Impact of Lactobacillus crispatus-containing oral and vaginal probiotics on vaginal health: a randomised double-blind placebo controlled clinical trial.

Health of reproductive tract is tightly associated with balance of microbial communities in this area. Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) represent common disturbances of vaginal communities. Vaginal discharge due to BV or VVC is a very frequent reason for visiting gynaecologist. We aimed to evaluate the impact of the novel evidence-based probiotics on BV and VVC patients. The study group included 89 BV and 93 VVC patients (aged 18-50 years) who were recruited into randomised double-blind placebo-controlled two-arm parallel trial. The patients of each diagnosis group received oral or vaginal probiotic capsules, or placebo capsules during 3 months. A probiotic capsule contained two (DSM32717 and DSM32720, in case of BV) or three (DSM32720, DSM32718 and DSM32716, in case of VVC) Lactobacillus crispatus strains. Vaginal, intestinal and general health was monitored weekly by questionnaire. Blood analyses were done in the beginning and at the end of trial. Vaginal samples were collected monthly, microscopic and molecular analyses were performed. The study revealed that both oral and vaginal capsules reduced the signs and symptoms in BV patients. Remarkable improvement was noted in Nugent score, amount and smell of discharge, but also in itching/irritation. Consumption of vaginal probiotics significantly increased the lactobacilli counts in their vagina while mean proportion of some BV-related bacteria decreased. In VVC patients, both oral and vaginal capsules lowered the combined score of two most important symptoms, amount of discharge and itching/irritation. In conclusion, the novel formulations of evidence-based well-focused probiotic L. crispatus strains are effective against BV and VVC being suitable for both vaginal and oral administration. Clinical trial registration: ISRCTN34840624, BioMed Central.

Potential vaginal probiotics: safety, tolerability and preliminary effectiveness.

Vaginal discharge is one of the common reasons for gynaecologist consultation, as bacterial vaginosis and candidiasis are the main causes of discharge. These patients frequently experience numerous problems due to recurrent infections, side effects and drug resistance therefore alternative drugs are needed. Our primary aim was to evaluate safety and tolerability of the potentially probiotic Lactobacillus crispatus strains in volunteer women considering themselves healthy. We also monitored the effects of these strains on vaginal health parameters and lactobacilli counts in vagina and intestine. Forty women were recruited into trial. Absence of chronic diseases was confirmed by questionnaire and blood analysis at screening visit. In randomised double-blind placebo-controlled crossover study the eligible participants were randomly allocated to one of four groups and had to consume one of the two study products (Pro I or Pro II) - a capsule containing 3 strains, 109 cfu per strain, or placebo for 1 week. Treatment period was followed by 2-week washout period and continued with second treatment and washout period. Individuals receiving firstly probiotic, received later placebo and vice versa. Blood, vaginal and faecal samples were collected, and self-reported questionnaires were applied. Thirty subjects completed the trial. The probiotic capsules were well-tolerated. The Pro II intake resulted in a significant decrease in Nugent score (from median 3.0 to 2.0, mean 3.9 to 2.6, P=0.002) and reduction in Gardnerella vaginalis counts (log10 3.57 to 2.38; P=0.027). Reduction of total vaginal bacterial counts was revealed in Pro I group (log10 7.99 to 7.72; P=0.048). In conclusion, the selected vaginal L. crispatus strains are well tolerable and Pro II mixture is prospectively effective in reducing Nugent score and vaginal counts of G. vaginalis. Therefore, these strains seem to be promising candidates for development of novel evidence-based well-focused probiotics to target female urogenital tract disorders.

Assessment of phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus sp.

AIM: To assess antibiotic susceptibility of vaginal lactobacilli strains and provide the data required for assessing the potential of antibiotic resistance risk of new strains selected as probiotic. METHODS AND RESULTS: Potential probiotic vaginal lactobacilli used in the study included 31 vaginal strains of Lactobacillus crispatus (n = 27), Lactobacillus gasseri (n = 3) and Lactobacillus jensenii (n = 1) obtained from the collection of Competence Centre on Health Technologies. Two commercial probiotic strains were used as controls (Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14). The phenotypic and genotypic antibiotic resistances of the strains were determined by E-test and PCR methods. The location (chromosomal DNA or plasmid) of antibiotic resistance genes was also detected. All lactobacilli strains expressed high level of resistance to kanamycin, metronidazole, norfloxacin and trimethoprim/sulphamethoxazole. Some of the strains also expressed resistance to other antibiotics (chloramphenicol, vancomycin) indicating acquired resistance. I class integrons were found in 20% (6/31) of the strains. The RPP (ribosomal protection protein) gene was found to be positive in 30% (9/31) of the strains. Only one L. jensenii strain was determined with tet(M) gene. The tet(K) gene was positive in 26·7% (8/31) and erm(B) gene in 43·3% (13/31) of strains. Three RPP and both four tet(K) and erm(B) genes were located in plasmids. CONCLUSIONS: High antibiotic resistance to clinically important antibiotics was demonstrated, including metronidazole, sulphonamides, aminoglycoside and quinolones. In addition, acquired tetracycline and erythromycin resistance genes were detected in either plasmid or chromosomal DNA of certain isolates, in some of the cases for the first time in the literature. It appears that antibiotic resistance genes erm(B) and tet(K) are widely spread in vaginal lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new data about antimicrobial resistance and genotypic diversity of vaginal Lactobacillus isolates. In addition, it provides data assessing the potential of antibiotic resistance risk of new strains selected as probiotic.

