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J Med Microbiol 52 (2003), 1117-1124; DOI: 10.1099/jmm.0.05155-0
© 2003 Society for General Microbiology
ISSN 0022-2615

Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli

María Silvina Juárez Tomás1, Virginia S. Ocaña1, Birgitt Wiese2 and María E. Nader-Macías1

1CERELA-CONICET (Centro de Referencia para Lactobacilos), Chacabuco 145, 4000, Tucumán, Argentina 2Institute of Biometrics, University Hospital, Hannover, Germany

Correspondence María E. Nader-Macías fnader{at}cerela.org.ar

Received December 16, 2002
Accepted September 5, 2003

Lactic acid-producing lactobacilli were selected from 134 human vaginal isolates by testing their capability to inhibit the growth of different pathogenic micro-organisms. Lactobacillus acidophilus CRL 1259 (from the CERELA Culture Collection) was selected to study the effects of temperature, pH and culture medium on growth and lactic acid production. Growth parameters were estimated by using the model of Gompertz. Kinetics of inhibition of uropathogenic Escherichia coli were evaluated in mixed cultures of the pathogen and L. acidophilus. Optimal conditions for growth and lactic acid production by L. acidophilus were pH 6.5 or 8.0 and 37 °C. Under these conditions, growth was higher in LAPTg (yeast extract/peptone/tryptone/Tween 80/glucose) broth than in MRS (De Man–Rogosa–Sharpe) broth. However, lactic acid production was more efficient in MRS broth. Under optimal conditions for lactic acid production, L. acidophilus inhibited the growth of E. coli. These results suggest that inclusion of L. acidophilus CRL 1259 in probiotic products for vaginal application would be beneficial.

Abbreviation: LDH, lactic acid dehydrogenase.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
In the vaginal tract, high levels of oestrogens stimulate the deposit of glycogen in the epithelia, which is then fermented to acetic and lactic acids by epithelial cells and/or vaginal flora (Paavonen, 1983). Recent studies support the hypothesis that vaginal bacteria are the primary source of lactic acid in the vagina (Boskey et al., 1999, 2001). Lactobacilli have been recognized as the predominant microflora of the healthy human vagina to maintain a pH of < 4.5 (Redondo-López et al., 1990). This low pH reduces the risk of colonization by pathogens (Stamey & Kaufman, 1975; Stamey & Timothy, 1975; Hanna et al., 1985; Tevi-Bénissan et al., 1997). Bacterial vaginosis, the most common vaginal pathology worldwide, is characterized by a vaginal pH of > 4.5 and by an overgrowth of anaerobic bacteria (Eschenbach, 1993). An increase in vaginal pH is detrimental to the survival of lactobacilli; therefore, local acidification with lactic acid or lactobacilli is useful for restoration of the vaginal ecosystem (Melis et al., 2000).

The characteristics of lactobacilli, i.e. their ability to colonize different hosts (Kotarski & Savage, 1979), led to the isolation of strains from the human vagina (Ocaña et al., 1999a) and their use in vaginal probiotic products (Ocaña et al., 1999b, c, d). Optimal culture conditions to obtain the highest growth of the selected micro-organisms (Juárez Tomás et al., 2002a), as well as a higher degree of bacteriocins (Juárez Tomás et al., 2002b), were reported.

Lactic acid production by lactobacilli that are used by food industries has been studied extensively (Passos et al., 1994; Kylä-Nikkilä et al., 2000). However, there are only a few reports concerning the growth and lactic acid production by vaginal lactobacilli (Boskey et al., 1999, 2001). In this paper, the capability of autochthonous strains of vaginal lactobacilli to inhibit growth of different pathogenic micro-organisms was analysed. Lactobacillus acidophilus CRL 1259 was selected to study the effects of different culture conditions on biomass and lactic acid production. The inhibitory effect of lactic acid produced by this strain on the growth of a human uropathogenic Escherichia coli strain was also determined.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Micro-organisms and culture media.

