Dobroslava Bujňáková

Developmental Changes of Gut Microflora and Enzyme Activity in Rat Pups Exposed to Fat-rich Diet

The aim of this study was to investigate the effect of a high‐fat (HF)/energy diet on the intestinal microbiota, the alkaline phosphatase (AP) activity, and related parameters of growth and obesity during the suckling and weaning periods in male Sprague‐Dawley rats. From birth, nutrition in suckling pups was manipulated by feeding rat dams either HF or a standard diet, and then after weaning, by exposure of experimental pups to the HF, and control rats to normal diet. On days 15, 20, 40 the numbers of 2 microbial groups, i.e., Bacteroides/Prevotella (BAC) and the Lactobacillus/Enterococcus (LAB) in the jejunum, were determined by fluorescent in situ hybridization technique, and the AP activity was assayed histochemically. During all investigated periods HF pups gained body fat more rapidly than control animals, but from weaning they displayed significantly stunted growth resulting in final body weight loss. Obesity in HF rats was also accompanied by higher LAB and lower numbers of BAC and with permanently higher AP activity. Correlation of these data showed significant negative correlation between LAB, AP, and weight gain and energy efficiency, and significant positive correlation of BAC and AP activity with body fat. These data support the concept that postnatal nutritional experience represents an important factor affecting the ontogeny of intestinal microbial communities and intestinal function. These acquired changes could be a component of regulatory mechanisms involved in adverse and/or positive consequences of HF diet for adiposity, body weight, and energy‐balance control in later life.

Effect of oregano essential oil on chicken lactobacilli andE. coli

The effect of the oregano essential oil (EO) on lactobacilli andE. coli isolated from chicken was showed in the series ofin vitro andin vivo experiments. Minimum inhibitory concentrations of amikacin, apramycin, streptomycin, neomycin againstE. coli strains increased after oral application of EO.

Intestinal microflora and obesity in rats

The relationship was evaluated between early nutritional experiences, the intestinal microflora and the small intestinal functions in the mechanism of predisposition to obesity development. Male Sprague-Dawley rats were used in which the quantity of nutrition was manipulated from birth to weaning (day 30) by adjusting the number of pups in the nest to 4 small litters (SL) and 10 normal litters (NL) and fed a standard diet from days 30 to 40 of age. After 40 d, the postnatally overfed SL pups became heavier, displayed significantly enhanced adiposity, body mass gain and food intake as well as a significantly higher jejunal alkaline phosphatase and maltase activity than in rats nursed in NL nests. The effect of different early nutrition was also accompanied by the appearance of significantly decreased Bacteroides and significantly increased enterococci and lactobacilli of obese rats than in lean NL rats. The amounts of Bacteroides were negatively correlated with fat pad mass, body mass, body-mass gain and food intake whereas enterococci and lactobacilli were correlated positively with the same parameters. Our results demonstrate that postnatal nutritional experience may represent a predisposing factor influencing ontogeny of small intestine function and development of intestinal microbial communities. The acquired changes and associated alterations in food digestion could be a component of regulatory mechanisms contributing to the development of obesity and its maintenance in later life.

Development of gut microflora in obese and lean rats

The influence was evaluated of post-weaning normal nutrition and over-nutrition upon the development of the intestinal microbiota, the alkaline phosphatase activity (AP) and occurrence of obesity in male Sprague-Dawley rats (from days 21 to 40 the control rats were submitted to ad libitum intake of a standard laboratory diet whereas overfed rats received the same diet supplemented with milk-based high fat liquid diet). The jejunal numbers of two dominant divisions of bacteria, i.e. Firmicutes (Lactobacillus/ Enterococcus — LAB) and the Bacteroidetes (Bacteroides/Prevotella — BAC), were determined using the fluorescent in situ hybridization (FISH) method, and the jejunal AP activity was assayed histochemically. On day 40, the overfed rats in comparison with control animals displayed increased adiposity accompanied by enhanced AP activity, abundance of LAB, lower amounts of BAC and, thereafter, higher LAB/BAC ratio (L/B). The numbers of LAB and L/B index positively correlated with body fat, energy intake and AP activity, whereas numbers of BAC showed an opposite tendency. These results revealed the significance of nutritional imprint upon the post-weaning development of intestinal microbial and functional axis and contribute to better understanding of their involvement in energy-balance control and in adverse and/or positive regulation of adiposity.