A combination of the probiotic and prebiotic product can prevent the germination of Clostridium difficile spores and infection.

Clostridium difficile infection (CDI) is one of the most prevalent healthcare associated infections in hospitals and nursing homes. Different approaches are used for prevention of CDI. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients. Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic Lactobacillus plantarum Inducia (DSM 21379) to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum Inducia, prebiotic xylitol (Xyl) and their combination as a synbiotic (Syn) product; c) the suppression of germination of C. difficile spores in vitro and in vivo in animal model of C. difficile infection with Inducia, Xyl and Syn treatment. The VPI strain 10463 (ATCC 43255), epidemic strain (M 13042) and clinical isolates (n = 12) of C. difficile from Norway and Estonia were susceptible and contrarily L. plantarum Inducia resistant to vancomycin, metronidazole and ciprofloxacin. The intact cells of Inducia, natural and neutralized cell free supernatant inhibited in vitro the growth of tested C. difficile reference strain VPI and Estonian and Norwegian clinical isolates of C. difficile after co-cultivation. This effect against C. difficile sustained in liquid media under ampicillin (0.75 µg/ml) and Xyl (5%) application. Further, incubation of Inducia in the media with 5% Xyl fully stopped germination of spores of C. difficile VPI strain after 48 h. In infection model the 48 hamsters were administered ampicillin (30 mg/kg) and 10-30 spores of C. difficile VPI strain. They also received five days before and after the challenge a pretreatment with a synbiotic (single daily dose of L. plantarum Inducia 1 ml of 1010 CFU/ml and 20% xylitol in 1 ml by orogastric gavage). The survival rate of hamsters was increased to 78% compared to 13% (p = 0.003) survival rate of hamsters who received no treatment. When administered Xyl the survival rate of hamsters reached 56% vs.13% (p = 0.06). In both Syn (6/9, p = 0.003) and Xyl (3/9, p = 0.042) groups the number of animals not colonized with C. difficile significantly increased. In conclusion, the combination of xylitol with L. plantarum Inducia suppresses the germination of spores and outgrowth into vegetative toxin producing cells of C. difficile and reduces the colonization of gut with the pathogen. Putative therapeutical approach includes usage of the synbiotic during antimicrobial therapy for prevention of CDI and its potential to reduce recurrences of CDI.

Comparison of detection methods for vaginal lactobacilli.

Vaginal lactobacilli offer protection against microbiota imbalance and genitourinary tract infections. We compared vaginal lactobacilli in 50 Estonian women of child-bearing age applying culture-based methods, quantitative PCR and next-generation sequencing (NGS). The culture-based methods found three different lactobacilli: Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri. Using NGS revealed the presence of L. crispatus in 76%, Lactobacillus iners in 52%, L. jensenii in 47% and L. gasseri in 33% of the samples. According to qPCR, L. iners was present in 67% and L. crispatus in 64% of the samples. The proportions of L. crispatus revealed by qPCR and NGS were in good correlation (R=0.79, P<0.001), while that of L. iners correlated poorly (R=0.13, P>0.05). Good concordance for L. crispatus was also found between the results of the culture-based method and qPCR. Finally, good overlap between the results of the culture-based method and NGS was revealed: in case of a positive NGS result for L. crispatus, the same species was isolated in 95% of samples. The corresponding percentages were 82% for L. jensenii and 86% for L. gasseri. Our data indicate fairly general concordance of the three methods for detecting vaginal lactobacilli, except for L. iners. This points out the importance of standardisation of techniques, and the respective studies should involve cultures applying a medium suitable for the fastidious L. iners.

Safety of a probiotic cheese containing Lactobacillus plantarum Tensia according to a variety of health indices in different age groups.