Vaginal lactobacilli strains (n = 134) that had been isolated previously from vaginal samples of healthy women from 19 to 45 years old from Tucumán, Argentina (Ocaña et al., 1999a), were studied. The following human uropathogenic micro-organisms were employed: E. coli, Klebsiella sp., group B Streptococcus sp., Enterococcus faecalis, Staphylococcus aureus, Neisseria gonorrhoeae, Candida sp. and Gardnerella sp. (provided by the Instituto de Microbiología ‘Luis Verna’ of the Universidad Nacional de Tucumán) and Streptococcus agalactiae ATCC 27956 (CRL 1022) (from the American Type Culture Collection). The strain of E. coli that was used for mixed cultures had the following urovirulence characteristics: type P fimbriae (as demonstrated by the haemagglutination test), production of haemolysins and pyelonephritogenic effects, as tested in mice (Silva-Ruiz et al., 2001).

All micro-organisms were stored in milk/yeast extract (130 g non-fat milk, 5 g yeast extract and 10 g glucose l-1) at -20 °C, except for N. gonorrhoeae and G. vaginalis, which were used as soon as they had been isolated. Stored lactobacilli and pathogens were subcultured three times for 12 h in LAPTg (yeast extract/peptone/tryptone/Tween 80/glucose) broth (Raibaud et al., 1973), prior to screening for production of inhibitory substances.

Before the growth experiments, L. acidophilus CRL 1259 was subcultivated in either MRS (De Man–Rogosa–Sharpe; De Man et al., 1960) broth (Biokar Diagnostics) or LAPTg broth. The inoculum was prepared as described previously (Juárez Tomás et al., 2002a).

Screening for production of inhibitory levels of antagonistic substances.

The effects of supernatant fluid of 134 strains of vaginal lactobacilli on the growth of uropathogens were studied by employing the plate-diffusion technique (Jack et al., 1995). Briefly, LAPTg agar plates (standardized volume, 15 ml LAPTg broth with 1 % agar) with 106–107 c.f.u. ml-1 of each pathogen were prepared, as described previously (Ocaña et al., 1999b). Standardized aliquots (25 µl) of non-treated and neutralized supernatant of lactobacilli were placed into holes (standardized diameter, 4 mm) in the pathogen-inoculated plates. The plates were incubated for 5 h at room temperature and then for 24 h at 37 °C. A clear inhibition zone of >= 6 mm diameter was defined as a positive result. Control assays with the culture medium (LAPTg broth, pH 4 or 6.5) were also performed.

Growth and lactic acid production by L. acidophilus CRL 1259.

Combinations of two culture media (LAPTg or MRS broth), three temperatures (30, 37 or 44 °C) and three initial pH values (5.0, 6.5 or 8.0) were evaluated. Growth experiments, including preparation of culture media, pH determination and quantification of c.f.u. ml-1, were performed as described previously (Juárez Tomás et al., 2002b).

Amounts of D- and L-lactic acid produced during growth were analysed enzymically by using a lactic acid dehydrogenase (LDH) commercial test kit (Boehringer Mannheim). The assay was performed on supernatant fluids of lactobacilli cultures that were obtained by centrifugation at 5000 r.p.m. for 10 min.

Estimation of growth curves.

Growth parameters, estimated by using the four-parameter Gompertz model, are: log (c.f.u. ml-1)t (cell concentration at time t); log (c.f.u. ml-1)0 (cell concentration at time zero); A [increase of biomass between log (c.f.u. ml-1)0 and log (c.f.u. ml-1)max]; µ [maximum specific growth rate (h-1)]; and {upsilon} [duration time of lag phase (h)] (Zwietering et al., 1990; Juárez Tomás et al., 2002a).

Standard errors (SE) of the growth parameters were calculated by the bootstrapping method (Efron, 1982; Huet et al., 1996; Juárez Tomás et al., 2002b).

To determine the statistical significance of the effects of each growth medium (LAPTg or MRS broth) on growth parameters, the differences between parameters were included directly in the equation of the model, in order to estimate confidence intervals (data not shown).

To evaluate multivariate effects of different conditions (temperature, initial pH and culture medium) on growth parameters, the non-linear mixed-effects model [as proposed by Lindstrom & Bates (1990)] was applied by using restricted maximum-likelihood.

