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Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H2O2, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H2O2 concentrations. Generation of H2O2 was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H2O2 only occurred upon agitation of the cultures, but the H2O2-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H2O2 production, while Cu2+ inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe3+, hemin, and hemoglobin did not accumulate H2O2. Fe3+ activated an extracellular peroxidase that destroyed the H2O2 being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe3+ ions being a necessary cofactor of the reaction.
The vaginal microbiota of healthy, fertile women is dominated by lactobacilli. Their proportion in this habitat is consistently higher than 70%, in some cases being practically exclusive (8, 20, 26). This abundance contrasts with their scarcity on other mucosae; for example, they never account for more than 1% of the intestinal microbiota and, in many cases, they are not found in feces (35).
Upon first isolation, the vaginal lactobacilli behave as fastidious microorganisms, whose recovery and growth are improved by the addition of a heme source, such as hemin or hemoglobin, to the medium and by incubation under an atmosphere enriched to 10% CO2 or even under anaerobic conditions. The identification of newly isolated strains leads to their ascription to Lactobacillus acidophilus or L. fermentum when culture-dependent methods are used (1, 30). However, genotyping reveals a predominance of L. crispatus, L. gasseri, and L. jensenii, with L. iners and L. vaginalis also being found (5, 6, 34).
The dominance of the lactobacilli in the vagina suggests that they play a fundamental role in the protection of the cavity against the generation of pathological conditions. Among them are those caused by the excessive proliferation of indigenous microorganisms, such as Gardnerella vaginalis, whose dominance may lead to bacterial vaginosis, and colonization by pathogens, such as Candida spp. and Trichomonas vaginalis, which may induce vaginitis or, less frequently, cervicitis and other regional and systemic problems (15, 24, 28).
The antagonistic properties of the vaginal microbiota have two complementary aims: promotion of specific adherence to the epithelial cells and production of antimicrobial substances. Of these, the generation of organic acids and hydrogen peroxide is of paramount importance. Lactobacilli present a strictly fermentative metabolism of sugars, with production of mainly lactic acid (4). As a consequence, the physiological pH of the vagina is around 4, which is inhibitory to the growth of many potential colonizers. In fact, a differential symptom of bacterial vaginosis is an exudate with a pH close to neutral (24, 28).
Hydrogen peroxide generation appears to be common among vaginal lactobacilli, while it is exceptional in strains that usually live in the intestines or in the external environment, such as L. plantarum and L. paracasei, even if they are isolated from the vagina (29). Hawes et al. (13) have shown that the frequency of vaginosis is lower among women that harbor H2O2-producing lactobacilli (3%) than among those colonized by H2O2-negative strains (25%) or by other bacterial species (46%). However, not all H2O2-positive lactobacilli appear to be equally protective, with the most efficient strains belonging to the species L. crispatus and L. jensenii (2).
The bactericidal effect of H2O2 operates through the generation of oxidizing metabolites, such as the radical OH− that introduces breaks in the DNA of the cell. This effect is notably enhanced in the acidic vaginal environment by the presence of myeloperoxidase and halides, which are very abundant in the uterine mucus, especially during ovulation (17, 18).
Given the apparent importance of hydrogen peroxide in the maintenance of vaginal homeostasis, we wanted to know the capacity for generation of this compound of a collection of vaginal lactobacilli, as part of selecting those with the best probiotic properties (19). Previously, we compared the sensitivity and range of measurable concentrations provided by three published H2O2 quantification methods (11, 22, 25). We have also studied the influence of a series of environmental conditions on the production and persistence of H2O2 in the cultures and how H2O2 affects the viability of the lactobacilli producing it.
In this study, 45 vaginal isolates of Lactobacillus, obtained from healthy premenopausal women, were used. They were previously classified as L. jensenii (17 strains), L. crispatus (21 strains), L. gasseri (6 strains), and L. plantarum (one strain) (19). L. jensenii CECT 4306 was used as a positive control for H2O2 generation. The bacteria were grown on MRS agar (Oxoid) supplemented with 1% hemoglobin (Sigma) and incubated at 37°C under a 10% CO2 atmosphere. Liquid cultures were performed in MRS medium and incubated under the same conditions without agitation. Inhibition of protein synthesis was performed with 100 μg/ml chloramphenicol (it was previously determined that the MIC was 1.5 μg/ml).
