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American Journal of Obstetrics & Gynecology, 185(2):375-379, August 2001

Defense factors  of vaginal lactobacilli

Aroutcheva, Alla MD, PhD; Gariti, Dominique MD; Simon, Melissa MD; Shott, Susan PhD; Faro, Jonathan BA; Simoes, Jose A. MD, PhD; Gurguis, Alfred MD; Faro, Sebastian MD, PhD

ABSTRACT

Objective: To determine the antagonistic relationship between vaginal lactobacilli and endogenous vaginal microflora.

Study Design: Twenty-two Lactobacillus strains were studied for the production of lactic acid, hydrogen peroxide, and bacteriocin.

Results: Under standardized growth conditions, most strains increased their biomass by more than 4 times. Lactobacillus species grew best at a pH >= 4.5, and growth was retarded at a pH < 4.5. Lactic acid levels were 0.68 to 2.518 mg/mL and were not related to the number of cells or the pH of media. The pH of the media was caused by the secretion of lactic and other organic acids. Approximately 80% of the strains produced H₂O₂ and were graded as 2+ in one third of the strains and 1+ in others. No statistical correlation was found between H₂O₂ lactic acid and bacteriocin production. Bacteriocin activity was tested on 4 strains of Gardnerella vaginalis. Approximately 80% of the lactobacilli tested produced bacteriocin that inhibited growth of G vaginalis. Six of the strains did not produce bacteriocin. Thirteen strains produced all 3 defense factors, whereas the others lacked 1 or 2 properties.

Conclusions: Lactobacillus species grow best at a pH > 4.5. The pH of the media is dependent on the cell mass and on all organic acids produced by Lactobacillus species. Although all species produce organic acids, not all produce H₂O₂ and bacteriocin. Not all strains of G vaginalis can be inhibited by lactobacilliproducing bacteriocin. (Am J Obstet Gynecol 2001;185:375-9)

INTRODUCTION

A healthy vaginal ecosystem is dominated by certain species of Lactobacillus, which exert a significant influence on the microbiology of the vagina. [1,2] Some species of Lactobacillus suppress the growth of other endogenous bacteria in the vagina through the production of organic acids such as lactic acid, H₂O₂, and bacteriocins or lactocins. The maintenance of a healthy vaginal ecosystem is important to both the patient's vaginal and general health. Understanding the microbial physiology of the vaginal ecosystem will lead to better prevention of infections of the reproductive tract.

The production of organic acids maintains the vaginal pH at < 4.5, thereby creating an inhospitable environment for the growth of most endogenous pathogenic bacteria. In addition to lactic acid, the combination of H₂O₂ and lactocins further suppresses the endogenous pathogenic bacteria to maintain a healthy vaginal ecosystem. [3] The growth of Lactobacillus species maintains a higher oxidation-reduction potential in the vaginal environment, inhibiting the growth of the obligate anaerobic bacteria.

Ninety-six percent of Lactobacillus species found in a healthy vaginal ecosystem produced H₂O₂, whereas only 6% of the lactobacilli recovered from women with bacterial vaginosis (BV) produced H₂O₂. [4] Therefore, the ability to produce lactic acid is not the only requirement to maintain a healthy vaginal ecosystem. It was postulated that H₂O₂ inhibits the growth of vaginal microorganisms either directly or through the enhancement of the enzyme peroxidase-halide. [4,5] H₂O₂ produced by lactobacilli was found to be lethal to Gardnerella vaginalis, Bacteroides bivia, and Escherichia coli. [6,7,8] Hawes et al [9] found that vaginal colonization with H₂O₂-producing lactobacilli was associated with a decrease in the occurrence of BV.

A third component to this system is the production of bacteriocin or lactocin, which are proteins that have bactericidal activity. The range of inhibitory activity by bacteriocins varies from narrow to diverse. Bacteriocins with a narrow range of bactericidal activity inhibit the growth of closely related Lactobacillus species. [10] Bacteriocins with a broad range of antibacterial activity inhibit a diverse group of bacteria, including G vaginalis. [11-13]

The aim of this study was to investigate the relationship between major inhibitory factors produced by vaginal lactobacilli isolated from women with healthy vaginal ecosystems.

MATERIALS AND METHODS

Vaginal specimens were obtained from 22 women between the ages of 25 and 43 years with healthy vaginal ecosystems. Specimens were obtained by swabbing the lateral vaginal wall with sterile cotton-tipped applicators (Copan Diagnostics Inc, Corona, Calif). The vaginal smear underwent Gram staining and was evaluated by using Nugent criteria. [14] Lactobacilli were isolated by inoculating MRS agar and broth (Remel, Lenexa, Kan), incubated anaerobically at 36°C for 24 to 48 hours. G vaginalis was isolated by inoculating the vaginal specimens on V agar (Becton Dickinson, Baltimore, Mass) and incubating in 5% CO₂ atmosphere at 37°C for 48 hours.

