Vladimír Kmeť

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.

Aggregation of Lactobacilli and Bifidobacteria with Escherichia coli O157.

A total of 5Bifidobacterium spp. isolated from pig and children’ feces and 6Lactobacillus spp. from chicken feces were examined for expression of aggregation, cell surface hydrophobicity (CSH) and adherence to intestinal mucin. Co-aggregation activity was seen in 3 strains of auto-aggregative bifidobacteria and 4 auto-aggregative strains ofLactobacillus spp. with 2 enterohemorrhagicE. coli (O157). CSH correlated withLactobacillus auto-aggregating activity and adherence to mucin but the correlation betweenBifidobacterium adherence to mucin and CSH was not confirmed.

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.

Antimicrobial susceptibility of microflora from ovine cheese.

Strains identified in ovine cheese and bryndza by matrix-assisted laser desorption/ionization time-of-flight analysis belonged to ten species of non-enterococcal lactic acid bacteria and included Lactobacillus casei/Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici. The susceptibility toward antibiotics was determined in lactobacilli, lactococci and pediococci and also in Escherichia coli for comparison. Analysis of L. fermentum and pediococci revealed the presence of non-wild-type epidemiological cut-offs in streptomycin, clindamycin or gentamicin. E. coli were resistant to ampicillin, tetracycline, enrofloxacin and florfenicol. No extended spectrum β-lactamases were detected.

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.

Bifidobacterium animalis subsp. lactis strains isolated from dog faeces

The aim of the study was to identify and characterize dog bifidobacterial isolates and compare them with commercial probiotic strains. Sixteen isolates of Bifidobacterium animalis ssp. lactis from dog faeces (German Shepherd Dog) were identified by subspecies-specific PCR, MALDI-TOF MS and sequencing. This study is the first describing B. animalis ssp. lactis occurring within the intestinal tract of dogs. Our dog isolates showed slightly different fingerprinting profiles obtained by RAPD-PCR and REP-PCR from those isolated from yogurt and type strains of B. animalis ssp. lactis. Both, dog and yogurt origin strains indicated survival in the simulated in vitro digestion assay and were resistant to low pH and bile salts. Moreover, strong auto-aggregation activity was observed only in dog origin B. animalis ssp. lactis strains. Dog strains showed good properties predicting their survival ability in GIT and could be tested as a potential new probiotics for dogs or other hosts.

Antimicrobial resistance and species identification of staphylococci isolated from the meat of wild rabbits (Oryctolagus cuniculus) in Slovakia

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%).

Species Identification and Antimicrobial Resistance of Coagulase‐Negative Staphylococci Isolated from the Meat of Sea Fish

UNLABELLED Seventy-eight isolates of staphylococci obtained from the meat of Theragra chalcogramma, Scomber scombrus, and Clupea harengus were identified and tested in this study. 16S rDNA sequence specific for the genus Staphylococcus was detected in all isolates with the help of PCR method. All of 78 isolates were coagulase-negative, and DNAse activity was only confirmed in 4 of them. The following species of staphylococci were identified using MALDI-TOF mass spectrometry: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Antimicrobial resistance to 7 antibiotics was determined in each isolate with the help of agar dilution method. In general, resistance against ampicillin was observed in majority of isolates (87%). On the contrary, the best sensitivity of CoNS was determined to gentamicin (96%). Only 1 S. warneri strain showed resistance to cefoxitin. Furthermore, 83% of staphylococcal isolates were simultaneously resistant to 2 or more antibiotics. PRACTICAL APPLICATION This study confirmed the need of monitoring antimicrobial resistance in coagulase-negative staphylococci not only in the meat of slaughter animals but also in retail marine fish. The results showed that MALDI-TOF mass spectrometry is useful, accurate, and rapid method for species identification of food pathogens including Staphylococcus spp.