Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies.

Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

Biodiversity of Intestinal Lactic Acid Bacteria in the Healthy Population.

The complex ecosystem of the gastrointestinal tract involves tight interrelations among host cells, diet, and billions of microbes, both beneficial and opportunistic pathogens. In spite of advanced genomic, metagenomic, and metabonomic approaches, knowledge is still quite limited regarding the biodiversity of beneficial microbiota, including Lactobacillus spp., and its impact on the main biomarkers of general health. In this paper, Lactobacillus biodiversity is demonstrated through its taxonomy, function, and host-microbial interactions. Its prevalence, composition, abundance, intertwined metabolic properties, and relation to host age, genotype, and socioeconomic factors are reviewed based on the literature and original research experience. The species richness, e.g., the biodiversity of gut microbiota, provides the host with a variety of metabolically active species and strains that predict their response for different health conditions and extrinsic interventions. Metabolically active and safe Lactobacillus species and specific strains with particular functional properties increase the biodiversity of the whole intestinal microbiota. The elaborated principles for effective application of probiotics are discussed, aimed at regulating the composition of microbiota simultaneously with blood and urine biomarkers at the borderline of normality. This approach targets the impact of probiotic strains to maintenance of health with anti-infectious, cardiovascular, and metabolic support.

Higher blood glucose level associated with body mass index and gut microbiota in elderly people.

BACKGROUND: Some dominant bacterial divisions of the intestines have been linked to metabolic diseases such as overweight and diabetes. OBJECTIVE: A pilot study aimed to evaluate the relations between the culturable intestinal bacteria with body mass index (BMI) and some principal cellular and metabolic markers of blood in people older than 65. DESIGN: Altogether 38 generally healthy elderly people were recruited: ambulatory (n=19) and orthopedic surgery (n=19). Questionnaires on general health, anthropometric measurements, routine clinical and laboratory data, and quantitative composition of cultivable gut microbiota were performed. RESULTS: Blood glucose level was positively correlated with BMI (r=0.402; p=0.014). Higher blood glucose level had negative correlation with relative share of intestinal anaerobic bacteria such as bacteroides (r=-0.434; p=0.0076) and gram-positive anaerobic cocci (r=-0.364; p=0.027). In contrast, the relative share of bifidobacteria (r=0.383; p=0.019) and staphylococci (r=0.433; p=0.008) was positively correlated to blood glucose level. In elderly people, a higher blood glucose concentration was predicted by the reduction of the anaerobes' proportion (adj. sex, age, and BMI R(2)=0.192, p=0.028) and that of Bacteroides sp. (adj. R(2)=0.309, p=0.016). CONCLUSION: A tight interplay between increased BMI, level of blood glucose, and the reduced proportion of cultivable bacteroides is taking place in the gut microbiota of elderly people.

Hypocaloric diet supplemented with probiotic cheese improves body mass index and blood pressure indices of obese hypertensive patients--a randomized double-blind placebo-controlled pilot study.

BACKGROUND: Gut lactobacilli can affect the metabolic functions of healthy humans. We tested whether a 1500 kcal/d diet supplemented with cheese containing the probiotic Lactobacillus plantarum TENSIA (Deutsche Sammlung für Mikroorganismen, DSM 21380) could reduce some symptoms of metabolic syndrome in Russian adults with obesity and hypertension. METHODS: In this 3-week, randomized, double-blind, placebo-controlled, parallel pilot study, 25 subjects ingested probiotic cheese and 15 ingested control cheese. Fifty grams of each cheese provided 175 kcal of energy. Blood pressure (BP), anthropometric characteristics, markers of liver and kidney function, metabolic indices (plasma glucose, lipids, and cholesterol), and urine polyamines were measured. Counts of fecal lactobacilli and L. plantarum TENSIA were evaluated using molecular methods. The data were analyzed by t-test for independent samples and Spearman's partial correlation analysis. RESULTS: The probiotic L. plantarum TENSIA was present in variable amounts (529.6 ± 232.5 gene copies) in 16/25 (64%) study subjects. Body mass index (BMI) was significantly reduced (p = 0.031) in the probiotic cheese group versus the control cheese group. The changes in BMI were closely associated with the water content of the body (r = 0.570, p = 0.0007) when adjusted for sex and age. Higher values of intestinal lactobacilli after probiotic cheese consumption were associated with higher BMI (r = 0.383, p = 0.0305) and urinary putrescine content (r = 0.475, p = 0.006). In patients simultaneously treated with BP-lowering drugs, similar reductions of BP were observed in both groups. A positive association was detected between TENSIA colonization and the extent of change of morning diastolic BP (r = 0.617, p = 0.0248) and a trend toward lower values of morning systolic BP (r = -0.527, p = 0.0640) at the end of the study after adjusting for BMI, age, and sex. CONCLUSION: In a pilot study of obese hypertensive patients, a hypocaloric diet supplemented with a probiotic cheese helps to reduce BMI and arterial BP values, recognized symptoms of metabolic syndrome.

