Selection of Vaginal H2O2-Generating Lactobacillus Species for Probiotic Use

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H₂O₂. This paper describes the selection of H₂O₂-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H₂O₂ level, was chosen to study: (a) the kinetics of H₂O₂ production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H₂O₂ in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H₂O₂L⁻¹) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H₂O₂. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H₂O₂ since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract. Received: 17 November 1998 / Accepted: 17 December 1998     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

In vitro inhibitory activity of human vaginal lactobacilli against pathogenic bacteria associated with bacterial vaginosis in Kenyan women

Lactobacilli have been shown to inhibit in vitro growth of many pathogens and have been used as probiotics to treat a broad range of gastrointestinal and/or vaginal disorders. We sought to determine the in vitro inhibitory potential of lactobacilli of vaginal origin to some bacteria associated with bacterial vaginosis (BV), to characterize the inhibitory substances produced by these lactobacilli and to assess H₂O₂ production. Vaginal specimens were obtained by swabbing the lateral vaginal walls from 107 women two months following BV treatment. One hundred and fifty eight Lactobacillus spp. were isolated in 82 of the 107 women. Lactobacillus jensenii was the predominant strain isolated among these women (29/158; 18.4%). Among 158 culture supernatants tested for antibacterial activity against BV-associated bacteria, none inhibited the growth of Bacteroides fragilis while 23% (37/158), 28% (45/158) and 29% (46/158) inhibited the growth of Prevotella bivia, Gardnerella vaginalis and Mobiluncus spp. respectively. The lactobacilli produced supernatants with a pH range between 2.62 and 6.71; the highly acidic (pH 2–3.99) supernatants were more inhibitory to the indicator strains. There was significant reduction in the mean zones of inhibition following chemical and physical treatment of the supernatants (p = 0.0025). Acid, bacteriocins and H₂O₂ demonstrated potential for antagonism of the bacterial pathogens. These substances may augment each other rather that each working independently on the pathogens.     

Consistent Condom Use Increases the Colonization of Lactobacillus crispatus in the Vagina

Background

Non-hormonal contraception methods have been widely used, but their effects on colonization by vaginal lactobacilli remain unclear.

Objective

To determine the association between non-hormonal contraception methods and vaginal lactobacilli on women’s reproductive health.

Methods

The cross-sectional study included 164 healthy women between 18–45 years of age. The subjects were divided into different groups on the basis of the different non-hormonal contraception methods used by them. At the postmenstrual visit (day 21 or 22 of the menstrual cycle), vaginal swabs were collected for determination of Nugent score, quantitative culture and real-time polymerase chain reaction (PCR) of vaginal lactobacilli. The prevalence, colony counts and 16S rRNA gene expression of the Lactobacillus strains were compared between the different groups by Chi-square and ANOVA statistical analysis methods.

Results

A Nugent score of 0–3 was more common in the condom group (93.1%) than in the group that used an interuterine device(IUD) (75.4%), (p = 0.005). The prevalence of H₂O₂-producing Lactobacillus was significantly higher in the condom group (82.3%) than in the IUD group (68.2%), (p = 0.016). There was a significant difference in colony count (mean ± standard error (SE), log₁₀colony forming unit (CFU)/ml) of H₂O₂-producing Lactobacillus between condom users (7.81±0.14) and IUD users (6.54±0.14), (p = 0.000). The 16S rRNA gene expression (mean ± SE, log₁₀copies/ml) of Lactobacillus crispatus was significantly higher in the condom group (8.09±0.16) than in the IUD group (6.03±0.18), (p = 0.000).

Conclusion

Consistent condom use increases the colonization of Lactobacillus crispatus in the vagina and may protect against both bacterial vaginosis (BV) and human immunodeficiency virus (HIV).     