For analyses and graphical presentations, the statistical programs SAS 8.2, SPSS 10 and S-Plus 2000 were used.

Mixed cultures of L. acidophilus CRL 1259 and E. coli.

Mixed cultures of L. acidophilus CRL 1259 and E. coli were performed in LAPTg broth at 37 °C. MRS broth was not used, as E. coli grew slowly in this medium. Inocula contained 105–106 c.f.u. ml-1 for E. coli and 106–107 c.f.u. ml-1 for lactobacilli. Viable cell counts were determined by the plate-dilution method by using selective culture media: MacConkey agar for E. coli and lactobacillus selective medium (LBS) for lactobacilli. MacConkey and LBS plates were incubated at 37 °C for 48 h under aerobic and microaerophilic conditions, respectively.

The pH values and levels of D- and L-lactic acids in pure and mixed cultures were determined as described above. All experiments were performed in triplicate. Means of the data are represented in the graphs.

Determination of the MIC of lactic acid.

The diffusion method was applied to agar plates that were prepared as described above and contained uropathogenic E. coli. Decreasing concentrations of lactic acid were evaluated (111–1.1 mM). The MIC was defined as the lowest amount of lactic acid that produced a clear inhibition zone.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Inhibition of pathogens by lactobacilli supernatants

Among the 134 strains of vaginal lactobacilli isolated previously (Ocaña et al., 1999a), only Lactobacillus brevis CRL 1335 and L. acidophilus strains CRL 1259, CRL 1307, CRL 1320 and CRL 1324 were able to inhibit the growth of E. coli, Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis, Klebsiella sp., N. gonorrhoeae and G. vaginalis. Inhibition haloes were shown to be produced by the low pH of the lactobacilli supernatants, as they disappeared when the supernatants were neutralized. L. acidophilus CRL 1259 produced bigger inhibition haloes on the pathogen plates (data not shown).

Lactobacillus salivarius subsp. salivarius CRL 1328 was able to inhibit the growth of E. coli, Klebsiella sp., G. vaginalis, Staphylococcus aureus and Streptococcus agalactiae by the effect of pH, and N. gonorrhoeae and Enterococcus faecalis by a bacteriocin-like substance that was reported previously (Ocaña et al., 1999d). Lactobacillus crispatus CRL 1266 only inhibited the growth of S. aureus by the effect of H2O2 (a catalase-sensitive metabolite) (Ocaña et al., 1999b).

Optimization of growth conditions of L. acidophilus CRL 1259

Fig. 1 shows the growth and pH decrease of L. acidophilus CRL 1259 in LAPTg and MRS broth under different combinations of initial pH and temperature. At 44 °C, the viability of the micro-organisms decreased after a short time. In this case, growth-parameter estimation and lactic acid determination were not performed.



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  		Fig. 1. Kinetics of growth and decrease in pH of L. acidophilus CRL 1259 under different culture conditions. Log c.f.u. ml-1 in LAPTg broth (•) and MRS broth ({bigcirc}); pH modifications in LAPTg broth ({blacktriangleup}) and MRS broth ({triangleup}).

 

Values of the growth parameters obtained varied with the culture conditions tested (Table 1). For all conditions tested, LAPTg broth supported higher growth than MRS broth, but this was statistically significant only at an initial pH of 8.0 and 37 °C. For all growth media and pH values assayed, growth rates were higher at 37 °C. Length of lag phases was inversely related to temperature. When the two types of broth were incubated at the same temperature, lag phases were longer at an initial pH of 8.0.


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  		Table 1. Estimation of growth parameters of L. acidophilus CRL 1259 under different growth conditions by application of the Gompertz model Parameters of the Gompertz model (± SE): log (c.f.u. ml-1)0, initial biomass; A, increase between initial and final biomass; µ, maximum specific growth rate; {lambda}, lag phase.
 

According to statistical analysis performed with the non-linear mixed-effects model, initial pH of the culture medium and temperature of incubation showed significant effects (P < 0.05) on all growth parameters tested (increase of biomass, growth rate and lag phase). However, culture medium only affected the final biomass significantly.