Safety of the probiotic Lactobacillus plantarum strain Tensia (DSM 21380) was tested in vitro, in semihard Edam-type cheese, in an animal model and after consumption of the probiotic cheese in double-blind randomized placebo-controlled human intervention studies with different age groups. The susceptibility of L. plantarum Tensia to 8 antibiotics, and the presence of tetracycline (tet M, S, O, K, L) genes and class 1 integron was assessed by applying epsilometer-test and PCR-based methods. Production of biogenic amines by the probiotic strain in decarboxylation medium containing 1% of l-histidine, l-glutamine, l-ornithine, l-arginine, or l-lysine and in cheese was tested by gas chromatography. The biosafety of L. plantarum Tensia was evaluated on National Institutes of Health-line mice fed cheese containing Tensia at a concentration of 9.6 log cfu/g for 30 consecutive days. In human intervention trials in adults and the elderly, the effects of different doses of Edam-type cheese and the probiotic bacterium on BW, gut functionality indices, and host metabolism were evaluated. The strain L. plantarum Tensia was susceptible to all tested antibiotics and did not possess the tetracycline resistance-determining genes tet(L), tet(S) and tet(O), nor did it contain the integron (Int1) gene. However, the strain was tet(K) and tet(M) positive. Lactobacillus plantarum Tensia did not produce potentially harmful biogenic amines, such as histamine or cadaverine. The amount of tyramine produced in the cheese environment during ripening and after 15 wk of storage was below the clinically significant content. In the animal model, no translocation of the administered strain or other microbes into the blood or organs of mice was detected. No harmful effect was observed on body mass index, inflammatory markers, or serum lipidograms during human intervention trials with different age groups at a daily dose of 10.3 or 8.17 log cfu/serving for 3 wk. No negative effect on gastrointestinal welfare was observed, but the consumption of 100g/d for 3 wk caused hard stools from the second week of the trial. The content of total lactobacilli increased in feces, and the presence of the ingested probiotic strain was confirmed after the consumption of cheese. Thus, L. plantarum strain Tensia is suitable for generally recognized as safe (GRAS) and qualified presumption of safety (QPS) criteria because it did not have any undesirable characteristics. The regular semihard Edam-type cheese (fat content of 26%) with the probiotic additive at a daily dose of 50 g or in excess (100g) and with a probiotic daily dose of 10 log cfu for 3 wk was safe.

[Effect of probiotic Heart cheese "Harmony" comprising bacteria L. plantarum TENSIA DSM 21380 on blood pressure and metabolic health indices].

The probiotic L. plantarum strain TENSIA (DSM 21380) is a novel microorganism having antimicrobial and antihypertensive properties. The aim of the study was to test the efficacy of the consumption of the cheese, comprising the novel strain TENSIA on multiple health markers of humans. Human intervention trial showed that the blood pressure lowering effect of cheese, comprising L. plantarum TENSIA was evident in healthy volunteers with high normal blood pressure up to normal values. The 3-week consumption of the prohiotic cheese did not increase the CVD risk factors like BMI, the level of plasma lipids and glucose as well as inflammatory and immunological markers of human body.

Amniotic fluid microflora in asymptomatic women at mid-gestation.

The presence and composition of amniotic fluid (AF) microflora, as well as AF glucose concentration and white blood cell (WBC) count, were investigated in 22 consecutive asymptomatic women with intact membranes at mid-gestation. AF was retrieved by trans-abdominal amniocentesis. Three of the 22 women (13.6%) had microorganisms in their AF: Chlamydia trachomatis in 2 and both Corynebacterium group absolute nonfermenter (ANF) group and Propionibacterium spp. in 1. No differences were found in clinical characteristics, glucose concentration or WBC count in patients with and without microorganisms in their AF.

Translocation of indigenous microflora in an experimental model of sepsis.

Translocation of viable bacteria from gut to bloodstream and other sterile body sites during shock has been demonstrated in several experimental and clinical studies. The factors causing translocation and its incidence at different stages of shock are not known. The aim of the study was to evaluate the importance of several factors causing translocation of indigenous microflora in an experimental model of septic shock based on intraperitoneal Escherichia coli sepsis in rats. Counts of inoculated E. coli and translocated bacteria in different locations, gut morphology and haematological values were evaluated at different stages of sepsis. Sepsis developed in all animals and E. coli achieved the highest counts in blood 6 h after inoculation. Translocation was commonest at 6 and 12 h after inoculation. Frequently translocating bacteria were lactobacilli, bifidobacteria, bacteroides and peptostreptococci. In early sepsis, translocation was associated with high E. coli counts in blood, yet in late sepsis the opposite correlation was present. Low infiltration by neutrophils in the ileum and decreased mitotic activity in the colon were associated with a high translocation rate. In early sepsis, translocation was associated with low lymphocyte counts, but in late sepsis, with low neutrophil counts. Translocation of bacteria (including anaerobes) that colonise the gut in high counts takes place during sepsis. Putative influencing factors such as activity of the primary disease (bacterial counts in blood), gut morphology or haematological values seem to have different impacts on translocation, depending on the stage of the disease.