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Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture

Fabrice Atassi, Dominique Brassart, Philipp Grob, Federico Graf, Alain L. Servin
DOI: http://dx.doi.org/10.1111/j.1574-695X.2006.00162.x 424-432 First published online: 1 December 2006

Abstract

The purpose of this study was to investigate how human vaginal isolates of Lactobacillus acidophilus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus crispatus inhibit the vaginosis-associated pathogens Gardnerella vaginalis and Prevotella bivia. Results show that all the strains in coculture condition reduced the viability of G. vaginalis and P. bivia, but with differing degrees of efficacy. The treatment of G. vaginalis- and P. bivia-infected cultured human cervix epithelial HeLa cells with L. gasseri strain KS120.1 culture or cell-free culture supernatant (CFCS) results in the killing of the pathogens that are adhering to the cells. The mechanism of the killing activity is not attributable to low pH and the presence of lactic acid alone, but rather to the presence of hydrogen peroxide and proteolytic enzyme-resistant compound(s) present in the CFCSs. In addition, coculture of G. vaginalis or P. bivia with L. gasseri KS120.1 culture or KS120.1 bacteria results in inhibition of the adhesion of the pathogens onto HeLa cells.

Keywords
  • Prevotella bivia
  • Gardnerella vaginalis
  • Lactobacillus
  • antibacterial

Introduction

Urogenital infections, such as yeast and bacterial vaginosis, and urinary tract infections (UTI) are major medical problems affecting millions women every year. Antimicrobial therapy is generally an effective way of eradicating these infections, but the increasing emergence of microorganisms resistant to antimicrobial agents is currently reducing its effectiveness. Moreover, some women experience frequent symptomatic recurrences.

Bacterial vaginosis is a polymicrobial syndrome that is characterized by a high vaginal pH and overgrowth of a variety of mostly anaerobic pathogenic bacteria (Pybus & Onderdonk, 1999; Sobel, 2000; Wang, 2000). Gardnerella vaginalis (Sehgal & Nalini, 1990; Mikamo et al., 2000; Aroutcheva et al., 2001b) and Prevotella sp. (Egwari et al., 1995; Pybus & Onderdonk, 1997) are of particular importance in the etiology of bacterial vaginosis. The optimum pH for the growth of G. vaginalis and of Prevotella bivia is c. pH 7 (Boskey et al., 1999). Consistent with this, shifts in vaginal pH have long been associated with a switch in the bacterial flora from one in which Lactobacillus species dominate to one in which G. vaginalis and anaerobic bacteria, such as P. bivia, dominate (Sehgal & Nalini, 1990; Chaudhuri & Chatterjee, 1996; Mikamo et al., 2000; Aroutcheva et al., 2001b). Moreover, it has recently been demonstrated that a commensal, symbiotic relationship between G. vaginalis and P. bivia may be significant for bacterial vaginosis (Pybus & Onderdonk, 1997).

The vaginal microbial flora may play a role in maintaining human health (Pybus & Onderdonk, 1999; Sobel, 2000). Lactobacillus species that have been identified in the human vagina include L. jensenii, L. gasseri, L. acidophilus, L. fermentum, L. plantarum, L. casei, L. cellobiotus, L. oris, L. reuteri, L. ruminis, L. crispatus, L. iners, and L. vaginalis, and L. crispatus (Antonio et al., 1999). The presence and/or dominance of Lactobacillus in the vagina has been associated with a reduced risk of bacterial vaginosis. The antibacterial mechanisms of action of the resident vaginal strains of Lactobacillus appear to involve a lowering of the pH, the production of metabolites, such as hydrogen peroxide and lactic acid, and of antibacterial molecules, including bacteriocins (Reid & Burton, 2002; Servin, 2004). For example, Antonio (2005) recently observed that colonization of the vagina and rectum by hydrogen peroxide-producing Lactobacillus was associated with a lower prevalence of bacterial vaginosis, whereas women colonized either vaginally only, or rectally only, or at neither site faced a progressively increasing risk of bacterial vaginosis. We report that Lactobacillus strains isolated from the vaginas of healthy women develop antagonistic activities against vaginosis-associated G. vaginalis and P. bivia. Moreover, the mechanism by which Lactobacillus strains antagonize G. vaginalis and P. bivia was investigated, showing that the killing activity results from hydrogen peroxide and proteolytic enzyme-resistant compound(s) present in the cell-free culture supernatant (CFCS).