The concentration of H2O2 was determined colorimetrically through detection of the absorbance induced with the following chromophores: N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64) (Wako), 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma), and o-dianisidine (Sigma) (11, 22, 25). The reaction mixtures were set up as follows: a commercial H2O2 stock solution (30%, corresponding to 9.812 M) was diluted with 100 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6.5) or MRS broth to 1 M. The working concentrations were prepared by diluting this solution with PIPES buffer by a factor of at least 10 (to avoid any color interference by the MRS broth), and 100-μl amounts of the resulting solutions were mixed with 100 μl of the corresponding chromophore (10 mM, final concentration) and 2 μl of a horseradish peroxidase solution (1 mg/ml). After incubation for 10 min at 16°C, the absorbance was measured at 405, 620, and 727 nm for o-dianisidine, tetramethylbenzidine, and DA-64, respectively.
Cultures were set up in 10 ml of liquid MRS medium by inoculation with 1 ml of a 48-h-old culture. Incubation proceeded for several time periods (usually 8 or 16 h) at 37°C under a 10% CO2 atmosphere or anaerobically, without agitation. The following cofactors (2 mM, final concentration) were added to the cultures to determine their influence on H2O2 production: MgCl2, MnSO4, FeCl3, CaCl2, ZnCl2, and CuSO4 (Sigma). Hemoglobin and hemin (1% and 30 mM final concentration, respectively) (Sigma) were also tested. At the end of the incubation period, H2O2 generation was induced by placing the cultures in 100-ml Erlenmeyer flasks, which were subjected to agitation at 300 rpm for up to 3 h at 37°C. Samples (220 μl) were taken every 30 min and centrifuged for 2 min at 12,000 rpm, and the supernatants stored at 4°C until the end of the experiment. The concentration of H2O2 was immediately determined as described in the previous paragraph, using at least three replicates from each sample.
Cultures of L. jensenii CECT 4306 were set in liquid MRS medium with or without 2 mM FeCl3. After incubation for 16 h at 37°C, the culture grown without added Fe cations was divided into two aliquots and one of them was adjusted to 2 mM Fe3+. Fifteen minutes later, H2O2 (2.5 mM final concentration) was added to the three cultures and incubation continued at 37°C for 3 h. Samples were taken every 15 min, and the residual H2O2 concentration was quantified with tetramethylbenzidine.
H2O2 in solution was quantified by measuring the degree of color generation by oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, and DA-64 in the presence of horseradish peroxidase (Fig. (Fig.11).
The procedure using DA-64 turned out to be the most sensitive at low concentrations of hydrogen peroxide (Fig. (Fig.1,1, inset). However, the system became saturated at relatively low H2O2 concentrations. In contrast, the reaction mixtures set with TMB kept the linearity of the determination up to at least 100 mM, irrespective of whether the determination was made in PIPES or in 1/10 volume of MRS (diluted in PIPES).
As part of a previous communication, the isolation of 45 strains of lactobacilli from the vaginas of an equal number of healthy, fertile women was reported (19). Liquid cultures of all these strains were taken during the exponential and stationary phases and subjected to strong agitation under a normal aerobic atmosphere for 3 h, and the generation of H2O2 was quantified at 30-min intervals, using DA-64 and TMB to cover the whole range of production shown by the strains.
The kinetics of production was similar for all producing strains, i.e., H2O2 started to accumulate after 30 min of aeration, with no production in static cultures (Fig. (Fig.22).
The data obtained indicate that the majority of strong producers were L. jensenii strains, followed by L. crispatus isolates (Table (Table1).1). The cultures of strains that achieved a high H2O2 concentration at 8 h of incubation (exponential phase) remained so or with slightly lower values at 16 h (stationary phase). Some others reached levels of 0.5 mM or more only after 16 h of incubation.
Cultures of L. jensenii CECT 4306 (a strong H2O2 producer, which was chosen for further work) treated with chloramphenicol prior to the agitation period produced concentrations of H2O2 similar to those in the untreated cultures (Fig. (Fig.2),2), indicating that the specific H2O2-generating system was already present in the cells before the culture was subjected to aeration and, consequently, that this treatment did not act as an inducer of the system.
To determine whether H2O2 generation could be improved, different cofactors were added to cultures of L. jensenii CECT 4306 at the beginning of the incubation. Supplementation with Mg2+, Mn2+, Ca2+, and Zn2+ affected the production of H2O2 only slightly with respect to the levels in cultures grown in MRS without additions (Fig. (Fig.3).3). Cu2+ inhibited bacterial growth. Surprisingly, cultures containing Fe3+, hemin, or hemoglobin did not accumulate H2O2 upon agitation (Fig. (Fig.3),3), while the growth of the strain was generally slightly improved (data not shown, but see Fig. Fig.77).