Optimal conditions for the growth of L. acidophilus were LAPTg broth with an initial pH of 6.5 and at 37 °C. Under these conditions, the highest biomass and growth rates, together with shorter lag phases, were obtained. Similar growth was observed in LAPTg broth at 37 °C and an initial pH of 8.0.

pH decrease by L. acidophilus CRL 1259 under different growth conditions

Decrease in pH and acidification rates were significantly higher in LAPTg broth than in MRS broth, due to the higher ion content and buffering capacity of the latter medium (Fig. 1). The difference between initial and final pH of L. acidophilus cultures was related directly to initial pH when LAPTg or MRS broth was incubated at the same temperature. The same behaviour was observed with acidification rates. The largest decrease in pH was obtained in LAPTg broth at an initial pH of 6.5 or 8.0 and at 37 °C. This effect was also observed at 30 °C, but after a longer incubation time.

Lactic acid production by L. acidophilus CRL 1259

Relative proportions of D- and L-lactic acid varied according to the growth medium used (Fig. 2). In general, levels of the D-isomer produced in LAPTg (expressed as g l-1; Fig. 2) were higher than those of the L-isomer. An inverse relationship was observed in MRS broth.



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  		Fig. 2. Kinetics of growth and lactic acid production by L. acidophilus CRL 1259 under different culture conditions. Log c.f.u. ml-1 in LAPTg broth (•) and MRS broth ({bigcirc}); levels of D-lactic acid in LAPTg broth ({blacktriangleup}) and MRS broth ({triangleup}); levels of L-lactic acid in LAPTg broth ({diamondsuit}) and MRS broth ({blacksquare}).

 

In both growth media at different initial pH levels, production of the L- and D-isomers was maximal at 37 °C. When the two types of broth were incubated at the same temperature (except for LAPTg broth at 37 °C), higher amounts of D- and L-lactic acid (expressed as g l-1) were observed at pH 6.5. Maximal concentrations of D-lactic acid were obtained in LAPTg broth at 37 °C and pH 6.5 (5.09 g l-1 after 12 h culture) or 8.0 (5.64 g l-1 after 24 h). The best conditions for production of L-lactic acid were MRS broth at an initial pH of 6.5 and 30 or 37 °C (5.04 and 4.57 g l-1, respectively, both after 24 h culture).

Levels of D-, L- and total lactic acid produced by 107 c.f.u. were higher in MRS broth than in LAPTg broth (Table 2). This indicates that L. acidophilus is more active, from a metabolic point of view, in MRS broth.


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  		Table 2. Mean values of maximal lactic acid concentration produced by L. acidophilus CRL 1259 under different culture conditions
 

Mixed cultures of L. acidophilus CRL 1259 and E. coli

Results from mixed cultures of L. acidophilus CRL 1259 and E. coli are shown in Fig. 3. When using an E. coli inoculum of 1.01x106 c.f.u. ml-1, complete inhibition of pathogen growth was observed after 21 h, whereas when the inoculum of E. coli was 2.4x105 c.f.u. ml-1, 100 % inhibition of pathogen growth was observed after 15 h.



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  		Fig. 3. Pure and mixed cultures of L. acidophilus CRL 1259 and E. coli. Log c.f.u. ml-1, pH or lactic acid levels of E. coli in pure ({blacksquare}) or mixed ({square}) cultures; log c.f.u. ml-1, pH or lactic acid levels of L. acidophilus in pure (•) or mixed ({bigcirc}) cultures; pH or lactic acid levels in mixed cultures ({blacktriangleup}).

 

Levels of L- and D-lactic acid produced by lactobacilli, either in pure or mixed culture, were two times higher than those produced by pure E. coli cultures at both inoculum levels. In mixed cultures, the concentrations were 5.5 g l-1 for D-lactic acid and 2.8 g l-1 for L-lactic acid.

Determination of MIC

The MIC of lactic acid for E. coli was 55.49 mM (equivalent to 5.0 g l-1). This value was lower than the lactic acid levels produced by L. acidophilus CRL 1259 after 9 h in mixed cultures, when pathogen viability decreased.