Differences in TLR9-dependent inhibitory effects of H2O2-induced IL-8 secretion and NF-kappa B/I kappa B-alpha system activation by genomic DNA from five Lactobacillus species

Lactic acid bacteria (LAB) show anti-inflammatory effects, and their genomic DNA was identified as one of the anti-inflammatory components. Despite the differences in anti-inflammatory effects between live LAB dependent not only on genus but also species, this effect has not been compared at the genomic DNA level. We compared the anti-inflammatory effects of the genomic DNA from five Lactobacillus species—Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, and Lactobacillus reuteri—using Caco-2 cells. To evaluate anti-inflammatory effects, decreases in H₂O₂-induced IL-8 secretion and inhibition of H₂O₂-induced NF-κB/IκB-α system activation were examined. All LAB genomic DNAs dose-dependently decreased H₂O₂-induced IL-8 secretion and inhibited H₂O₂-induced NF-κB/IκB-α system activation. Comparison of these effects between Lactobacillus species showed that the anti-inflammatory effects of L. acidophilus genomic DNA are lower than those of the other species. Furthermore, suppression of Toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, expression by RNAi abolished the decrease of H₂O₂-induced IL-8 secretion and inhibition of H₂O₂-induced NF-κB/IκB-α system activation by LAB genomic DNA. Our results demonstrated that the anti-inflammatory effects of genomic DNA differ between Lactobacillus species and TLR9 is one of the major pathways responsible for the anti-inflammatory effect of LAB genomic DNA.     

Beneficial lactobacilli: effects on the vaginal tract in a murine experimental model

Vaginal probiotics containing lactic acid bacteria with activity towards pathogenic microorganisms that cause urogenital tract infections have been proposed as a valid strategy for their prophylaxis and therapy. A murine experimental model was set up to evaluate the colonization capability of beneficial human lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells. Five Lactobacillus strains were intravaginally inoculated into previously estrogenized BALB/c mice. The significance of the effects observed in the vaginal tract was determined by analysis of variance using the general linear model. The numbers of viable vaginal lactobacilli were significantly higher at proestrous–estrous than those at the metaestrous–diestrous phase and decreased markedly on the days after inoculation. Lactobacilli inoculation did not cause cytological or histological modifications of the murine vaginal tract. Moreover, the intravaginal administration of Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos culture collection) 1328 and Lactobacillus gasseri CRL 1263 did not affect the amounts of granulocytes and macrophages present in vaginal washings. In conclusion, the results demonstrate that vaginal lactobacilli did not produce adverse effects on the murine vaginal tract. Therefore, they could be proposed as safe probiotic candidates to promote a balanced microbiota in the urogenital tract.     

H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H₂O₂, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H₂O₂ production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The K_m for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H₂O₂ production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H₂O₂ production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H₂O₂ production in L. johnsonii.     

Changes in the phase variant spectra in the populations of lactic acid bacteria under antibiotic treatment

Effect of the antibiotics kanamycin and ampicillin on the growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M76AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT. The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.     

Modeling of lactic acid fermentation of leguminous plant juices

Lactic acid fermentation of leguminous plant juices was modeled to provide a comparative efficiency assessment of the previously selected strains of lactic acid bacteria as potential components of starter cultures. Juices of the legumes fodder galega, red clover, and alfalfa were subjected to lactic acid fermentation in 27 variants of the experiment. Local strains (Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, and Lactobacillus sp. RS 4) and the collection strain Lactobacillus plantarum BS 933 appeared the most efficient (with reference to the rate and degree of acidogenesis, ratio of lactic and acetic acids, and dynamics of microflora) in fermenting fodder galega juice; Lactobacillus sp. RS 1, Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, Lactobacillus sp. RS 4, and L. plantarum BS 933 were the most efficient for red clover juice. Correction of alfalfa juice fermentation using the tested lactic acid bacterial strains appeared inefficient, which is explainable by its increased protein content and a low level of acids produced during fermentation.     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.     