The lack of H2O2 in the supernatants of cultures grown in the presence of Fe3+ might be due to inability to generate the compound or to its subsequent destruction. To discriminate between these possibilities, two complementary experiments were performed. In the first, cultures were set up with increasing iron concentrations (from 25 nM to 2 mM), and their capacity to generate H2O2 after 16 h of incubation was tested. An inverse relationship was found between the concentrations of Fe3+ added to the cultures and those of H2O2 recovered (Fig. (Fig.4).4). In the second, cultures were set up and H2O2 was added to them after growth for 16 h. The culture medium with or without Fe3+ and the cultures grown without an excess of the cation did not destroy H2O2. Conversely, Fe3+-containing cultures eliminated the exogenously added H2O2, as did the cultures grown in the absence of added Fe3+ upon addition of the cation (Fig. (Fig.55).
To determine whether Fe3+ was an inducer of the synthesis of a peroxidase or simply acted as a cofactor for a preformed enzyme, chloramphenicol was added to aliquots of 16-h-old cultures grown in MRS medium with or without Fe3+, to inhibit their protein synthesis. After 30 min, Fe3+ was added to the latter cultures and incubation continued for 30 min before the cells were subjected to forced aeration. The cultures with Fe3+ did not accumulate H2O2, irrespective of the moment of its addition and whether chloramphenicol was present or not, while parallel controls without Fe3+ did. This indicates that iron was a necessary cofactor for the enzymatic activity of a constitutively synthesized peroxidase (Fig. (Fig.66).
Cultures of L. jensenii were inoculated into liquid MRS medium with or without Fe3+, containing increasing concentrations of H2O2. Incubation proceeded for 6 h at 37°C. The data obtained (Fig. (Fig.7)7) indicate that Fe3+ slightly improved the growth of the strain (cultures without H2O2) and that this compound, at concentrations equal to or greater than 2.5 mM, completely inhibited the development of L. jensenii. However, complete inhibition of growth was observed at 1.25 mM H2O2 in the cultures devoid of Fe3+, while those containing the cation were only marginally inhibited.
Usually, determination of the ability of vaginal lactobacilli to generate H2O2 is performed by semiquantitative methods based mainly on the growth of the strains under anaerobiosis on the surface of solid medium containing a chromophore and a peroxidase (8, 25). The resulting colonies are exposed to air to allow color development by the positive strains. The intensity of the color is the clue for classification of the lactobacilli as non-, weak, or strong H2O2 producers. However, a more precise determination is desirable to allow for the selection of probiotic precursor strains with the most significant properties for protection of the vaginal environment. In this respect, it must be taken into account that H2O2-producing strains appear to be more stable in the vaginal environment (32) and to effectively antagonize undesirable microorganisms (3, 8, 17, 32), including those that produce sexually transmitted infections, such as Neisseria gonorrhoeae (31).
We chose to test the three selected H2O2 quantification methods because they use similar technologies, thus allowing an easy comparison. Other methods, such as iodination (10) and oxidation of 4-amino-antipyrine (12), were discarded because they were considered to have a low sensitivity (11) or because there was some interference with components of MRS medium (21), the medium used almost universally to grow lactobacilli. The data obtained make the use of both the DA-64- and the TMB-based procedures advisable. The first will allow a precise quantification at low H2O2 concentrations due to its extreme sensitivity, while the second maintains its usefulness through a wide range of concentrations, allowing discrimination between high producers.
The data obtained by applying these procedures to our 45 strains were only partially coincident with those reported after testing H2O2 production on TMB-plus solid medium (19, 25). In both cases, L. jensenii accounted for the highest proportion of producing strains, but some that were considered to be strong producers on TMB-plus medium behaved differently after growth in liquid medium and vice versa.
The application of these methods also enabled the study of the characteristics of H2O2 generation and degradation by L. jensenii CECT 4306, a vaginal isolate that was chosen as a model.