Materials and methods

Bacterial strains and culture conditions

The Lactobacillus strains were isolated from the vaginal flora of healthy women (Department of Obstetrics and Gynecology, Zurich University Hospital, Switzerland) (Atassi et al., 2006). All the Lactobacillus strains were grown in De Man, Rogosa, Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for 18 h at 37°C. For the selection of hydrogen peroxide-producing strains, the amount of hydrogen peroxide present in the overnight cultures of the Lactobacillus strains was determined using Merchoquant Peroxide testing sticks.

CFCSs were obtained by centrifuging the Lactobacillus 18-h cultures at 10 000 g, for 30 min at 4°C. CFCSs were passed through a sterile 0.22-µm Millex GS filter unit (Millipore, Molsheim, France). Separated bacteria were washed three times with sterile MRS and resuspended in fresh MRS.

Gardnerella vaginalis DSM 4944 and Prevotella bivia CI-1 strains came from Medinova (Zurich, Switzerland). The G. vaginalis and P. bivia strains were grown on Gardnerella agar plates purchased from BioMerieux (Lyon, France). Bacteria were suspended in pH 7.0 buffered sodium chloride–peptone solution at about 106 CFU mL−1. Five hundred microliters of the prepared suspension was spread on each agar plate. The inoculated plates were dried under a sterile laminar airflow. The agar plates were then incubated under anaerobic conditions using a sealed anaerobic jar (Becton Dickinson, USA) at 37°C for up to 36 h. Before being used, the G. vaginalis and P. bivia strains were subcultured in Brain Heart Infusion (BHI) supplemented with yeast extract (1%), maltose (0.1%), glucose (0.1%) and horse serum (10%), under anaerobic conditions using a sealed anaerobic jar at 37°C for up to 36 h. The bacterial cultures were centrifuged at 5500 g for 5 min at 4°C before being used. The culture medium was discarded, and the bacteria were washed once with phosphate-buffered saline (PBS) and then resuspended in PBS.

Determination of lactic acid and hydrogen peroxide

A commercial d- and l-lactic acid determination kit (Test-Combination d-lactic acid/l-lactic acid UV-method, Boehringer Mannheim GmbH, Germany) was used to determine the concentration of lactic acid in the Lactobacillus cultures (Table 1).

View this table:
Table 1

Production of lactic acid and hydrogen peroxide by the Lactobacillus vaginal isolates

StrainsLactic acid (mM)Hydrogen peroxide (mM)
L. acidophilus KS10965 ± 80.06 ± 0.02
L. acidophilus KS124.180 ± 100.06 ± 0.02
L. gasserii KS114.1100 ± 150.19 ± 0.05
L. gasseri KS120.165 ± 110.19 ± 0.05
L. gasseri KS123.133 ± 100.12 ± 0.03
L. gasseri KS124.358 ± 120.60 ± 0.15
L. crispatus KS116.190 ± 130.06 ± 0.03
L. crispatus KS119.482 ± 110.08 ± 0.03
L. crispatus KS127.154 ± 100.02 ± 0.02
L. jensenii KS119.160 ± 80.63 ± 0.11
L. jensenii KS121.161 ± 110.03 ± 0.01
L. jensenii KS122.185 ± 120.03 ± 0.01
L. delbrueckii KS122.582 ± 90.03 ± 0.01
  • Lactobacillus 18-h culture

