Why do lactobacilli dominate the human vaginal microbiota?

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1 DOI: / Review article Why do lactobacilli dominate the human vaginal microbiota? SS Witkin, a IM Linhares b a Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, NY, USA b Department of Gynecology and Obstetrics, University of Sao Paulo Medical School, Sao Paulo, Brazil Correspondence: Dr SS Witkin, Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medicine, 1300 York Avenue, Box 35, New York, NY 10065, USA. switkin@med.cornell.edu Accepted 8 September Published Online 7 November Lactobacilli are the most abundant vaginal bacteria in women. They inhibit binding of other bacteria to epithelial cells and produce lactic acid that kills or inhibits the growth of many other bacteria. Lactic acid blocks histone deacetylases, thereby enhancing gene transcription and DNA repair. Lactic acid induces autophagy in epithelial cells to degrade intracellular microorganisms and promote homeostasis. Lactobacilli are tolerated by vaginal epithelial cells and inhibit induction of pro-inflammatory cytokines. Emotional stress may reduce lactobacilli abundance in the vaginal microbiota and enhance inflammation. The ability of lactobacilli to inhibit infection without inducing inflammation may maximise fecundity and successful pregnancy outcome in women. Keywords Immune tolerance, lactobacilli, pregnancy, vaginal microbiome. Tweetable abstract Lactobacilli prevent infection without inducing inflammation to maximise fertility and pregnancy outcome. Please cite this paper as: Witkin SS, Linhares IM. Why do lactobacilli dominate the human vaginal microbiota? BJOG 2017;124: Lactobacillus species, notably Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri and Lactobacillus jensenii, are the most abundant bacteria in the vagina in the majority of reproductive age women. 1 This differs from what is present in the vagina of all other species, including nonhuman primates. 2,3 The relative abundance of vaginal lactobacilli in women increases further during pregnancy. 4,5 The reason(s) for this unique colonisation have largely been unexplored. In this communication, based on recent studies of the properties of vaginal bacteria, their interactions with host epithelial cells and lactic acid influences on mammalian cell functions, possible selective advantages for reproductive age women of having lactobacilli as the most abundant genus in their vaginal microbiota are presented and evaluated. Properties of lactobacilli Under the influence of oestrogen, glycogen is deposited in the human vagina. a-amylase, the enyzme that degrades glycogen to maltose, maltotriose and alpha dextrines, is also present. 6,7 Lactobacilli utilise these glycogen breakdown products to produce lactic acid. This acidification of the vagina to a ph of results in inhibition of the growth of other bacteria Lactobacilli bind to the surface of vaginal epithelial cells and compete with other microorganisms to prevent them from attaching to and infecting these cells. They also release soluble components that inhibit other bacteria from associating with the epithelial cell membrane. 11 The production by lactobacilli of compounds called bacteriocins, which kill other bacteria, also contributes to their dominance. 12,13 Thus, by a number of mechanisms lactobacilli in a woman s vagina may inhibit the proliferation of other bacteria that might be present in low levels as well as protect against acquisition of bacteria deposited in the vagina from exogenous sources. It is not generally appreciated that ejaculated human semen contains a higher number of bacteria than of spermatozoa. 14 Thus, the abundance of vaginal lactobacilli increases resistance to infection and contributes to the well-being of pregnant and non-pregnant women. Lactic acid production A major characteristic of lactobacilli is their ability to metabolise glycogen-derived products under anaerobic conditions to produce lactic acid. This property is likely one of the major reasons for the emergence of lactobacilli as the 606 ª 2016 Royal College of Obstetricians and Gynaecologists

2 Why do lactobacilli dominance? most abundant genus in the vagina of the majority of healthy reproductive age women. In a minority of women who do not have a lactobacilli-dominated vaginal microbiota, other lactic acid-producing bacteria such as Atopobium, Megasphaera and Leptotrichia, may fulfill this function. 15 It should be noted that lactic acid is also produced by vaginal epithelial cells under the control of oestrogen. 16,17 The human vaginal mucosa resides in a relatively anaerobic environment due to the existence of a limited blood supply. Thus, anaerobic glucose metabolism results in lactic acid production by the vaginal epithelial cells. The lactic acid then diffuses out of the cells into the vaginal lumen. 18 However, it appears likely that bacteria, and not host epithelial cells, contribute most of the lactic acid to the vagina. 19,20 Although lactobacilli produce both the D- and L-chiral isomers of lactic acid, 20 humans produce only the L-isomer, with the possible exception of a small quantity of the D-isomer released via the methylglyoxal pathway. 21 D- and L-Lactic acid isomers Among the four most common lactobacilli species in the human vagina only L. iners lacks the gene coding for D- lactic acid dehydrogenase. 12 Therefore, unlike L. crispatus, L. gasseri and L. jensenii, L. iners only produces the L-lactic acid isomer. 22 It is of interest to point out that L. iners is unique among the vaginal lactobacilli in being frequently detected in women in whom lactobacilli are not the most abundant constituents of the vaginal microbiota. 