Identification of Lactobacillus species was conducted by colony morphology, Gram stain, catalase activity, and the Biolog Microplate One metabolic test system (Biolog Inc, Hayward, Calif).

Lactobacillus organisms were grown in MRS broth by inoculating 5 mL of medium with 0.1 mL of a 2.0 MacFarland density solution of a single species of Lactobacillus. Growth was determined by optical density measurements. Bacteria were grown in micro-wells, incubated at 37°C for 24 hours in a Bioscreen automated optical density monitoring system (Labsystems, Helsinki, Finland). Growth was continuously measured during the prescribed period of incubation.

The hydrogen ion concentration was determined by measuring the pH of MRS broth cultures after 24 hours (pH meter, Orion Research, Inc, Beverly, Mass). Lactic acid concentrations were determined by using the Sigma lactate kit (Sigma, St Louis, Mo). A calibration curve was prepared by adding different concentrations of lactic acid to the MRS broth to achieve final concentrations of 0.25, 0.5, 0.7, 1.0, 1.5, 2.0, and 3.0 mg/mL. All measurements were determined at 340 nm by using a Beckman DU 7500 spectrophotometer (Beckman Instrument Inc, Fullerton, Calif). The calibration curve was designed with the use of the Table Curve 2D program (SPSS Inc, Chicago, Ill).

Detection of hydrogen peroxide was carried out in MRS agar with 5 mg/mL of hemin, 1 mg/mL of vitamin K, 0.01 mg/mL of horseradish peroxide, and 0.25 mg/mL of tetramethylbenzidine as indicator (Sigma). [15] One loop of each Lactobacillus isolate was spotted on that agar and incubated anaerobically at 37°C for 48 hours. The cultures were subsequently exposed to ambient air at room temperature within 24 hours. The horseradish peroxidase oxidized the tetramethylbenzidine in the presence of H₂O₂ to form a blue pigment. Colonies that did not produce H₂O₂ did not form a blue color in the medium. The color intensity was graded as 2+, 1+, and 0.

The presence of bacteriocin was demonstrated by the inhibition of the growth of 4 strains of G vaginalis (254, 340, 415, and 4). The test strains of G vaginalis were isolated from patients with BV. To detect the presence of bacteriocin, a modification of the method described by Kekessy and Piguet was used. [16] MRS agar was streaked with a 10-µL loop containing an 18-hour culture of Lactobacillus and incubated anaerobically for 18 hours. A disk 11 mm in diameter and 4 mm deep was cut out from the MRS Lactobacillus culture. A second culture, G vaginalis, was prepared by inoculated (10⁵ bacteria/mL) V agar on the surface. After inoculating the V agar with G vaginalis and allowing the surface to dry for 5 to 10 minutes, a layer of plain soft agar (0.8%) was placed on top of the inoculated V agar. Five agar disks, 1 control and 4 different Lactobacillus species, were inverted so the surface containing the bacteria was touching the plain agar. The control disk did not contain bacteria.

The bioassay plates were incubated in 5% CO₂ at 37°C for 24 hours. The bacteriocin production was quantified by measuring a zone of inhibition in millimeters around the agar disk.

Statistical analysis was performed by calculation of the nonparametric correlations by using Spearman correlation coefficients, the Friedman test, and the Mann-Whitney test. No one-sided tests were done, and a 0.05 significance level was used.

RESULTS

The ecosystem was characterized as healthy by using the following criteria: discharge was white to slate gray, no odor was detected either in its natural state or when tested with 10% potassium hydroxide, pH was <= 4, squamous epithelial cells were well estrogenized, no clue cells were found either on wet preparation or Gram stain, and white blood cell counts were less than 4 per high-power field.

Lactobacilli were isolated from each patient, and the species are listed in Table I. Lactobacillus acidophilus was isolated in a total of 5 patients , Lactobacillus casei in 5, Lactobacillus gasseri in 3, Lactobacillus paracasei in 3, Lactobacillus plantarum in 2, Lactobacillus jensenii in 1, Lactobacillus bifermentans in 1, Lactobacillus vaginalis in 1, and Lactobacillus pentosus in 1. L acidophilus, L casei, and L gasseri were the most frequently isolated species. The fact that L jensenii and Lactobacillus crispatus were not isolated as frequently in this study as compared with other reports may be because polymerase chain reaction was not performed to separate the isolates.

Table I. Association between amount of cells, pH, lactic acid, and H₂O₂
 

After 24 hours of incubation and growth, Lactobacillus species exhibited different increases in their biomass. The different species displayed 2 patterns of growth. Nineteen species exhibited a growth rate with an optical density of 2.0, whereas 3 species had a growth rate with an optical density of 1.5 (P < .05).