The amount of hydrogen peroxide was determined using the assay described by Yap & Gilliland (2000). Lactobacillus were cultured overnight in MRS containing 5 mM glucose. A sample of CFCS (5 mL) was mixed with 0.1 mL of 1% aqueous O-dianisidine (Sigma Chemical Co, St Louis, MO), and 1 mL of 0.001% aqueous peroxidase (Horseradish Type VI-A; Sigma). After incubation for 10 min at 37°C, the reaction was stopped by adding HCl (0.2 mL, 4N) and the absorbance reading (A400nm) was determined (Table 1). Hydrogen peroxide content was determined by comparison with a standard curve.

Effect on the viability of pathogens

Pathogens (108 CFU mL−1, 500 µL) were incubated with the Lactobacillus culture (108 CFU mL−1, 500 µL) at 37°C. The incubating medium was cultured as indicated above, with each pathogen examined. Aliquots were removed at the outset and at predetermined intervals, serially diluted, and plated on appropriate media as described above to determine the bacterial colony counts of the pathogen.

To test the killing effect of hydrogen peroxide, CFCS was treated at 37°C for 1 h with catalase (from bovine liver, Sigma) at a final concentration of 5 µg mL−1 (van de Guchte et al., 2001). To test the sensitivity of killing activity to proteases, CFCS was incubated at 37°C for 1 h with and without the proteolytic enzymes pronase (200 µg mL−1), trypsin (200 µg mL−1), proteinase K (100 µg mL−1), and pepsin (200 µg mL−1) (Bernet-Camard et al., 1997; Coconnier et al., 1997). Enzyme activities were controlled with hydrogen peroxide solution for catalase and with bovine serum albumin for proteolytic enzymes.

Adhesion assay

Human cervical HeLa cells were cultured at 37°C in a 5% CO2–95% air atmosphere in RPMI 1640 (Invitrogen, France) supplemented with 10% heat-inactivated (30 min, 56°C) fetal calf serum (FCS; Boehringer, Mannheim, Germany), as previously described (Guignot et al., 2001). The culture medium was changed daily. To investigate the adhesion of Lactobacillus strains, postconfluent HeLa cells were washed twice with PBS. For each adhesion assay, 500 µL of the Lactobacillus suspension (bacteria with spent broth culture supernatant) was mixed with Dulbecco's modified Eagle's medium (DMEM) (500 µL), and then added to each well of the tissue-culture plate (24 wells), which was then incubated at 37°C for 3 h in 10% CO2–90% air. For each assay, after incubating, the monolayers were washed five times with sterile PBS, the cells were lysed with sterile H2O, and appropriate dilutions were then plated on tryptic soy agar (TSA) to determine the number of viable cell-associated bacteria by bacterial colony counts. Each cell-association assay was performed at least in triplicate, with three successive cell passages. Results were expressed as CFU well−1 of cell-associated bacteria.

Inhibition of the adhesion of G. vaginalis and P. bivia onto epithelial HeLa cells

For cell monolayer infection, the pathogens were cultured at 37°C for 18 h in appropriate culture media, as described above. Prior to infection, postconfluent cells prepared in 24-well TPP tissue-culture plates (ATGC, Paris, France) were washed twice with PBS. Infecting bacteria were suspended in the culture medium, and a total of 500 µL of DMEM+250 µL of culture pathogen (1 × 108 CFU mL−1)+250 µL of Lactobacillus culture (1.5 × 109 CFU mL−1) or 250 µL of separated and washed Lactobacillus bacteria in fresh MRS (1.5 × 109 CFU mL−1) were added to each well of the tissue-culture plate. The plates were incubated at 37°C in 10% CO2–90% air for 1 h, and then washed three times with sterile PBS; HeLa cells were then lysed with sterile H2O. Dilutions were plated on appropriate media as described above, to determine the number of viable cell-associated bacteria by bacterial colony counts. Each assay was conducted in triplicate, with three successive passages of HeLa cells. Results were expressed as CFU well−1 of cell-associated bacteria.