The association of gut microbiota with body weight and body mass index in preschool children of Estonia.

BACKGROUND: The gut microbiota has been shown to affect both fat storage and energy harvesting, suggesting that it plays a direct role in the development of obesity. The aim of this study was to investigate whether intestinal colonization by particular species/groups of the intestinal microbiota is related to body weight values in Estonian preschool children born in different years during the entire 1990s. METHODS: Body weight, height, body mass index (BMI), and quantitative composition of cultivable gut microbiota (staphylococci, enterococci, streptococci, enterobacteria, lactobacilli, anaerobic gram-positive cocci, bifidobacteria, eubacteria, bacteroides, clostridia, and candida) were studied in 51 healthy 5-year-old children (40 were born between 1993 and 94 and 11 were born between 1996 and 97). RESULTS: At the age of 5 years, median weight was 19.5 kg and median BMI was 15.3 kg/m(2). Significantly higher BMI (p=0.006) was found in 5-year-old children born in late versus early 1990s during the development of socioeconomic situation of Estonia (2% rise in gross domestic product). The counts of the different gut bacteria did not show any association with weight and BMI in the 5-year-old children. However, the BMI values were in positive correlation with a relative share of anaerobic gram-positive bacteria, for example, bifidobacteria when adjusted for sex and year of birth (adj R(2)=0.459, p=0.026) and eubacteria (adj R(2)=0.484, p=0.014) in the community of cultured intestinal microbiota. The relative share of bacteroides showed a negative correlation with the childrens' weight (adj R(2)=- 0.481, p=0.015). CONCLUSION: The body weight indices of preschool children of the general population are associated with the proportion of anaerobic intestinal microbiota and can be predicted by sex and particular socioeconomic situation from birth to 5 years of age.

Comparative Analysis of Gut Microbiota in Centenarians and Young People: Impact of Eating Habits and Childhood Living Environment.

The composition of centenarians' gut microbiota has consistently been used as a model for healthy aging studies. However, there is an incomplete understanding of how childhood living conditions and eating habits affect the development and composition of gastrointestinal microbiota in centenarians with good cognitive functions. We compared the gut microbiota as well as the living and eating habits of the oldest-old group and the young people group. The richness and diversity of microbiota and the abundance of hereditary and environmental microbes were higher in people with longevity than young people. People with longevity ate more potatoes and cereal products. In their childhood, they had more exposure to farm animals and did not have sewers compared with young people. Young people's gut microbiota contained more butyrate-producing bacteria and bacteria that characterized an animal-based Western diet. These results expand our understanding of the effects of childhood environment and diet on the development and stability of the microbiota in people with longevity.

Diversity and metabolic impact of intestinal Lactobacillus species in healthy adults and the elderly.

The present study aimed at assessing the counts and species distribution of intestinal lactobacilli and exploring if the data are associated with BMI and blood glucose level in healthy adults and elderly persons. The BMI (P < 0·01), the level of fasting blood glucose (P < 0·001) and the total counts of lactobacilli (P < 0·01 by bacteriology; P < 0·001 by real-time PCR) were higher in the elderly. The number of species in adults was lower (P < 0·05), who were more often colonised with Lactobacillus acidophilus (P = 0·031) and L. helveticus (P < 0·001). In contrast, L. plantarum (P = 0·035), L. paracasei (P < 0·001) and L. reuteri (P = 0·031) were more prevalent in the elderly. L. rhamnosus was detected in adults (P < 0·001), but not in any elderly person. BMI was associated with counts of lactobacilli, adjusted for age and sex (P = 0·008). The higher BMI in both groups of persons was associated with the presence of obligate homofermentative lactobacilli and L. sakei, both adjusted for age and sex. Plasma glucose values were positively correlated with BMI and negatively correlated with colonisation with L. paracasei (P = 0·0238) in adults and on the borderline with L. fermentum (P = 0·052) in the elderly. Thus, the species-specific PCR analysis of Lactobacillus sp. combined with viable plating data indicates substantial age-related structural differences in the intestinal lactobacilli communities. The higher counts of intestinal Lactobacillus sp. are associated with higher BMI and blood glucose content, while their specific fermentative groups and species of lactobacilli appear at different glucose levels both in adults and in the elderly.