Effect of lactobacilli onE. coli adhesion to caco-2 cellsin Vitro

Inhibitory effect of various lactobacilli against pathogenic strains ofE. coli in model system Caco2 cells was determined by enumerating the number of adheringE. coli after pre-incubation (exclusion), post-incubation (displacement) or co-incubation (competition) with lactobacilli. PorcineE. coli strain F107 (F18ab, Stx2v) in the competition assay with porcine lactobacillus strain P10 gave bacterial counts 7.25 (log CFU per well); in the exclusion test it was only 7.05 while in displacement test it reached 7.29. The lowestE. coli counts adhering to Caco-2 cells were in exclusion assay (pre-incubation,Lactobacillus inoculated as the first). Pre-treatment ofE. coli with our lactobacilli strains reduced the cultivableE. coli numbers.

Enumeration, isolation, and identification of Bifidobacteria from infant feces

Thirty-three fully breast-fed infants aged between 1 and 12 weeks were screened for bifidobacteria in feces. Bifidobacteria counts in most fecal samples determined both by TPY agar and FISH procedure ranged from 108 to 1011 CFU/g. Three infants did not contain any bifidobacteria in their fecal samples. One child was delivered by caesarean section and the other two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. In bifidobacteria-positive samples,B. longum (57.9 % of the samples) was the most frequently found species, followed byB. adolescentis (31.6 %),B. bifidum (21.0 %),B. breve (10.5 %),B. pseudocatenulatum (5.3 %), andB. dentium (5.3 %).

The anti-translocation and anti-inflammatory effect of cinnamon oil in mice with TNBS induced colitis

The bacterial translocation induced by colitis may cause the organ failure and sepsis. Therefore, it is necessary to find new possibilities for prevention and therapy of this problem. The purpose of this study was to examine Escherichia coli anti-translocation activity of cinnamon oil and its ability to reduce colonic damage in mice with TNBS (2,4,6-trinitrobenzenesulfonic acid) induced colitis. Mice received cinnamon essential oil in four various concentrations (0.5%, 0.25%, 0.125% and 0.063%) in the powdery commercial rodent diet, starting 21 days before induction of TNBS colitis. The colonic damage was assessed using the colon macroscopic scoring system (Wallace score). E. coli translocation to the mesenteric lymphatic nodules was evaluated by serial dilutions method for counting bacteria. Bacterial translocation was significantly reduced in first and third group (15.2% or 42.8% in cinnamon oil groups versus 100% in TNBS group). Cinnamon oil was effective also against the colonic damage in all cinnamon oil groups (macroscopically scores of grade 9 in TNBS group versus 5.25, 5.63, 5.13 and 3.25 in cinnamon oil groups). Our results confirmed that dietary administration of cinnamon oil could possess potential therapeutic effects on bacterial translocation and intestinal wall injury in colitis.

Inhibitory potential of lactobacilli against Escherichia coli internalization by HT 29 cells

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.

Characterisation of lactobacilli from ewe's and goat's milk for their further processing re-utilisation.

Raw ewes and goats milk is a good source for isolation of wild lactobacilli which are able to bring unique processing properties in development of dairy products - cheeses or fermented dairy products. 34 strains of lactobacilli were isolated, purified and identified from fermented ewes and goats dairy products. These products were processed without thermal treatment and without using of any commercial starters. After preliminary selection, the final collection of 5 strains was established. The strains were identified as: Lbc. plantarum (2), Lbc. paraplantarum (1), Lbc. paracasei (1) and Lbc. johnsonii (1). Except two strains, all were able to coagulate milk. After hydrolysis of lactose in milk, two strains were able to form sensorial attractive coagulate too. All of the strains were homofermentative, they produced lactic acid but they did not produce CO2. Their ability to produce diacetyl was low. They did not show strong proteolytic activity. All strains grew at 30 °C and 37 °C, however Lbc. johnsonii much slower at 30 °C than the others. Except Lbc. johnsonii , all strains tolerated 2% concentration of NaCl and even in presence of 5% concentration of NaCl their growth was inhibited only moderately. All of characterized strains can be provisionally used as starter or starter adjuncts in dairy technology, during production of cheeses or fermented milk products from pasteurised milk. These results will be used in further processing studies of isolated strains and will be supplemented with other properties e.g. safety, probiotic and antimicrobial properties.