Killing of G. vaginalis and P. bivia adhering to epithelial HeLa cells

Prior to infection, the post-confluent HeLa cells prepared in 24-well TPP tissue-culture plates (ATGC) were washed twice with PBS. The cells were infected with P. bivia or G. vaginalis (250 µL, 1 × 108 CFU mL−1) at 37°C in 10% CO2–90% air for 90 min. The plates were then washed five times with sterile PBS to remove any non-adhering bacteria. Infected cells were exposed to Lactobacillus cultures (250 µL, 1.5 × 109 CFU mL−1), or Lactobacillus CFCS (250 µL), or separated and washed Lactobacillus bacteria in fresh MRS (250 µL, 1.5 × 109 CFU mL−1) at 37°C in 10% CO2–90% air for 90 min. The plates were then washed three times with sterile PBS, and HeLa cells lysed with sterile H2O. Dilutions were plated on appropriate media as described above, to determine the number of viable cell-associated bacteria by bacterial colony counts. Each assay was conducted in triplicate, with three successive passages of HeLa cells. Results were expressed as CFU well−1 of cell-associated bacteria.

Statistical analysis

Results are expressed as the mean±standard error of the mean (SD). Student's t-test was used for statistical comparisons (P<0.05).

Results

Killing activity against G. vaginalis and P. bivia

A collection of Lactobacillus vaginal isolates were examined for their killing activity against G. vaginalis strain DSM 4944 and P. bivia strain CI-1. Killing activity was measured after 4 h of coculture as previously described (Fayol-Messaoudi et al., 2005). The results reported in Table 2 show that some of the human vaginal isolates of L. acidophilus, L. gasseri, L. jensenii and L. crispatus reduced the viability of G. vaginalis and P. bivia with different levels of efficacy. The strain showing the highest activity against both G. vaginalis and P. bivia was L. gasseri KS120.1, and for this reason it was chosen for further study of the mechanism of killing activity, the inhibition of adhesion of G. vaginalis and P. bivia onto cervix epithelial HeLa cells, and the killing of these two pathogens when they are adhering to preinfected HeLa cells.

View this table:
Table 2

Killing effect of Lactobacillus vaginal isolates on the viability of Gardnerella vaginalis DSM strain 4944 and Prevotella bivia strain Cl-1 in co-culture conditions

G. vaginalis (log CFU mL−1)P. bivia (log CFU mL−1)
Control8.10 ± 0.608.35 ± 0.50
L. acidophilus KS1098.45 ± 0.146.65 ± 2.00*
L. acidophilus KS124.18.31 ± 0.198.50 ± 0.15
L. gasserii KS114.13.32 ± 1.10*5.44 ± 0.13*
L. gasseri KS120.12.15 ± 0.20*2.67 ± 0.58*
L. gasseri KS123.17.80 ± 0.607.84 ± 0.10
L. gasseri KS124.36.20 ± 0.13*5.45 ± 0.35*
L. crispatus KS116.16.76 ± 0.32*2.30 ± 0.35*
L. crispatus KS119.45.20 ± 0.70*4.30 ± 2.17*
L. crispatus KS127.17.50 ± 0.206.49 ± 0.20*
L. jensenii KS119.15.30 ± 0.30*5.15 ± 0.80*
L. jensenii KS121.16.35 ± 0.50*2.00 ± 0.40*
L. jensenii KS122.16.82 ± 0.785.77 ± 0.75*
L. delbrueckii KS122.58.30 ± 0.178.31 ± 0.08
  • The inoculum of G. vaginalis or P. bivia contained 108 CFU mL−1. The control consisted of the pathogen co-cultured in the presence of non-cultured MRS adjusted to pH 4.5 with HCl. Pathogens were incubated with the Lactobacillus strains at 37°C for 4 h, and the CFU mL−1 of each pathogen was determined by plating on appropriate media. Each value shown is the mean ± SD from three experiments. Values that are significantly different from the control value are indicated by an asterisk (P<0.05).