Human microbial ecology: lactobacilli, probiotics, selective decontamination.

Health care-associated infections are closely associated with different medical interventions which interrupt the balance of human microbiota. The occasional predominance of opportunistic pathogens may lead to their translocation into the lymph nodes and bloodstream, causing endogenous (primary or secondary) hospital infections. The question is raised as to if there is a possibility for prevention of the imbalance of GI microbiota during medical interventions in critically ill patients. Prophylactic selective decontamination of the digestive tract (SDD) simultaneously applies three to four different antimicrobials for the suppression of enteric aerobic microbes, which are potentially pathogenic microorganisms. However, there is no convincing evidence that the indigenous beneficial intestinal microbiota are preserved, resulting in reduced mortality of high-risk patients. In this overview, we have evaluated the antimicrobial treatment guidelines of the Infectious Diseases Society of America (IDSA) for intra-abdominal infections in adults and seniors according to their safety for different Lactobacillus spp. The data from our group and in the literature have shown that all tested lactobacilli strains (nearly one hundred) were insusceptible to metronidazole while different species of lactobacilli of the three fermentation groups expressed particular antibiotic susceptibility to vancomycin, cefoxitin, ciprofloxacin and some new tetracyclines. We have relied on microbial ecology data showing that the GI tracts of adults and the elderly are simultaneously colonised at least with several (four to a maximum of 12) Lactobacillus species expressing variable intrinsic insusceptibility to the aforementioned antimicrobials, according to the provided data in table. This finding offers the possibility of preserving the colonisation of the intestine with some beneficial lactobacilli during antimicrobial treatment in critically ill patients with health care-associated infections. Several probiotic Lactobacillus spp. strains are intrinsically resistant to antimicrobials and can be used during antibacterial therapy, however, their application as an additive to antimicrobial treatment in critically ill patients needs to be investigated in well-designed clinical trials.

Antigenic proteins of Lactobacillus acidophilus that are recognised by serum IgG antibodies in children with type 1 diabetes and coeliac disease.

Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

Screening and evaluation of human intestinal lactobacilli for the development of novel gastrointestinal probiotics.

The aim of this study was to screen intestinal lactobacilli strains for their advantageous properties to select those that could be used for the development of novel gastrointestinal probiotics. Ninety-three isolates were subjected to screening procedures. Fifty-nine percent of the examined lactobacilli showed the ability to auto-aggregate, 97% tolerated a high concentration of bile (2% w/v), 50% survived for 4 h at pH 3.0, and all strains were unaffected by a high concentration of pancreatin (0.5% w/v). One Lactobacillus buchneri strain was resistant to tetracycline. None of the tested strains caused lysis of human erythrocytes. Six potential probiotic strains were selected for safety evaluation in a mouse model. Five of 6 strains caused no translocation, and were considered safe. In conclusion, several strains belonging to different species and fermentation groups were found that have properties required for a potential probiotic strain. This study was the first phase of a multi-phase study aimed to develop a novel, safe and efficient prophylactic and therapeutic treatment system against gastrointestinal infections using genetically modified probiotic lactobacilli.

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people.

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2-10.8 vs. median 6.3, range 3.3-9.7 log cfu/g; p=0.005), adjusted (OR=1.71, CI95 1.04-2.82; p=0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r=-0.367, p=0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR=0.16, CI95 0.04-0.73; p=0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r=-0.309, p=0.062) adjusted for BMI (Adj. R(2)=0.181; p=0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r=0.434, p=0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R(2)=0.193, p=0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = -0.389, p = 0.016; Adj. R(2)=0.184 p=0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).

Persistence of Escherichia coli clones and phenotypic and genotypic antibiotic resistance in recurrent urinary tract infections in childhood.