23 D-Lactic acid down-regulates the rate of matrix metalloproteinase (MMP)-8 production by its ability to modulate extracellular matrix metalloproteinase inducer activity. 22 MMP-8 is capable of degrading the cervical plug that prevents bacteria from entering the upper genital tract. 24 Thus, a high D-lactic acid level, present when L. crispatus, L. jensenii and/or L. gasseri is abundant in the vagina, may lower the likelihood of bacterial transport to the uterus that can negatively influence pregnancy outcome as well as initiate an upper genital tract infection in non-pregnant women. Domination by a single Lactobacillus species Four Lactobacillus species L. crispatus, L. iners, L. gasseri and L. jensenii have evolved to specifically inhabit the human vagina. However, only one of these lactobacilli is present in most reproductive age women, to the complete or near total exclusion of other members of this genus. 1,12,25 Explanations for this selective abundance in individual women remain a matter of speculation. A comparative analysis of the genomes of 25 lactobacilli revealed that the four vaginal inhabitants possessed a smaller genome and a lower GC content than did the non-vaginal lactobacilli species. Among the vaginal species, L. crispatus had the largest and L. iners the smallest genome. Although there appeared to be no proteins that were produced only by the vaginal species, differences were noted relative to the other species in the frequency of expression of many protein families. Lastly, each of the four vaginal species differed in protein expression, suggesting that variations in host metabolites and/or the composition of other bacterial components of the vaginal microbiota might influence which lactobacillus species predominates. 12 Lactic acid transport and histone acetylation Intracellular lactic acid is an epigenetic regulator of gene activity. 26 Specifically, both D- and L-lactic acid inhibit histone deacetylase activity, resulting in increased acetylation of histones on the surface of DNA and thereby facilitating gene transcription. An in vitro investigation revealed that the concentration of lactic acid that is normally present in the vagina hyperacetylated histone H3 and histone H4 in three different cervical cancer-derived cell lines, facilitated the repair of damaged DNA, and enhanced cell survival. 27 Interestingly, the two lactic acid isomers appeared to have different activities, with D-lactic acid being the more potent histone deacetylase inhibitor and a better activator of DNA repair enzymes. If these in vitro activities also apply to normal vaginal epithelial cells, an attractive but yet unproven possibility, it strongly suggests that the production of lactic acid, including the bacteria-specific D-isomer, enhances the ability of these cells to survive insults that alter DNA integrity as well as promote gene transcription. These properties might be especially beneficial for cells in the vagina since they are exposed to chemicals present in the external environment as well as to compounds in semen from male partners. The deactivation of host histone deacetylase by lactic acid would allow the transcription of genes that were previously blocked. This may facilitate the release of innate immune system components that inhibit growth of bacteria other than lactobacilli. A possible example of this property may be the induced production of neutrophil gelatinaseassociated lipocalin (NGAL) by vaginal epithelial cells in response to lactobacilli. 28 NGAL inhibits the activity of siderophores, iron-sequestering compounds released by bacteria, and therefore its presence inhibits growth of bacteria that require this element. 29 Iron is not required for growth of lactobacilli 30 but Gardnerella vaginalis, a microbe whose presence in the vagina is associated with an increased susceptibility to various pathological conditions, depends on the availability of exogenous iron for its proliferation. 31 ª 2016 Royal College of Obstetricians and Gynaecologists 607

3 Witkin, Linhares Lactic acid and autophagy Another novel property of lactic acid is its ability to promote autophagy. 32 Autophagy is a basic cell survival mechanism conserved throughout evolution that maintains intracellular homeostasis. 33 Aged or defective organelles such as mitochondria, dysfunctional proteins as well as intracellular bacteria, viruses and protozoa and their components become sequestered within a double membrane structure called an autophagosome. The autophagosome fuses with a lysosome and the enclosed products are degraded by lysosomal enzymes. The intracellular accumulation of lactic acid inhibits production of cyclic adenosine monophosphate (camp), leading to the up-regulation of autophagy. 34 Thus, the activation of autophagy by an abundance of lactic acid-producing bacteria in the vagina would promote the clearance of intracellular pathogens from vaginal epithelial cells, protect the cells from the consequences of oxidative stress due to the presence of defective mitochondria, and remove proteins with dysfunctional activity. We also have recent evidence that autophagy activity in vaginal epithelial cells is highest when L. crispatus is the most abundant bacterium in the vaginal microbiota (Nasioudis and Witkin, in prep.). The ability of group B Streptococci to inhibit autophagy activity in vaginal epithelial cells from pregnant women has been shown recently to be a mechanism that may facilitate its intracellular survival. 