Mechanism of killing activity

In order to characterize better how the killing activity of L. gasseri strain KS120.1 develops, a set of additional experiment was conducted, with G. vaginalis strain DSM 4944 chosen as the pathogen test strain. As shown in Fig. 1, the viability of the pathogen decreased as a function of the time of coculture. A dramatic decrease of 6.7 logs in the viability of G. vaginalis bacteria was rapidly obtained after 1 h of coculture, and the activity did not change thereafter, despite continued coculture with the Lactobacillus strain.

Figure 1

Effect of Lactobacillus gasseri strain KS120.1 on the viability of the Gardnerella vaginalis DSM 4944 strain as a function of the time of coculture. The inoculum of G. vaginalis contained 108 CFU mL−1. The control consisted of the pathogen cocultured in the presence of non-cultured MRS. The pathogen was incubated without or with Lactobacillus culture at 37°C for 4 h, and at intervals the CFU mL−1 was determined by plating on an appropriate medium. Each value shown is the mean±SD from three experiments. Values that were significantly different from the control value are indicated by an asterisk (P<0.05).

KS120.1 bacteria alone have no killing activity against G. vaginalis (control: 8.8±0.4; KS120.1 bacteria: 8.5±0.5 CFU mL−1 after 1 h of coculture). In contrast, we found that the killing activity of the KS120.1 CFCS against G. vaginalis was similar to that of the KS120.1 culture, indicating that the killing activity was attributable to compound(s) located in the CFCS (control: 8.8±0.4; KS120.1 culture: 2.4±0.2; KS120.1 CFCS: 2.8±0.4 CFU mL−1 after 1 h of incubation).

We investigated whether pH, lactic acid, and/or hydrogen peroxide play a role in the killing activity against G. vaginalis described above. Neither MRS nor MRS at pH 4.5 (MRS-HCl) produced any killing activity compared with the control (Fig. 2). dl-lactic acid at the concentration present in the CFCS of an 18-h culture of strain KS120.1 (MRS-LA, 65 mM, pH 4.5) demonstrated no killing activity against G. vaginalis (Fig. 2). Hydrogen peroxide at the concentration present in the CFCS of an 18-h culture of strain KS120.1 (MRS-hydrogen peroxide, 0.2 mM, pH 4.5) demonstrated a significant killing activity against G. vaginalis (Fig. 2).

Figure 2

Effect of pH, lactic acid and hydrogen peroxide on the viability of Gardnerella vaginalis DSM 4944. The inoculum of G. vaginalis contained 108 CFU mL−1. The pathogen was incubated without or with MRS, MRS-HCl, MRS-LA or MRS-hydrogen peroxide at 37°C for 4 h, and the CFU mL−1 was determined by plating on an appropriate medium. Each value shown is the mean±SD from three experiments. Values that were significantly different from the control value are indicated by an asterisk (P<0.05).

Catalase treatment of KS120.1 CFCS abolished only 37% of the killing activity, suggesting that other molecule(s) exert a killing activity (Fig. 3a). In contrast, the killing activity of the KS120.1 CFCS was not modified by proteolytic enzyme treatment (Fig. 3b). Adding NaOH to increase the pH of the KS120.1 CFCS to pH 5.5 reduced the killing activity by c. 50% (Fig. 3c). At pH 6.5, only c. 20% of the initial killing activity remained present (Fig. 3c).

Figure 3

Effect of catalase and proteolytic treatments, and pH neutralization on the killing effect of Lactobacillus gasseri strain KS120.1 against Gardnerella vaginalis DSM 4944. The inoculum of G. vaginalis contained 108 CFU mL−1. The pathogen was incubated without or with untreated or treated KS120.1 CFCS at 37°C for 4 h, and the CFU mL−1 was determined by plating on an appropriate medium. (a) The effect of catalase. (b) The effect of proteolytic enzymes. (c) The effect of pH neutralization. Each value shown is the mean±SD from three experiments. Values that were significantly different from the control value are indicated by an asterisk (P<0.05).