We assessed the clonality of consecutive Escherichia coli isolates during the course of recurrent urinary tract infections (RUTI) in childhood in order to compare clonality with phenotypic antibiotic resistance patterns, the presence of integrons, and the presence of the sul1, sul2, and sul3 genes. Altogether, 78 urinary E. coli isolates from 27 children, who experienced recurrences during a 1-year follow-up after the first attack of acute pyelonephritis, were investigated. The MICs of sulfamethoxazole, trimethoprim-sulfamethoxazole (SXT), ampicillin, cefuroxime, cefotaxime, and gentamicin and the presence or absence of the intI gene for class 1 integrons and the sulfamethoxazole resistance-encoding genes sul1, sul2, and sul3 were determined. All E. coli strains were genotyped by pulsed-field gel electrophoresis. There were no significant differences in the prevalences of resistance to beta-lactams and SXT between initial and consecutive E. coli isolates (41 versus 45% and 41 versus 29%, respectively). However, the E. coli strains obtained after SXT administration more frequently carried two or more sul genes than the nonexposed strains (9/21 [43%] versus 11/57 [19%], respectively; P = 0.044). In 78% of the patients, the recurrence of unique clonal E. coli strains alone or combined with individual strains was detected. Phenotypic resistance and the occurrence of sul genes were more stable in clonal strains than in individual strains (odds ratios, 8.7 [95% confidence interval {95% CI}, 1.8 to 40.8] and 4.4 [95% CI, 1.1 to 17.7], respectively). Thus, in children with RUTIs, the majority of E. coli strains from consecutive episodes are unique persisting clones, with rare increases in the initially high antimicrobial resistance, the presence of sul genes, and the presence of integrons.

The occurrence of antimicrobial resistance and class 1 integrons among commensal Escherichia coli isolates from infants and elderly persons.

BACKGROUND: The aim of our study was to compare the presence of the intI1 gene and its associations with the antibiotic resistance of commensal Escherichia coli strains in children with/without previous antibiotic treatments and elderly hospitalized/healthy individuals. METHODS: One-hundred-and-fifteen intestinal E. coli strains were analyzed: 30 strains from 10 antibiotic-naive infants; 27 from 9 antibiotic-treated outpatient infants; 30 from 9 healthy elderly volunteers; and 28 from 9 hospitalized elderly patients. The MIC values of ampicillin, cefuroxime, cefotaxime, gentamicin, ciprofloxacin, and sulfamethoxazole were measured by E-test and IntI1 was detected by PCR. RESULTS: Out of the 115 strains, 56 (49%) carried class 1 integron genes. Comparing persons without medical interventions, we found in antibiotic-naive children a significantly higher frequency of integron-bearing strains and MIC values than in healthy elderly persons (53% versus 17%; p < 0.01). Evaluating medical interventions, we found a higher resistance and frequency of integrons in strains from hospitalized elderly persons compared with non-hospitalized ones. Children treated with antibiotics had strains with higher MIC values (when compared with antibiotic-naive ones), but the integron-bearing in strains was similar. In most cases, the differences in resistance between the groups (integron-positive and negative strains separately) were higher than the differences between integron-positive and negative strains within the groups. CONCLUSION: The prevalence of integrons in commensal E. coli strains in persons without previous medical intervention depended on age. The resistance of integron-carrying and non-carrying strains is more dependent on influencing factors (hospitalization and antibiotic administration) in particular groups than merely the presence or absence of integrons.

Lactobacillus fermentum ME-3 - an antimicrobial and antioxidative probiotic.