The pH of MRS broth inoculated with Lactobacillus species decreased according to the amount of growth that occurred. The change in pH was dependent upon the number of bacteria (P = .012) and not the concentration of lactic acid. The growth rate or the number of bacteria produced was significant in decreasing the pH (eg, the starting pH of the MRS broth was 5.8, and after inoculating with a standard inoculum, the range of pH was 5.13 to 4.0). Species that had a fast rate of growth produced a higher number of cells and lowered the pH to 4.0. Species that had a slower rate of growth lowered the pH to 5.13. The lactic acid concentration produced in this study ranged from 0.68 to 2.518 mg/mL after 24 hours of anaerobic incubation at 37°C (Table I). Two strains were found that were considered low producers of lactic acid, strain 232 L plantarum (0.68 mg/mL) and strain 377 L gasseri (0.88 mg/mL).

A total of 18 (81.8%) of the strains isolated produced H₂O₂. Semiquantitative determinations revealed that 7 strains (38.9%) exhibited high production (2+) of H₂O₂. The relationships between parameters measured, measurement of growth, pH achieved, lactic acid, and H₂O₂ production are presented in Table II. No statistically significant relationship was found between the final pH of the medium and H₂O₂ production. The statistical difference between pH and lactic acid production was borderline, with a P value of .053. Although this is not statistically significant, it may indicate that a relationship does exist between the production of lactic acid (as well as other acids) and the pH of the environment. H₂O₂ production did not correlate with any of the parameters measured.

Table II. Nonparametric correlations (P value)
 

Table III presents the zones of inhibition exhibited by Lactobacillus bacteriocin against G vaginalis. The testing of all 22 strains of Lactobacillus revealed that they could be divided into 3 groups: high, moderate, and nonproducers. High producers included 4 strains of L acidophilus (348, 319, 160, and 29), 3 strains of L gasseri (377, 149, and 155), 4 strains of L casei (40, 66, 102, and 153), 1 strain of L paracasei (366), 1 strain of L jensenii (43), and 1 strain of L bifermentans (168).

Table III. Lactobacilli bacteriocin inhibition effect
 

Moderate bacteriocin effect was found in 1 strain of L casei (364) and in 1 strain of L vaginalis (179).

No bacteriocin inhibitory effect (zone size < 1 mm) was observed in the following:L plantarum (232 and 228), L paracasei (188), L pentosus (345), and L acidophilus (431).

Among the 4 test strains of G vaginalis, strain 254 was the most sensitive to Lactobacillus bacteriocin . G vaginalis strains 340 and 415 exhibit moderate sensitivity to bacteriocin. Strain 4 was not affected and was resistant to Lactobacillus bacteriocin.

No statistical correlation was found between bacteriocin activity, lactic acid, and hydrogen peroxide production (P > 0.05). L gasseri (377) and L plantarum (232) were similar in that they both were graded low producers of lactic acid and high producers of H₂O₂ but were different with respect to bacteriocin activity. Strain 377 was deemed to have a bacteriocin that exhibited a high level of activity, and strain 232 did not demonstrate bacteriocin growth inhibition. Lactobacillus strains 228, 345, and 431 produced H₂O₂ but did not demonstrate any inhibitory effect.

COMMENT

The ability of lactobacilli to inhibit growth of other bacteria has been attributed to the secretion of lactic acid, hydrogen peroxide, and an inhibitory protein. Many investigators believe that lactic acid production is a primary mechanism in maintaining the equilibrium of a healthy vaginal ecosystem. However, it appears that lactobacilli exert their effect on the vaginal ecosystem through several different mechanisms, including the production of lactic and other organic acids, hydrogen peroxide, and bacteriocin. Several investigators have demonstrated that the activity of bacteriocin is influenced by the hydrogen ion concentration in the environment. [11,17] These investigators demonstrated that at a low pH the Lactobacillus bacteriocin was active and decreased dramatically as the hydrogen ion concentration decreased. The activity of H₂O₂ was also found to be pH dependent. Hydrogen peroxide was stable in an acid environment and degraded as the hydrogen ion concentration decreased. [18] Therefore, as the pH of the vagina increases, bacteriocin looses its effectiveness, hydrogen peroxide is degraded, and lactobacilli cannot compete against the other bacteria, such as G vaginalis.

