucosal surface remain to be elucidated. It is assumed that antiviral, antiyeast, and antibacterial effects shown in vitro[71, 80] also occur in vivo, but mechanistic and cause-and-effect studies are required to verify this hypothesis. A recent study on a dextran sulphate-induced mouse colitis model suggested that DNA extracted from probiotic organisms and E. coli could mediate anti-inflammatory activity and ameliorate disease through tolllike receptor 9 signaling.[85] However, the interpretation of the findings by authors and writers who have subsequently reported on the results[86] has been exaggerated and overextended. The experimental model is 1 way of creating and studying colitis, but using the same probiotic organisms, the principal author of the study did not find an effect of the extracted DNA using a different animal model of colitis.[87] Also, there are other ways to influence inflammatory responses; thus, even if it is possible to interfere with toll-like receptor 9 activity, it might not result in a clinically significant outcome for colitis patients. People consume bacteria and bacterial DNA on a daily basis, yet rates of inflammatory bowel disease increase. This latest study provides a useful insight into potential mechanisms of probiotic actions, but clearly much clinical investigation is needed before DNA can be recommended for symptomatic relief of disease in the gut or other mucosal sites. The actual mechanisms of action of probiotics in the vagina have not been proven and are probably multifactorial. The production of lactic acid, bacteriocins, and hydrogen peroxide seems to be important, but these substances have not been measured in healthy women and compared with those obtained in women immediately before and during a BV or UTI event. Modulation of immunity is another plausible mechanismfor example, in BV IL-1 and IL-8 levels are elevated compared with levels in healthy (lactobacilli-dominated) vaginas.[88] Lactobacilli have been shown to produce biosurfactants and collagen-binding proteins that

inhibit pathogen adhesion and to some extent displace pathogens.[89, 90] This might explain why vaginal mucosa dominated by lactobacilli but that has much of the surface uncovered by these organisms could still be less receptive to pathogens. Among Lactobacillusby-products, cell-signaling molecules have been discovered, which can downregulate pathogen virulence (unpublished findings). Thus, cell-tocell communication is another probable mechanism of action. This communication may also involve the signaling of mucus production, which acts as a barrier to pathogens,[91] or signaling of anti-inflammatory cytokine production.[92, 93] How normal flora can become conducive to BV so rapidly is still a mystery. Perhaps epithelial cell turnover is involved, exposing new surfaces to pathogens, or perhaps ascension of pathogens from the anus becomes so overwhelming that the microenvironment changes and the pathogens thrive due to rapid multiplication. Clearly, more research is needed. The concept of using nonviable bacteria as a vaccine in the vagina has also been explored in a study in which a combination of antigens from various uropathogens was tested for its ability to reduce risk for recurrent UTI.[94] The results are encouraging, but again more research is required to confirm the results and to elucidate the mechanisms involved. It seems unlikely that dead pathogens in a vaccine induce immune protection when live pathogens, present in the vagina in large numbers in patients with recurrent UTI, do not. Competitive exclusion also seems unlikely, because although dead pathogens could interfere with adhesion of live ones, the effect would not last long, as the vaccine components would degrade or be sloughed off. Vaccines that specifically interfere with adhesion, such as type 1 fimbriae of E. coli, may have some success. However, these organisms have several mechanisms of attaching to cells, and blocking 1 species may simply lead to infection by another if the environment is not conducive to restoration of a normal flora.

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Practical Steps for Clinical Practice

For the general practice physician, it is difficult to apply probiotic concepts to patient care because products especially designed for female urogenital health are not yet available. In terms of diagnosis of abnormal vaginal microbiota, a simple Gram stain of a vaginal swab can determine, with reasonable effectiveness, the extent to which the vagina contains or has depleted numbers of lactobacilli. The dominant presence of Gram-negative rods, yeast, or Gram-positive cocci is suggestive of an infectious or potentially infectious state. In many women, this

imbalance seems to self-correct on a regular basis for reasons not yet understood, but in others an asymptomatic condition progresses to a symptomatic one that will require treatment. The easily performed Gram stain can also indicate whether a patient is likely to have BV or yeast vaginitis. The antibiotic or antifungal to be prescribed should have limited adverse effect on the indigenous bowel and vaginal microbiota. Such agents as nitrofurantoin and some newer classes of macrolides and cephalosporins may not induce a yeast vaginitis, a condition that has long been associated with antibiotic eradication of indigenous microflora.[95]

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Concluding Remarks
The first step in changing management paradigms is to understand the process of infection and the key factors associated with health maintenance. Lest we forget, studies from the 1970s documented the importance of the vaginal flora in both health and disease.[46, 96] The second step is to accurately diagnose problems. The expense, timeframe, often-inaccurate diagnostic systems, and efficacy of some drugs led to widespread use of empirical therapy and self-medication in the latter part of the 1990s. But the frequency with which infections recur and the emergence of multidrug-resistant bacteria, as well as patient dissatisfaction with side effects of drugs, has led to an increased interest in better ways to diagnose, treat, and prevent infection. With the advent of DNA-based technologies, it should soon be possible to quickly and precisely diagnose the cause of infection or inflammation. The final step is prevention or treatment. With few new classes of antimicrobial agents emerging, it becomes more difficult to provide new-and-improved management guidelines. The recurrent nature of UTI, BV, and yeast vaginitis remains a major problem for practitioners and their patients. Indeed, it is nothing short of a silent epidemic worldwide. Probiotics do not represent a magic bullet, but evidence is accumulating that the use of proven probiotic strains and manipulation of the host's own intestinal and vaginal/urethral microbiota will provide valuable options to help restore and maintain urogenital health. Once appropriate product formulations with supporting clinical data become available, it will be up to the physician to determine their place in patient management.