The paper lays out the short scientific history and characteristics of the new probiotic Lactobacillus fermentum strain ME-3 DSM-14241, elaborated according to the regulations of WHO/FAO (2002). L. fermentum ME-3 is a unique strain of Lactobacillus species, having at the same time the antimicrobial and physiologically effective antioxidative properties and expressing health-promoting characteristics if consumed. Tartu University has patented this strain in Estonia (priority June 2001, patent in 2006), Russia (patent in 2006) and the USA (patent in 2007). The paper describes the process of the identification and molecular typing of this probiotic strain of human origin, its deposition in an international culture collection, and its safety assessment by laboratory tests and testing on experimental animals and volunteers. It has been established that L. fermentum strain ME-3 has double functional properties: antimicrobial activity against intestinal pathogens and high total antioxidative activity (TAA) and total antioxidative status (TAS) of intact cells and lysates, and it is characterized by a complete glutathione system: synthesis, uptake and redox turnover. The functional efficacy of the antimicrobial and antioxidative probiotic has been proven by the eradication of salmonellas and the reduction of liver and spleen granulomas in Salmonella Typhimurium-infected mice treated with the combination of ofloxacin and L. fermentum strain ME-3. Using capsules or foodstuffs enriched with L. fermentum ME-3, different clinical study designs (including double-blind, placebo-controlled, crossover studies) and different subjects (healthy volunteers, allergic patients and those recovering from a stroke), it has been shown that this probiotic increased the antioxidative activity of sera and improved the composition of the low-density lipid particles (LDL) and post-prandial lipids as well as oxidative stress status, thus demonstrating a remarkable anti-atherogenic effect. The elaboration of the probiotic L. fermentum strain ME-3 has drawn on wide international cooperative research and has taken more than 12 years altogether. The new ME-3 probiotic-containing products have been successfully marketed and sold in Baltic countries and Finland.

Eradication of Salmonella Typhimurium infection in a murine model of typhoid fever with the combination of probiotic Lactobacillus fermentum ME-3 and ofloxacin.

BACKGROUND: The aim of the study was to detect whether in experimental Salmonella enterica Typhimurium infection the probiotic Lactobacillus fermentum ME-3 in combination with fluoroquinolone therapy would eradicate S. Typhimurium, prevent the development of liver and spleen granulomas and improve the indices of oxidative stress in the ileum mucosa. The selected bacteriological, histological and biochemical methods were applied. RESULTS: Combined treatment with L. fermentum ME-3 and ofloxacin eradicated Salmonella Typhimurium from blood, ileum and liver, decreased the number of animals with liver and spleen granulomas and reduced the value of lipid peroxides in the ileum mucosa. Higher total counts of intestinal lactobacilli in all experimental groups were associated with the absence of liver granulomas. CONCLUSION: The antimicrobial and antioxidative probiotic L. fermentum ME-3 combined with ofloxacin enhances the eradication of experimental S. Typhimurium infection. These observations on probiotic and antimicrobial co-action may serve as basis to develop new strategies for treatment of invasive bacterial infections of the gut.

Molecularly assessed shifts of Bifidobacterium ssp. and less diverse microbial communities are characteristic of 5-year-old allergic children.

The composition of intestinal microbiota and the Bifidobacterium group community in 20 allergic and 20 nonallergic 5-year-old children was visualized by PCR-denaturing gradient gel electrophoresis (DGGE). The number of dominant bands in the DGGE profiles was smaller in allergic children than in nonallergic children (P<0.001). Allergic children mainly formed a single group upon cluster analysis, whereas nonallergic children were divided between four different groups. In allergic children the Bifidobacterium adolescentis species prevailed, and in nonallergic children the Bifidobacterium catenulatum/pseudocatenulatum prevailed (P=0.01 and P=0.01, respectively). The less diverse composition of intestinal microbiota and prevalence of particular species of Bifidobacterium were characteristic of allergic children even at the age of 5 years.

Evaluation of the functional efficacy of an antioxidative probiotic in healthy volunteers.

BACKGROUND: In persons without clinical symptom it is difficult to assess an impact of probiotics regarding its effect on health. We evaluated the functional efficacy of the probiotic Lactobacillus fermentum ME-3 in healthy volunteers by measuring the influence of two different formulations on intestinal lactoflora, fecal recovery of the probiotic strain and oxidative stress markers of blood and urine after 3 weeks consumption. METHODS: Two 3-week healthy volunteer trials were performed. Open placebo controlled (OPC) study participants (n = 21) consumed either goat milk or by L. fermentum ME-3 fermented goat milk (daily dose 11.8 log CFU (Colony Forming Units). Double blind randomised placebo controlled (DBRP) study participants (n = 24) received either capsules with L. fermentum ME-3 (daily of dose 9.2 CFU) or placebo capsules. The faecal lactoflora composition, faecal ME-3 recovery, effect of the consumption on intestinal lactoflora, and oxidative stress markers of blood (total antioxidative activity; total antioxidative status and glutathione red-ox ratio) was measured. RESULTS: ME-3 was well tolerated and a significant increase in total faecal lactobacilli yet no predominance of ME-3 was detected in all study groups. Faecal recovery of ME-3 was documented by molecular methods only in fermented milk group, however the significant improvement of blood TAA (Total Antioxidative Activity) and TAS (Total Antioxidative Status) indices was seen both in case of fermented goat milk and capsules", yet glutathione re-ox ratio values decreased only in case of fermented by ME-3 goat milk. CONCLUSION: The functional efficacy of both consumed formulations of an antioxidative probiotic L. fermentum ME-3 is proved by the increase of the intestinal lactobacilli counts providing putative defence against enteric infections and by reduction of the oxidative stress indices of blood and urine of healthy volunteers. In non-diseased host the probiotic health claims can be assessed by improvement of some measurable laboratory indices of well-established physiological functions of host, e.g. markers of antioxidative defence system.