Adaptation and Probiotic Potential of Lactobacilli,Isolated from the Oral Cavity and Intestines of Healthy People

The present study shows that, from 300 Lactobacillus strains isolated from the oral cavity and large intestine of 600 healthy people, only 9 had high antagonistic activity against pathogens and opportunistic pathogens. All antagonistic strains of lactobacilli have been identified by 16S rRNA sequencing and assigned to four species: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei. In addition, these lactobacilli appeared to be nonpathogenic and had some probiotic potential: the strains produced lactic acid and bacteriocins, showed high sensitivity to broad-spectrum antibiotics, and were capable of forming biofilms in vitro. With the help of PCR and specific primers, the presence of genes for prebacteriocins in L. plantarum (plnEF, plnJ, plnN) and L. rhamnosus (LGG_02380 and LGG_02400) has been revealed. It was found that intestinal strains of lactobacilli were resistant to hydrochloric acid and bile. Lactobacilli isolated from the oral cavity were characterized by a high degree of adhesion, whereas intestinal strains were characterized by average adhesion. Both types of lactobacilli had medium to high rates of auto-aggregation and hydrophobicity and could coaggregate with pathogens and opportunistic pathogens. Additionally, the ability of the lactobacilli strains to produce gasotransmitters, CH₄, CO₂, C₂H₆, CO, and NH₃, has been revealed.     

Lactic Acid Bacteria in Durum Wheat Flour Are Endophytic Components of the Plant during Its Entire Life Cycle

This study aimed at assessing the dynamics of lactic acid bacteria and other Firmicutes associated with durum wheat organs and processed products. 16S rRNA gene-based high-throughput sequencing showed that Lactobacillus, Streptococcus, Enterococcus, and Lactococcus were the main epiphytic and endophytic genera among lactic acid bacteria. Bacillus, Exiguobacterium, Paenibacillus, and Staphylococcus completed the picture of the core genus microbiome. The relative abundance of each lactic acid bacterium genus was affected by cultivars, phenological stages, other Firmicutes genera, environmental temperature, and water activity (a_w) of plant organs. Lactobacilli, showing the highest sensitivity to a_w, markedly decreased during milk development (Odisseo) and physiological maturity (Saragolla). At these stages, Lactobacillus was mainly replaced by Streptococcus, Lactococcus, and Enterococcus. However, a key sourdough species, Lactobacillus plantarum, was associated with plant organs during the life cycle of Odisseo and Saragolla wheat. The composition of the sourdough microbiota and the overall quality of leavened baked goods are also determined throughout the phenological stages of wheat cultivation, with variations depending on environmental and agronomic factors.     

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10^?10 M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H₂O₂ and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.     

Impact of vaginal microbiome communities on HIV antiretroviral-based pre-exposure prophylaxis (PrEP) drug metabolism

Despite the efficacy of antiretroviral-based pre-exposure prophylactics (PrEP) in men who have sex with men, studies in women have produced widely varying outcomes. Recent evidence demonstrates that vaginal microbial communities are associated with increased HIV acquisition risk and may impact PrEP efficacy. Here, we investigate the mechanisms underlying how vaginal bacteria alter PrEP drug levels and impact HIV infection rates ex vivo. Using cervicovaginal lavages (CVLs) from women with or without bacterial vaginosis (BV), we identified microbial metabolism of PrEP drugs in BV samples through LC-MS/MS analysis of soluble drug levels and metabolite formation in dual T-cell cultures. CVL samples were assessed for microbiome analysis using sequencing of bacterial 16S rRNA genes. We also observed non-Lactobacillus bacteria that are associated with BV may potentially impact PrEP efficacy through increased HIV infection rates in co-cultures containing Lactobacillus or BV bacteria, PrEP drugs, CEM-GFP cells, and HIV-1_LAI virus. Finally, we used these data to develop a novel predictive mathematical simulation modeling system to predict these drug interactions for future trials. These studies demonstrate how dysbiotic vaginal microbiota may impact PrEP drugs and provides evidence linking vaginal bacteria to PrEP efficacy in women.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Characterization of vaginal lactobacilli in women after kidney transplantation

