Prevention and treatment of urogenital infections and complications: lactobacilli's multi-pronged effects

Abstract

The association between depletion of vaginal lactobacilli and increased risk of various urogenital infections and complications such as preterm labor, has been known for some time. Restoration of depleted lactobacilli by administration of probiotic strains has been demonstrated in humans using Lactobacillus rhamnosus GR-1 and L. reuteri RC-14. These organisms also augment antibiotic efficacy, lessen the side effects of these drugs, and alleviate diarrhea in AIDS patients. The mechanisms appear to be multi-factorial, and include production of: (i) anti-microbial factors such as lactic acid, bacteriocins, hydrogen peroxide, (ii) biosurfactants or other components that affect pathogen colonization and biofilm formation, (iii) signalling compounds that influence pathogen virulence expression, and (iv) signalling compounds that modulate immunity. Recombinant strains have been produced that inhibit or kill HIV, offering hope for microbicide applications that can be self-used by women. As more information becomes available about the ‘normal’ versus ‘disease prone’ vaginal microbiota and strains that confer the most benefits, current and new probiotic remedies will potentially provide improved restorative and therapeutic options to lower the one billion urogenital infections currently afflicting women around the world.

• probiotics
• lactobacillus
• urogenital infections
• mechanisms

Microbiota of the vagina

Our knowledge of the microbes that inhabit a healthy or infected vagina has in recent times changed significantly. The failures of the long used API 50 CH strips have been fully exposed [1], and the equally long-held view that Lactobacillus acidophilus is the most dominant vaginal organism, likewise is no longer true. The development of non-culture techniques has led to the detection of a number of species which have fastidious growth requirements and have therefore gone unnoticed until now. In particular, L. iners is commonly found in pre and postmenopausal women [2–4], while Atopobium vaginae is a newly discovered causative agent of bacterial vaginosis (BV)[5], [6]. In the case of BV, originally regarded as a condition associated with Gardnerella vaginalis, molecular methods have identified a broad range of organisms with up to 17 phylotypes identified in vaginal samples [7], [8]. This diversity is not found in healthy women, where single Lactobacillus species are often found [7], [8].

The factors which alter an acidic environment occupied by large numbers of lactobacilli, to a multi-speciated microbiota associated with amine production and elevated pH, remain somewhat of a mystery. Fluctuations in estrogen levels and glycogen, induced by estrogen stimulation are factors [9], as is the possible influx of phages [10] and production by pathogens of anti-lactobacilli bacteriocins [11]. The fact that women can have BV at different times of the menstrual cycle [12], suggests that hormones are not the main factor responsible for an altered microbiota. Clearly, studies are needed to uncover the mechanisms involved in the transition between health and disease. The increased risk of sexually transmitted diseases, preterm labour and other problems associated with BV [13–16], indicate that more research is needed urgently into how and why this condition arises.

Replenishment of lactobacilli

The depletion of vaginal lactobacilli in women who develop urogenital infection led to the concept that artificial administration of lactobacilli might be therapeutic. The question is which strains of lactobacilli should be used and how often do they need to be given? Prior to the first study of this type in 1987, the factors considered important in selecting lactobacilli were their ability to adhere to epithelial cells in vitro, as well as inhibit pathogen growth and adhesion. Strains, including the most common species known, at that time, to be found in the vagina, were tested and ranked, with the result that L. rhamnosus GR-1 showed the most promise [17]. In a small pilot clinical study in which GR-1 was suspended in milk and administered to the vagina, there was a resultant lengthening of time between episodes of urinary tract infection (UTI), but enterococci did not appear to be displaced [18]. This led to a search for lactobacilli that were particularly effective against gram positive cocci. Ultimately, two strains, L. acidophilus RC-14 and L. fermentum B-54 were chosen for further study. Of note, bacterial classification has evolved since 1987, and strain RC-14 was identified by ribotyping in 1998 as L. fermentum[19], then in 2005 by DNA-DNA hybridization as L. reuteri species, along with B-54. The studies prior to 2006 that reported clinical benefits using L. acidophilus RC-14, L. fermentum RC-14 and L. reuteri RC-14, were all performed with the same organism.