35 Tolerance and resistance Defence against the negative consequences of a microbial infection requires two distinct processes. Resistance is the capability to recognise the presence of an invading microorganism and mount an effective pro-inflammatory immune response to prevent its persistence. Tolerance is a mechanism that negates or minimises the consequences of invasion by a pathogen but without the induction of inflammation. Tolerance mechanisms have been shown to be up-regulated during pregnancy. 36,37 This is undoubtedly necessary due to the negative effects of inflammation on proper fetal development as well as the triggering of premature parturition. Thus, the ability to prevent the influx of pathogenic microorganisms into the vagina without the necessity of inducing pro-inflammatory immunity would appear to be advantageous for a successful pregnancy outcome. The concentration and persistence of lactobacilli in the vagina is increased during pregnancy. 4,5 The elevated release of oestrogen, leading to increased availability of glycogen, as well as alterations in the composition of cervico-vaginal secretions, may selectively favour lactobacilli proliferation. Lactic acid production is a major factor in preventing pathogenic microorganisms from infecting the female reproductive tract. Lactic acid selectively lyses bacteria other than lactobacilli. 9,10 In addition, the inactivation of Chlamydia trachomatis 38 and human immunodeficiency virus 39 by lactic acid has been reported. As mentioned above, lactobacilli-stimulated autophagy results in the engulfment and degradation of bacteria, viruses and protozoa that have entered into the epithelial cell cytoplasm. In vitro studies have demonstrated that L. crispatus and L. jensenii not only did not stimulate the release of pro-inflammatory mediators from vaginal epithelial cells but, in addition, inhibited their release when activators of innate immunity such as Tolllike receptor agonists were also present. 40 Thus, there are multiple lactic acid-mediated mechanisms to promote anti-microbial defense while not inducing immunemediated inflammation. Stress and lactobacilli Studies on the impact of environmental stress on the composition of the microbiome have almost exclusively been confined to the intestine. A variety of stressors have been shown to reduce the abundance of intestinal lactobacilli in humans, monkeys and laboratory animals. 41 A recent investigation on the influence of stress on the vaginal microbiome in pregnant mice revealed that there was a significant decrease in the abundance of lactobacilli following stress exposure. 42 One component of the stress response is activation of the sympathetic nervous system by the secretion of corticotropin-releasing hormone from the hypothalamus. This results in the discharge of norepinephrine (noradrenaline) from sympathetic nerve terminals. An elegant series of in vitro experiments by Brosnahan et al. 43 has demonstrated that two vaginal epithelial cell lines can bind exogenous norepinephrine as well as secrete both norepinephrine and dopamine. Furthermore, although norepinephrine by itself did not induce a pro-inflammatory immune response in the vaginal epithelial cells, it significantly enhanced the release of cytokines if immune system activators were also present. Thus, if these findings, plus the studies on the stress-induced loss of lactobacilli from the gut and vaginal microbiome, are translatable to the in vivo situation, it suggests the following scenario. Induction of a stress response leads to both a decrease in the abundance of lactobacilli in the vaginal microbiota and an increase in norepinephrine. Norepinephrine can enter the vagina from the systemic circulation by transduction and it is also produced locally by vaginal epithelial cells. A lowered lactobacilli abundance would increase the likelihood of proliferation of other bacteria and the increase in norepinephrine would potentiate the ability of these microorganisms to induce a magnified proinflammatory immune response. Thus, the severity and 608 ª 2016 Royal College of Obstetricians and Gynaecologists

4 Why do lactobacilli dominance? consequences of a vaginal infection may be magnified if the woman is also under stress. It is interesting to speculate whether individual variations in the extent of norepinephrine production and degradation in the context of various stressors as well as ethnic/racial differences in ease of induction of norepinephrine in response to specific situations may influence the composition of the vaginal microbiota and the extent of pathological consequences to any such alteration. Bacterial vaginosis Bacterial vaginosis (BV), purported to be the most frequent vaginal disturbance in reproductive age women, is characterised by a large decrease in lactobacilli and their replacement by high concentrations of other anaerobic and facultative bacteria. Some women with this diagnosis have a vaginal discharge and unpleasant vaginal odor, whereas others with the same microbiota are asymptomatic. 44 BV has been associated with an increased risk of acquiring a number of different sexually transmitted microorganisms human papillomavirus, 45 human immunodeficiency virus, 46 C. trachomatis and Neisseria gonorrhoeae 47 and a higher rate of pregnancy-related complications. 48,49 The magnitude of these adverse BV-related effects, although statistically significant, is mostly small and variable between populations. Individual variations between women in responses to external stressors, the extent and specificity of stress-related alterations in the vaginal microbiota, the presence of other lactic acid-producing bacteria in the vagina as well as ethnic/racial genetic differences in immune response genes 50,51 may explain why a vaginal microbiota consistent with a diagnosis of BV is not always associated with negative consequences. The over-diagnosis of BV in women with a paucity of vaginal lactobacilli, often determined at a single timepoint, and potential negative consequences of antibiotic treatment, have been the subject of recent reviews. 