Adhesion to HeLa cells

The adhesion of lactobacilli to epithelial cells has been described as the first step in the formation of a barrier to prevent undesirable microbial colonization. When a concentration of 9 logs CFU mL−1 of Lactobacillus strain KS120.1 was added to cultured, confluent human cervix HeLa cells, 7.54±0.7 logs CFU mL−1 were found adhering to the cells after 1 h of incubation.

Inhibition of the adhesion of vaginosis-associated pathogens to HeLa cells

Under control conditions, 7.0±0.6 logs CFU mL−1 of viable G. vaginalis bacteria adhered to the HeLa cells after 1 h of infection (Fig. 4a). No inhibition of adhesion was observed in the presence of MRS-LA (Fig. 4a). A significant decrease of c. 4 logs in adhering, viable G. vaginalis was observed when the cells were infected in the presence of KS120.1 culture (Fig. 4a). Similarly, a significant decrease of c. 4 logs in adhering, viable G. vaginalis was observed when the cells were infected in the presence of KS120.1 bacteria (Fig. 4a).

Figure 4

Effect of Lactobacillus gasseri strain KS120.1 on the adhesion of the Gardnerella vaginalis DSM 4944 and Prevotella bivia Cl-1 strains to HeLa cells. The inoculum of G. vaginalis or P. bivia contained 108 CFU mL−1. Infecting bacteria were suspended in the culture medium with or without KS120.1 culture, or KS120.1 bacteria alone. The plates were infected and incubated at 37°C in 10% CO2–90% air for 1 h. Infected cells were lysed with sterile H2O, and the CFU mL−1 was determined by plating on an appropriate medium. Each value shown is the mean±SD from three experiments. Values that were significantly different from the control value are indicated by an asterisk (P<0.05).

Under control conditions, 6.8±0.7 logs CFU mL−1 of viable P. bivia were adhering to HeLa cells after 1 h of infection (Fig. 4b). No inhibition of adhesion was observed in the presence of MRS-LA (Fig. 4b). A significant decrease of c. 2 logs in the cell association of viable P. bivia was observed in the presence of KS120.1 culture (Fig. 4b). A significant decrease of c. 2 logs in adhering, viable P. bivia was observed when the cells were infected in the presence of KS120.1 bacteria (Fig. 4b).

Killing of P. bivia and G. vaginalis adhering to HeLa cells

When P. bivia- or G. vaginalis-infected HeLa cells were subjected to treatment with KS120.1 culture, a significant decrease in the level of viable, adhering P. bivia and G. vaginalis was observed (Fig. 5a and b). An increased killing activity was observed when the P. bivia- or G. vaginalis-infected HeLa cells were subjected to treatment with the CFCS of strain KS120.1 as compared with KS120.1 culture (Fig. 5a and b). In contrast, no killing activity against adhering P. bivia and G. vaginalis was observed when the cells were subjected to treatment with KS120.1 bacteria (Fig. 5a and b). It was noted that MRS-LA displayed no killing activity against adhering P. bivia and G. vaginalis.

Figure 5

Killing effect of Lactobacillus gasseri strain KS120.1 against Gardnerella vaginalis DSM 4944 and Prevotella bivia Cl-1 bacteria that were adhering to pre-infected HeLa cells. The inoculum of G. vaginalis or P. bivia contained 108 CFU mL−1. The plates were infected and incubated at 37°C in 10% CO2–90% air for 90 min. Pre-infected cells were further incubated with or without KS120.1 culture, or KS120.1 CFCS, or KS120.1 bacteria alone at 37°C in 10% CO2–90% air for 90 min. The cells were lysed with sterile H2O, and the CFU mL−1 was determined by plating on an appropriate medium. Each value shown is the mean±SD from three experiments. Values that were significantly different from the control value are indicated by an asterisk (P<0.05).