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Primary selection of potentially probiotic strains must be performed through the application of ‘in vitro’ techniques. Production of antagonistic substances (organic acids, hydrogen peroxide or bacteriocins) against pathogens is a technique that is widely used (McLean & Rosenstein, 2000; Aroutcheva et al., 2001; Strus et al., 2002). Among 134 vaginal Lactobacillus strains isolated previously in our laboratory (Ocaña et al., 1999a), only six strains were able to inhibit all the pathogens under study, except for C. albicans. Inhibition of pathogenic micro-organisms that cause urogenital infections increases the relevance of these wild strains of Lactobacillus for use in probiotic products.

In this study, we employed two culture media that are commonly used for lactobacilli and pH levels other than 4 (the vaginal pH), instead of a chemically defined medium designed to simulate genital secretions (Geshnizgani & Onderdock, 1992). The objective of the present work was not to simulate vaginal conditions, but to assess the most favourable conditions to produce the highest biomass of L. acidophilus CRL 1259 in the shortest time and to evaluate factors that affect the production of lactic acid in laboratory assays.

Under conditions of good growth for L. acidophilus CRL 1259, the final pH values reached (3.5–4.6) were comparable to those determined in the healthy vagina (Andersch et al., 1986; Tevi-Bénissan et al., 1997). Boskey et al. (1999) reported that eight vaginal Lactobacillus strains acidified the growth medium to an asymptotic pH of 3.2–4.8. This fact suggests that lactobacilli create an acidic environment that can inhibit the growth of other micro-organisms.

Production of D- and L-lactic acid by L. acidophilus CRL 1259 was dependent on the three factors tested (growth medium, pH and temperature). Kylä-Nikkilä et al. (2000) reported that the level of production of each isomer only seemed to be dependent to a limited extent on change in expression of the genes responsible for D- and L-LDH. These authors observed different kinetics of production of D- and L-lactic acid by Lactobacillus helveticus CNRZ32 and suggested that different intracellular conditions can change either the catalytic activity of enzymes (D- or L-LDH) or their affinity for the substrate (pyruvate).

Optimal pH and temperature for maximum production of lactic acid were the same as those required for growth. Levels of total lactic acid produced by this micro-organism under different culture conditions (2.56–9.16 g l-1) were higher than those found in vaginal secretions of women (0.90–4.00 g l-1) (Boskey et al., 2001).

Mixed cultures showed that L. acidophilus CRL 1259 was able to inhibit the growth of E. coli at different incubation times, depending on the initial inoculum of pathogen. The final pH reached in mixed cultures was around 4.0. Stamey & Timothy (1975) observed that when the vaginal pH is < 4.5, colonization of the introitus by E. coli is not frequent, whereas the frequency of urinary tract infections is higher when the pH is > 4.5.

In vitro studies of interactions between organisms are over-simplified, compared with the complexity of human mucosal flora. Although its relevance to the in vivo situation is questionable, in vitro experimentation provides an approach for determination of the ability of lactobacilli to inhibit the growth of pathogens. In an animal model, L. fermentum CRL 1058 contained in agarose beads completely inhibited E. coli colonization of the urinary tract of mice (Silva-Ruiz et al., 1993, 1996; Nader de Macías et al., 1996). Reid et al. (1985) also reported that vaginal instillation of lactobacilli in mice protected against uropathogenic E. coli colonization and later reported similar observations for colonization of the human vagina (Reid et al., 1992).

In summary, the results of this study demonstrate that vaginal Lactobacillus strains isolated from Tucumán, Argentina, are able to inhibit the growth of uropathogens by the effect of lactic acid. The results of growth, lactic acid production and mixed cultures with E. coli strongly suggest that L. acidophilus CRL 1259, alone or combined with other strains of lactobacilli, can be used in probiotic products to prevent infections of the urogenital tract.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This paper was supported by Carrillo-Oñativia grants (from the Subsecretaría de Ciencia y Tecnología del Ministerio de Salud Pública de la República Argentina) with PID-BID 385 from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas –Argentina). B. W. is supported by BMBF, Germany, and by SETCIP (bilateral co-operation project ARG 99/025). We thank Elena Bru de Labanda for her help in the experimental design of this study.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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