It was assumed twenty or more years ago, that any Lactobacillus strain could colonize the host and protect against re-infection by producing acid and bacteriocins. On hindsight, the in vitro assays used to select the strains, were not sufficiently applicable to the in vivo setting, and their use could not definitively predict the probiotic efficacy of the organisms [20]. Still, they did provide some information about characteristics of the strains, and in the case of Lactobacillus GR-1 and B-54, there was some correlation between in vitro adhesion and an ability to colonize in the vagina [21], [22]. Not all strains appear suited for urogenital colonization and interference with the infection process in the vagina and bladder, as failed clinical studies with L. rhamnosus GG and L. acidophilus and those contained in a product called Fermalac have shown [23–26]. No studies have investigated why these organisms lack the attributes of strains GR-1, RC-14 and B-54.

The timing of replenishment of lactobacilli has not been studied. A case could be made for administering the organisms immediately following menses, as the lactobacilli count is low and subjects are at more risk of infection [27]. On the other hand, in mid cycle when estrogen levels are highest, lactobacilli appear to better adhere to epithelial cells in vitro[28], and potentially this could be an opportune time to replenish a vaginal area devoid of these organisms.

Clinical studies of probiotics for prevention and treatment of urogenital infections

The administration of exogenous lactobacilli to the host represents a probiotic application. Probiotics are defined as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” [20]. This definition emphasizes that a probiotic product can only be designated if the organisms and product formulation have been shown to confer physiological benefits in humans. Thus, many so-called probiotics are in fact not probiotic at all, at least not yet, as they have not been tested clinically and shown to provide a tangible benefit [29], [30]. This is an important point when analyzing the effects, or lack thereof, of products. Unreliable products often do not contain sufficient viable bacteria at time of use, some have contaminants and some have strains that may not be suited for the purpose at hand.

Of the probiotics shown to colonize the vagina, only four strains fulfil the Guidelines that were established by the Food and Agriculture Organisation of the United Nations (FAO) and World Health Organization (WHO)[31]: L. rhamnosus GR-1, L. reuteri RC-14 and B-54, and L. crispatus CTV05, which has shown promise. However, studies on other strains, such as the Polish product Lactovaginal, do not verify colonization or efficacy, while the French L. acidophilus Gynoflor product acts through an estrogen induced effect.

The efficacy of Lactobacillus GR-1 and RC-14 capsules has been reviewed previously [32], [33]. In brief, these organisms can colonize the vagina after direct instillation [34] or following oral intake and natural ascension from the rectal surface [35]. They create an environment that supports lactobacilli growth, even if the strains themselves are not always present [2]. The net effect on the woman is a reduction in the yeast and bacterial pathogens that ascend into the vagina [36], fewer episodes of BV [12] and UTI (strains GR-1 with B-54) [21], and even resolution of some cases of BV [37]. A recent study has confirmed these findings and shown cure of BV with intravaginal administration [38].

Recombinant strains

As our understanding of the vaginal microbiota and host factors improves, probiotic therapy for urogenital and reproductive health will be even more refined. This might include the use of recombinant strains targeted to confer specific effects, such as disruption of pathogenic biofilms, or killing of viruses, or modulating pH and immune responses. Three strains have been genetically modified for this purpose and shown in vitro to have potential for prevention and treatment of HIV. The first report used a strain of L. jensenii which secreted two-domain CD4 proteins that recognized a conformation-dependent anti-CD4 antibody and bound HIV type 1 (HIV-1) gp120 [39]. This intervention is designed to block the adhesion and invasion of the virus to vaginal cells. In the second study, a Lactococcus lactis strain, not known for its ability to colonize the vagina, was adapted to secrete Cyanovirin, an agent that binds to carbohydrate molecules on the HIV virus, in the hope of blocking a receptor used to infect the host [40]. In a collaborative study with Dr. Janice Lui in Seattle, we have used a chromosomal integration approach to create L. reuteri RC-14 strains that secrete a variety of microbicides, with the intent of reducing the ability of viral mutants to by-pass the treatment [41].