Hydrogen peroxide starts to be produced as a consequence of the aeration of the cultures, presumably because the static liquid cultures are virtually anaerobic, i.e., the limiting factor is the absence of O2 rather than the lack of the responsible enzymes. In fact, the H2O2 generation system was already present in the cultures prior to aeration, as was determined with cultures whose protein synthesis was inhibited before aeration started. This suggests that the production of H2O2 is constitutive upon exposure of the cells to oxygen. Synthesis of H2O2 in response to aeration is common not only among vaginal lactobacilli but in other lactic acid bacteria as well. However, instead of being considered a positive trait, it is perceived as detrimental for starters of food fermentation because it may inhibit their growth (21, 27, 33) and/or affect the sensory quality of the products; for example, H2O2 induces fat rancidity and discoloration of cured meats (23, 33).
Most probably, H2O2 generation proceeds through oxidation of NADH and/or pyruvate by the corresponding dehydrogenases, as happens in Lactobacillus delbrueckii (21) and Bifidobacterium bifidum (16). Furthermore, in the sequenced genomes of two strains of Lactobacillus acidophilus and L. gasseri, two species closely related to L. jensenii, there are genes that encode putative pyruvate dehydrogenases, and the first harbors an NADH oxidase as well. In addition, neither encodes sequence-recognizable superoxide dismutase determinants (GenBank accession numbers NC_006814 and NC_008530).
H2O2, either produced by L. jensenii or incorporated into the medium, was degraded when the cultures contained Fe3+ ions, added as part of an inorganic salt or as an iron-containing metabolite, such as hemin or hemoglobin. Even cultures grown without added Fe3+ showed this ability upon addition of the cation. This Fe3+-dependent, H2O2-degrading activity could be a consequence of a Fenton reaction (9) or an enzymatic process catalyzed by a peroxidase. We favor the second hypothesis because liquid MRS medium with Fe3+ did not destroy H2O2 (Fig. (Fig.5),5), indicating that the reaction is a consequence of the growth of the lactobacilli. Besides, when grown cultures were diluted and Fe3+ was subsequently added to them, the ability to destroy H2O2 decreased as a function of the dilution factor (data not shown). Finally, the activity became completely lost upon heating of the cultures to 60°C for 10 min. These findings suggest that the reaction is catalyzed by a heat-susceptible peroxidase that uses Fe3+ as a cofactor. Heme-dependent peroxidases have been found previously in Lactobacillus plantarum (14), Lactobacillus sakei (7), and other environmental, aerotolerant lactobacilli (33) but not in species that are normal inhabitants of the vagina, although it is known that their growth is improved by the addition of a heme source to the medium under partially aerobic conditions (19). However, a second, heme-independent catalase activity that is present in L. plantarum but not in other environmental species (14, 27) appears to also be absent in vaginal lactobacilli.
The addition of an iron source to the cultures did not induce the synthesis of the peroxidase, because cultures grown without added Fe3+, in which the protein synthesis was inhibited, destroyed exogenously added H2O2 upon addition of the cation. However, it cannot be ruled out that the iron included in the culture medium per se might induce the synthesis of the apoenzyme. In a previous study, it was found that Lactobacillus pentosus cells suspended in phosphate buffer degraded H2O2 following the addition of hematin (33). Our data indicate that under these nonproliferating conditions, hematin acted as an iron source to be used as a cofactor by a preexisting peroxidase.
The results obtained indicate that the processes of H2O2 generation and degradation may be part of the same detoxification mechanism and that this might occur on the vaginal mucosa. On one hand, the cavity is exposed to atmospheric oxygen, which might mediate deleterious oxidation of essential cell components, possibly explaining why most of the lactobacilli used in this work do not grow on the surface of solid media under aerobiosis (19). Besides that, the NADH oxidase activity postulated as the H2O2-generating system is energetically neutral (21), leaving detoxification as the only benefit of the reaction to the cell. On the other hand, the vaginal surface presumably contains an elevated concentration of Fe3+, which would be provided, among other sources, by menstruation. The benefit to the cell of the resulting H2O2-degrading activity is quite clear after having observed that the growth of the producing Lactobacillus is inhibited by H2O2. Thus, the MIC for L. jensenii became doubled in the presence of added Fe3+ with respect to MICs of cultures without any additions. It appears, then, that the protection provided by H2O2 against colonization by undesirable microorganisms would result from the equilibrium between its generation and destruction by the resident lactobacilli, to avoid the toxicity of O2 and of H2O2 in excess.
This work was supported by CICYT grants SAF2004-0033 and BFU2007-65781 from the Ministry of Science and Technology (Spain) and the FEDER Plan. R.M. is the holder of a scholarship from FICYT (Principado de Asturias).
Published ahead of print on 30 November 2009.