Discussion

The barrier effect to infection of Lactobacillus species in the female urogenital tract has been demonstrated to contribute to the control of vaginal microbiota, by competing with other microorganisms for adherence to epithelial cells (Boris & Barbes, 2000). The regulatory roles attributed to Lactobacillus species in the vaginal microbiota have attracted interest because of potential therapeutic applications (Pybus & Onderdonk, 1999; Reid et al., 2003). It has recently been reported that many Lactobacillus strains of human vaginal origin inhibit the growth of gram-positive aerobic cocci and gram-negative bacteria, such as Staphylococcus aureus, Bacteroides and Escherichia coli (Skarin & Sylwan, 1986; Eschenbach et al., 1989; Klebanoff et al., 1991; Ocana et al., 1999; McLean & Rosenstein, 2000; van de Guchte et al., 2001; Mastromarino et al., 2002; Juarez Tomas et al., 2003). Moreover, Lactobacillus inhibited facultative bacteria, such as G. vaginalis and the anaerobes P. anaerobius and P. bivia, whereas only a few of these Lactobacillus strains were able to inhibit the growth of Enterococcus faecalis or Streptococcus agalactiae (Skarin & Sylwan, 1986; Strus et al., 2002). The growth-inhibiting activity of Lactobacillus of vaginal origin has generally been attributed to the fact they lower the pH and/or produce lactic acid and hydrogen peroxide (Aroutcheva et al., 2001a).

The data reported here show that, after coculture, among the L. acidophilus, L. gasseri, L. jensenii and L. crispatus human vaginal isolates examined, some strains reduced the viability of G. vaginalis and P. bivia. However, among the strains exerting this activity, different levels of efficacy were observed. Interestingly, as previously observed by Ocana (1999), there is no correlation between the capability of exerting killing activity and a particular Lactobacillus species. Moreover, our results clearly show that, among the L. gasseri strains examined, a high variability of efficacy was present, with L. gasseri strain KS123.1 showing no killing activity and L. gasseri strain KS120.1 showing the highest activity. It is interesting to note that the killing activity of L. gasseri strain KS120.1 against G. vaginalis and P. bivia is in the range of killing activity stipulated for the lethal or bactericidal activity of an antimicrobial agent. Indeed, bactericidal activity is defined as the killing activity needed to kill most of (>99.9%) a test microorganism after being incubated with it for a fixed length of time under controlled conditions (N.C.C.L.S., 1999). The bactericidal activity observed for L. gasseri strain KS120.1 resembles the range of bactericidal activity displayed by selected Lactobacillus strains, including L. rhamnosus GG, L. acidophilus LB, L. casei Shirota YT9029, L. casei DN-114 001, L. helveticus KS300, L. rhamnosus GR-1 and L. johnsonii La1 against enterovirulent pathogens, uropathogenic E. coli and/or vaginosis-associated bacteria (Bernet-Camard et al., 1997; Coconnier et al., 1997; Hudault et al., 1997; Lievin-Le Moal et al., 2002; Fayol-Messaoudi et al., 2005; Atassi et al., 2006). Interestingly, we reported that when G. vaginalis- or P. bivia-preinfected HeLa cells were subjected to L. gasseri KS120.1 culture or L. gasseri KS120.1 CFCS treatments, a significant decrease in the viable, adhering G. vaginalis and P. bivia is observed compared with untreated, preinfected cells. However, it should be noted that the killing activity of L. gasseri strain KS120.1 against the adhering pathogens was lower when the pathogens were pre-adhering to HeLa cells as compared with the coculture condition without HeLa cells. This suggests that when the pathogens were in close contact with the host cells they were protected to some extent against the antibacterial treatment.