Limited number of publications described vaginal microflora after kidney transplantation. Our PubMed search revealed only 18 publications including words “vaginal bacteria &; kidney transplant” in the period of 1978–2011. The aim of this study was to characterize lactobacilli isolated from vaginal swabs of women after kidney transplantation, compared with healthy women. Eighteen renal transplant recipients (mean age 36.1) and 20 healthy women (mean age 36.0) were evaluated. Lactobacilli were cultured on MRS and Columbia blood agars. Biochemical identification with API 50 CHL (bioMerieux, Marcy L’Etoile, France) and multiplex PCR according to Song et al. was performed. Lactobacilli were tested for production of H₂O₂. Minimal inhibitory concentrations (MICs) of selected antimicrobial agents were determined with E-tests (bioMerieux, Marcy L’Etoile, France) and interpreted with CLSI and EUCAST criteria. No bacterial vaginosis was found among studied women. Two strains of group I were identified as Lactobacillus delbrueckii; 18 strains as Lactobacillus gasseri and 15 strains as Lactobacillus crispatus. Only 3 strains from group II were not identified by species-specific mPCR. Group IV was represented with 2 unidentified strains. Vaginal lactobacilli isolated from healthy women represented more homogenous group compared with heterogenous renal transplant recipients. Biochemical identification of lactobacilli by API 50 CHL kits was concordant with mPCR results only in 7 cases (17.5%), all 7 strains were identified as L. crispatus. Majority (93%) of lactobacilli were H₂O₂ producers. All isolated lactobacilli (100%) demonstrated high resistance to metronidazole (MIC > 256 μg/ml). Only 2 strains resistant to vancomycin (MICs: 32 and 256 μg/ml respectively), in the study and control group, and one to moxifloxacin (MIC = 32 μg/ml), were found. Resistance to metronidazole and vancomycin was concordant in CLSI and EUCAST (2010) criteria. Although significant differences between lactobacilli isolated from vaginas of kidney transplant and healthy women were not demonstrated, we demonstrated strains resistant to metronidazole, vancomycin and moxifloxacin in groups of examined women. Our study was performed on a small group of kidney transplant recipients and further more detailed molecular studies on a larger group of patients are required to confirm our results.     

Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.     

Autocrine factors in defense of cytotoxic CTLL-2 cells from oxidative stress

The role of pyruvate and autocrine polypeptide factors (APF) secreted by cytotoxic IL-2-dependent CTLL-2 cells in cell defense from oxidative stress was investigated. The addition of a conditioned medium (CM) containing pyruvate and APF into CTLL-2 cell cultures significantly increased the cell survival under oxidative stress conditions induced by hydrogen peroxide (H₂O₂). The kinetics of (H₂O₂) removal from cell cultures with added CM has been registered. It has been shown that, at the beginning of oxidative stress (less than 15 min), H₂O₂ was mostly removed by means of its reaction with pyruvate contained in CM. Pyruvate content in CM was estimated as 138 ± 7 μM. Gel filtration on a column with Bio-Gel P-10 was used to eliminate pyruvate from CM. Gel filtration resulted in three CM fractions (A, B, and C) corresponding to three chromatogram peaks. Pyruvate was not detected in any fraction. The fraction A was the first to be eluted from the column and contained the largest molecules. In the cell survival test, fraction B had the highest protective ability for CTLL-2 cells under oxidative stress. Fraction A supported cell survival to a lesser degree and fraction C did not show any protective abilities. Fraction B added to cells under oxidative stress kept intracellular ATP content at a significantly higher level then in control cells. Moreover, it was found that APF from fraction B was able to react with H₂O₂ directly and inactivate it in the absence of cells. APF from fraction A did not have such properties.     

Lactic acid production from agriculture residues

Various agriculture feedstock residues were evaluated for lactic acid production by simultaneous saccharification and fermentation (SSF) using Lactobacillus delbrueckii and Lactobacillus plantarum, without any additional nutrients. Lactic acid production was higher in alfalfa fiber and soya fiber compared to corncob (soft) and wheat straw. In Lactobacillus plantarum, the amount of lactic acid obtained from alfalfa fiber and soya fiber was 46 and 44 g/100 g fiber, respectively. However, in Lactobacillus delbrueckii, the lactic acid production in soya fiber was 44 g/100 g fiber and that of alfalfa was 32 g/100 g fiber. Small amounts of acetic acid were also produced from SSF of agricultural feedstocks residues. During SSF of alfalfa fiber, lactic acid production in both L. delbrueckii and L. plantarum was enhanced by adding pectinases and cellulases. Lactic acid production from alfalfa fiber did not change with increasing O₂ transfer rates in the fermentation medium, whereas acetic acid production in both Lactobacillus cultures increased with increasing O₂ transfer rates.