Selection of indicators for tonsillectomy in adults with recurrent tonsillitis.

BACKGROUND: We aimed to find some new indicators for tonsillectomy (TE) in adults with recurrent tonsillitis (RT) by exploring whether the frequency of tonsillitis episodes and the length of morbidity period are associated with the macroscopic signs of sclerotic process in tonsils and microbiological data assessed by culture, molecular (PCR) and transmission electron microscopy (EM) methods. METHODS: The study involved 62 RT patients admitted for TE (age range 15-35, median 22 years) and 54 healthy volunteers (age range 18-24, median 20 years). The index of tonsillitis (IT) was calculated by multiplying the number of tonsillitis episodes per year by the morbidity period in years. On oropharyngeal examination the presence or absence of three sclerotic signs was evaluated: tonsillar sclerosis, obstruction of tonsillar crypts and scar tissue on the tonsils. The occurrence of Streptococcus pyogenes was assessed by culture and PCR methods in 24 tonsillar core specimens. The samples for EM investigation of crypt epithelium were taken from 10 removed tonsils. RESULTS: The IT values were in positive correlation with the number of sclerotic signs on oropharyngeal examination (r = 0.325, P = 0.010). Based on the IT values and the presence or absence of tonsillar sclerosis and obstruction of tonsillar crypts the receiver-operating curve (ROC) was constructed. It revealed that an IT score of 36 is an optimal cut-off value for prediction of sclerotic type tonsils. S. pyogenes was never found by culture, but its presence by PCR in nearly one third (29%) of diseased tonsillar tissue specimens was tightly associated with longer morbidity. EM revealed coccoid forms of intracellular bacteria in the crypt epithelium, which was accompanied with the damage of tight junctions between epithelial cells. CONCLUSION: The index of tonsillitis > or = 36, being a combination between the frequency of tonsillitis and the length of morbidity period, predicts the sclerotic process in recurrently inflamed tonsils. Therefore, the high IT values could serve as an indicator for TE in adults. The correlation between the longer morbidity period and the presence of S. pyogenes by PCR suggests that persistent infection may have a role in maintenance of recurrent inflammation in tonsils.

New insights into the impact of Lactobacillus population on host-bacteria metabolic interplay.

We aimed at evaluating the association between intestinal Lactobacillus sp. composition and their metabolic activity with the host metabolism in adult and elderly individuals. Faecal and plasma metabolites were measured and correlated to the Lactobacillus species distribution in healthy Estonian cohorts of adult (n = 16; < 48 y) and elderly (n = 33; > 65 y). Total cholesterol, LDL, C-reactive protein and glycated hemoglobin were statistically higher in elderly, while platelets, white blood cells and urinary creatinine were higher in adults. Aging was associated with the presence of L. paracasei and L. plantarum and the absence of L. salivarius and L. helveticus. High levels of intestinal Lactobacillus sp. were positively associated with increased concentrations of faecal short chain fatty acids, lactate and essential amino acids. In adults, high red blood cell distribution width was positively associated with presence of L. helveticus and absence of L. ruminis. L. helveticus was correlated to lactate and butyrate in faecal waters. This indicates a strong relationship between the composition of the gut Lactobacillus sp. and host metabolism. Our results confirm that aging is associated with modulations of blood biomarkers and intestinal Lactobacillus species composition. We identified specific Lactobacillus contributions to gut metabolic environment and related those to blood biomarkers. Such associations may prove useful to decipher the biological mechanisms underlying host-gut microbial metabolic interactions in an ageing population.