The mechanism(s) underlying the killing activity of Lactobacillus strains appear to be multifaceted, and include the production of hydrogen peroxide, lactic acid, and antibacterial compounds including bacteriocins or bacteriocin-like molecules, nonbacteriocin molecules, and nonlactic acid molecules (Servin, 2004). The production of hydrogen peroxide has been previously reported for Lactobacillus of vaginal origin (Eschenbach et al., 1989; Klebanoff et al., 1991; Hillier et al., 1992, 1993; McLean & McGroarty, 1996; Ocana et al., 1999). Antonio (1999, 2005) found that hydrogen peroxide was produced by 95% of L. crispatus and 94% of L. jensenii vaginal isolates. Aroutcheva (2001a) reported that c. 80% of the strains of vaginal origin that inhibited growth of G. vaginalis produced hydrogen peroxide. McLean & McGroarty (1996) observed that hydrogen peroxide-producing Lactobacillus strains demonstrated bactericidal activity against metronidazole-susceptible and metronidazole-resistant G. vaginalis. Moreover, L. rhamnosus strain GR-1 of vaginal origin produced a heat-labile bioactive substance, which was not precipitated by up to 80% ammonium sulfate, was extractable in chloroform, and had a molecular weight greater than 12 000–14 000 Da (McGroarty & Reid, 1988). Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, including hydrogen peroxide and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa (van de Guchte et al., 2001). Our findings are consistent with these previous reports because we observed that the killing activity displayed by L. gasseri strain KS120.1 is not attributable to low pH and the presence of lactic acid, but rather to the presence of hydrogen peroxide and proteolytic enzyme-resistant compound(s) located in its CFCS. It should be noted that, as previously reported (Bernet-Camard et al., 1997; Coconnier et al., 1997; Hudault et al., 1997), the killing activity exerted by the compounds(s) present in the KS120.1 CFCS develops in acidic conditions. For the majority of known selected probiotic Lactobacillus strains, the compound(s) exerting killing activity against uropathogenic, enterovirulent or vaginosis-associated bacterial pathogens have not been characterized (Servin, 2004). The molecular nature of the proteolytic enzyme-resistant compound(s) present in L. gasseri KS120.1 CFCS and exerting antagonistic activity against vaginosis-associated P. bivia and G. vaginalis remains to be determined.

It has previously been reported that the strains of L. acidophilus, L. gasseri, and L. jensenii isolated from the vaginas of healthy premenopausal women adhere to epithelial vaginal cells, displacing G. vaginalis (Boris et al., 1998). Gardnerella vaginalis, a heavily pilated, gram-negative bacterium (Boustouller et al., 1987) that produces cytolysin (Cauci et al., 1993), uses an adhesin-receptor mechanism to attach itself to human red blood cells and cultured epithelial McCoy cells (Scott & Smyth, 1987; Scott et al., 1987). This attachment develops by means of distinct adhesins, and adhesion to red blood cells, but not to McCoy cells, is inhibited by galactose, lactose, N-acetylneuraminic acid, and phosphatidylserine. The mechanism of P. bivia attachment remains unknown. Our results demonstrate that the KS120.1 bacteria were adhering to HeLa cells. Consistent with the steric-hindrance inhibition described by Reid et al. (Chan et al., 1985; Reid et al., 1987), when a competitive experiment was conducted, the results showed that both the L. gasseri KS120.1 culture and the L. gasseri KS120.1 bacteria inhibited similarly the adhesion of G. vaginalis and P. bivia to HeLa cells.

Acknowledgements

This work was supported by a research contract between the Institut National de la Santé et de la Recherche Médicale (INSERM) and Medinova AG (Zurich, Switzerland). We are grateful to R. Amsellem for her expert assistance with the cell cultures. We are also indebted to U. Lauper (Department of Obstetrics and Gynecology, Zurich University Hospital, CH-8091 Zurich, Switzerland), who isolated clinical strains of Lactobacillus.

Footnotes

  • Editor: Alex van Belkum

References

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