44,52,53 Human behaviour Unlike the overwhelming majority of mammals where sexual intercourse appears to occur only when the female is ovulating as well as not during pregnancy, humans engage in this activity at any time during the menstrual cycle as well as during gestation. This increased exposure to the male ejaculate escalates a woman s frequency of exposure to potential pathogenic microorganisms as well as to spermatozoa and other non-infectious components of semen. Sperm-specific protein components are not produced until puberty, which is way past the time for development of tolerance to selfantigens. Therefore, spermatozoa are viewed as foreign entities by the female. Frequent exposure to semen, therefore, increases the risk of development of antibodies to spermatozoa that can interfere with fertility. 54 A lactobacillirich vaginal microbiota appears to favour the establishment of tolerance that would diminish the likelihood of development of antisperm immunity. The immunosuppressive properties of seminal fluid 55 also most likely contribute to the inhibition of sperm antibody formation. Although seminal fluid-invoked immunosuppression may enhance the likelihood of male-to-female sexual transmission of pathogenic bacteria, this may be negated or minimised by lactic acid-induced effects. Numerous studies have demonstrated that women with a lactobacilli-dominated vaginal microbiota are at lower risk for a variety of infections than are women with a vaginal microbiota dominated by other bacteria. In addition, development of antisperm antibodies in women as well as the failure of in vitro fertilisation have been linked to the absence of lactobacilli in the vagina. 56 It has not been well studied but it is also possible that the unique sexual behaviour of human males results in their acquisition of microorganisms not encountered by other primates. The human vaginal microbiota, therefore, may have evolved to best cope with the distinctive set of microbes that it encounters. It will be informative to determine whether differences in the vaginal microbiota between individual women may be related to variations in the bacterial composition of her partner s semen and/or to the number of sexual encounters with different partners. It is interesting to note that a lactobacilli-dominated vaginal microbiota is less common in Hispanic and Black women than in Whites and Asians. 1 Possibly, differences in the core semen microbiota between races/ethnicities may partially explain this observation. Conclusions The emergence of lactobacilli as the most abundant bacteria in the human vagina in the majority of reproductive Table 1. Properties of vaginal lactobacilli that protect against infection without inducing inflammation Interference with the binding of other microorganisms to epithelial cells Release of bacteriocins that kill other bacteria Down-regulation of the pro-inflammatory innate immune response to microbial antigens Production of D- and L-lactic acid isomers, which acidifies the vagina and prevents the growth of other microorganisms Lyses bacteria that are associated with bacterial vaginosis Inhibits histone deactylases and increases gene transcription and DNA repair Inhibits cyclic AMP leading to induction of autophagy to kill intracellular microorganisms and protect against oxidative stress and induction of inflammation ª 2016 Royal College of Obstetricians and Gynaecologists 609

5 Witkin, Linhares age women provides multiple unique mechanisms to lessen the negative impact of exposure to potentially pathogenic microorganisms and maximise maternal health, fertility potential as well as promoting proper fetal development and the initiation of parturition only at term (Table 1). A major advantage conferred by the anti-microbial properties of lactobacilli, largely but not exclusively related to lactic acid production, is effective anti-microbial activity that does not elicit pro-inflammatory immunity. It is interesting to speculate that this protection against invasion of the lower female genital tract by viruses, bacteria and other sexually transmitted parasites, coupled with the dominance of immune tolerance mechanisms, may be a consequence of sexual behaviours that are unique to man. Further comparative studies on human and non-human primates are clearly necessary to investigate this hypothesis further. The occurrence of a presumed vaginal lactic acid deficiency detected by an elevated vaginal ph in pregnant women followed by an appropriate treatment has been shown to improve pregnancy outcome. 57 Further investigations are needed to determine whether it might be advantageous to test pregnant women or those seeking to conceive for vaginal D- and L-lactic acid levels and, for those who are deficient, to provide either exogenous lactic acid, a prebiotic that selectively promotes the growth of lactic acid-producing bacteria, or lactic acid-producing bacteria that will successfully colonise the vagina. This alternative to antibiotic treatment might improve pregnancy outcomes in women with a sub-optimal vaginal microbiota. Disclosure of interests None declared. Completed disclosure of interests form available to view online as supporting information. Contribution to authorship SSW and IML both participated in the development and revisions of the manuscript. 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