The findings in this report suggest that the number of bacteria (biocell mass) in the vaginal environment is responsible for pH changes. The biocell mass or numbers of bacteria are significant with regard to the quantity of organic acids produced. Thus, the mere presence of lactobacilli and their ability to produce lactic and other organic acids is not the only important factor in maintaining a healthy vaginal ecosystem—also important is the active growth and the increase in number of bacteria present. In the present study, 2 patterns of growth were observed. Most strains increased the number of cells > 4 times in 24 hours, whereas others only doubled their number in 24 hours. The change in pH of the medium was closely correlated with the increase in the number of bacteria. It is possible that a similar type of growth exists in the vagina and that a reduction in growth of lactobacilli results in an elevation in the pH of the vagina. This results in an overgrowth of other bacteria (eg, G vaginalis) that leads to an imbalance within the endogenous vaginal microflora.

The oxidation-reduction potential of the environment is an important factor affecting which bacteria will grow. BV is defined, microbiologically, by a significant decrease in the growth of Lactobacillus and an increase in the growth of G vaginalis, followed by the growth of a variety of obligate anaerobic bacteria. [19-21] A low pH is not favorable for the growth of G vaginalis. G vaginalis grows best at pH 6.0 to 6.5. No significant growth occurs at pH 4.0, and growth is initiated at pH 4.5. [22] Our studies (A.A. et al, unpublished, 1998) have revealed that the growth of G vaginalis increases as the hydrogen ion concentration decreases, eg, pH 4.5, 5.0, 5.5, and 6.0. Obligate anaerobic bacteria, such as Prevotella bivia and Peptostreptococcus species, grow optimally at pH 5.5 to 6. When grown at pH 4.5, growth begins slowly after an initial incubation period of 24 hours. These bacteria do not grow at pH 3.5 to 4.0 (A.A. et al, unpublished data, 1998).

The concentration of lactic acid in the medium was not solely responsible for the pH change. Strains that acidified the medium to pH 4.0 generated different amounts of lactic acid, eg, 1.5 mg/mL and 2.259 mg/mL. This implies that the low pH was not dependent solely on the amount of lactic acid produced but on the production and secretion of other organic acids including acetic acid. [23] Adams and Hall [24] reported that lactic acid and acetic acid have the ability to inhibit bacterial growth and act synergistically. The activity of acetic acid is potentiated by a lower pH, whereas it is enhanced by lactic acid. Furthermore, the level of lactic acid in the medium did not depend on the number of bacteria in the medium (P > .05). This conclusion is supported by the comparison of 2 parameters: the measure of growth and the concentration of lactic acid. Three samples had a growth measurement of 2.5 with different concentrations of lactic acid, 1.513 mg/mL, 2.204 mg/mL, and 1.128 mg/mL.

Lactobacilli strains that produce hydrogen peroxide have been isolated from 79% to 96% of women with a healthy vaginal ecosystem. [4,25] In the present study, hydrogen peroxide–producing strains were isolated from 81.8% of women with a healthy vaginal ecosystem. Fontaine and Taylor-Robinson [18] measured the amount of hydrogen peroxide generated by Lactobacillus species in media and found that the range was 155 to 310 µg/mL. Our semiquantitative evaluation supports this report.

Bacteriocin produced by vaginal lactobacilli appears to have a broad spectrum of activity and inhibits a wide range of gram-positive and gram-negative bacteria. [12,25-27] McLean and McGroarty [28] showed a high sensitivity of G vaginalis to 23 strains of lactobacilli in the sandwich plate method. They suggested that lactic acid and low pH work synergistically with bacteriocin and may be more important than hydrogen peroxide in inhibiting the growth of G vaginalis. Skarin and Sylwan [11] concluded that low pH in the agar around lactobacilli correlated to wider zones of growth inhibition of G vaginalis and obligate anaerobes.

Among the strains studied in the present investigation, approximately 73% exhibited bacteriocin activity. In the present study, it was observed that not all strains of G vaginalis responded identically to the bacteriocinogenic activity of lactobacilli. One of 4 strains of G vaginalis was not inhibited by any of the Lactobacillus species assayed. This difference in the ability of G vaginalis to resist the action of bacteriocin may be responsible for the more resistant cases of BV and may be a clue to understanding the evolution of BV from a healthy vaginal ecosystem.

Approximately 60% of the isolates of Lactobacillus studied exhibited all 3 mechanisms of bacterial antagonism. The remaining strains lack one or two of the 3 characteristics studied.

The present study demonstrated that inhibitory substances produced by vaginal Lactobacillus species appear to be a primary factor in maintaining a healthy vaginal ecosystem. It is highly probable that these 3 factors, organic acids, bacteriocin, and hydrogen peroxide, act synergistically to suppress the growth of the endogenous pathogenic bacteria. Further understanding of the mechanism produced by Lactobacillus may enable us to understand how BV evolves from a healthy vaginal ecosystem and also shed light on the microbial pathophysiology of pelvic infections in healthy women.

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Key words: Lactobacillus species; lactic acid; hydrogen peroxide; bacteriocin; Gardnerella vaginalis; bacterial vaginosis

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