The overall strategy of these recombinant strains is to provide a method for women to self-reduce their risk of HIV. At a time when 7,000 females a day are becoming infected with the virus through sexual intercourse, such self-empowerment would be extremely valuable, if it works. This current infectivity rate places in jeopardy the very fibre of some nations, for example Botswana, where half the pregnant women are HIV positive. In the original FAO/WHO Expert Panel Report [20], a call was made for probiotics to be made available for developing countries and those where disease was particularly prevalent. The HIV epidemic meets this criterion, yet no probiotics have yet been used to alleviate complications of HIV/AIDS, or to reduce the incidence of infectivity [42]. If recombinant strains are proven to have efficacy in animal studies, and be safe for human use, the dilemma arises as to whether or not genetically modified organisms should be permitted for human use. A recent viewpoint recommended that all genetically modified probiotics should be banned [43]. The authors admitted to being ignorant about the gut ecology and clearly had no expertise in probiotics. In making their case, the authors failed to discuss risk-benefit issues, ignored the studies of Steidler [44] in which a suicide system has been developed to ensure no environmental release of modified organisms, and they did not mention the fact that mutations occur in nature, often induced by the action of humans, such as overuse of antibiotics. Their irrational presentation (use of words such as ‘rogue’, ‘dangerous’, ‘engineers’, ‘nasty’, ‘warfare’) and misrepresentation of information (wrong definition of probiotics, irrelevant reference to mousepox virus, presumption that scientists have not considered gene transfer between species, and implication that certain experimental studies on some bifidobacteria are designed to create dangerous human therapies) failed to offer a rational discussion on an important topic. Clearly, any human use of recombinant lactic acid bacteria must be carefully regulated and only permitted after stringent safety studies. Likely, a risk-benefit analysis will show that the benefits potentially accrued by the use of such organisms far outweigh the fear that they might somehow become pathogenic and environmentally uncontrollable. In addition to the detrimental effect of UTI, BV, yeast vaginitis and HIV infection to the lives of over a billion women, the cost of caring for these people far exceeds tens of billions of dollars. Any intervention that potentially provides relief or a reduction in cases of HIV/AIDS, must be considered carefully, and backed by thorough scientific and clinical testing.

Multi-factorial mechanisms

The mechanisms used by probiotic organisms to benefit the host are multi-factorial, and basically of three types.

1. Antimicrobial: For many years, in vitro growth inhibition assays have been used to describe strains as being probiotic. Of course, this is not sufficient without human studies, but the production of bacteriocins, organic acids and hydrogen peroxide may be factors which allow the strains to compete with pathogens and protect the vagina [34], [45]. The production of these substances has been demonstrated in the vagina [46], [47], and whilst the levels may be sufficient to kill some pathogenic bacteria, yeast and viruses, this does not guarantee that infection can be completely prevented. Clearly, some yeast and Gram positive cocci can tolerate acids and resist hydrogen peroxide action, while viruses can invade cells depending upon infective dose and time of exposure.

2. Biosurfactants: A number of lactobacilli strains, in particular L. reuteri B-54 and RC-14, produce mixtures of proteins and carbohydrates, loosely referred to as biosurfactants because of their ability to alter surface tension values. In vitro studies have shown that these substances can form a barrier to pathogen adhesion, potentially reducing the risk of infection [48]. The composition of the biosurfactants have only been partially dissected. The fact that the substance appears to remain on surfaces even when the lactobacilli have detached [49], may explain why relatively few lactobacilli can interfere with larger numbers of pathogens attempting to bind to the surface [50].

3. Anti-infective signalling compounds: The discovery that L. reuteri RC-14 produces a compound that down regulates superantigen-like protein 11 (SSL11) expression in Staphylococcus aureus[51] is exciting, as it demonstrates yet another mechanism whereby probiotic organisms can influence the infectious process. Although it is not yet known if such signalling can be effective in humans, one animal study has shown that exposure of RC-14 to a wound infected with S. aureus, does lead to significantly less inflammation and infectivity [52]. Similar anti-infective signalling has recently been reported against the expression of the flaA gene of Campylobacter jejuni[53].

4. Anti-inflammatory signalling compounds: The ability of Lactobacillus species to induce anti-inflammatory effects has been reported in the past few years. L. rhamnosus GG induces an IL-10 mediated effect [54], while L. rhamnosus GR-1 mediates an anti-inflammatory effect in macrophages through inhibition of c-Jun N-terminal kinases (JNKs) and activation of signal transduction and activator of transcription 3 (STAT3)[55]. Other studies have shown that L. reuteri upregulates an anti-inflammatory nerve growth factor, and inhibits NF-kappaB translocation to the nucleus [56].

In summary

There is growing recognition that microbial inhabitants of the urogenital tract play an important role in protecting the host from disease. As molecular tools help to identify the constituents of the microbiota in healthy women, and track the alterations that lead to infection, new interventional therapies will be conceived to maintain health. By understanding the mechanisms of action of bacteria in the vagina, a variety of probiotic strain by-products should become available to provide additional therapies. As with all approaches to disease treatment and prevention, determining the limitations, as well as the breadth of product effectiveness, will help to ensure that probiotics are used optimally.

Statement of Conflict

Dr. Reid has licensed the use of strains Lactobacillus GR-1 and RC-14 to Chr Hansen, Denmark.

The support of OMAF and the Ontario Neurotrauma Foundation are gratefully appreciated.