Viola Strompfová

Occurrence of the structural enterocin A, P, B, L50B genes in enterococci of different origin.

Enterococci are well-known producers of antimicrobial peptides--bacteriocins (enterocins) and the number of characterized enterocins has been significantly increased. Recently, enterocins are of great interest for their potential as biopreservatives in food or feed while research on enterocins as alternative antimicrobials in humans and animals is only at the beginning. The present study provides a survey about the occurrence of enterocin structural genes A, P, B, L50B in a target of 427 strains of Enterococcus faecium (368) and Enterococcus faecalis (59) species from different sources (animal isolates, food and feed) performed by PCR method. Based on our results, 234 strains possessed one or more enterocin structural gene(s). The genes of enterocin P and enterocin A were the most frequently detected structural genes among the PCR positive strains (170 and 155 strains, respectively). Different frequency of the enterocin genes occurrence was detected in strains according to their origin; the strains from horses and silage showed the highest frequency of enterocin genes presence. All possible combinations of the tested genes occurred at least twice except the combination of the gene of enterocin B and L50B which possessed neither strain. The gene of enterocin A was exclusively detected among E. faecium strains, while the gene of enterocin P, B, L50B were detected in strains of both species E. faecium and E. faecalis. In conclusion, a high-frequency and variability of enterocin structural genes exists among enterococci of different origin what offers a big possibility to find effective bacteriocin-producing strains for their application in veterinary medicine.

Oral application ofEnterococcus faecium strain EE3 in healthy dogs

The ability of canine strainEnterococcus faecium EE3 to survive in healthy dogs and its effect on microbiological and biochemical parameters was determined. The strain was individually applied to 11 dogsper os at a dose of 109 CFU/mL (differed from 2 to 3 mL) for 1 week and persisted in feces for 3 months after cessation of its administration (reaching average concentration of 6.83±0.95 log CFU/g). Seven d after administration, a decrease in staphylococci and a significant decrease inPseudomonas-like bacteria was observed. On the other hand, concentration of lactic acid bacteria increased but the growth ofE. coli was not influenced. In the blood samples of dogs after 0–1 d (before application) and the blood samples 1 week after application, total lipids decreased in 8 dogs; the total protein also decreased. The levels of cholesterol were brought to the physiological level,i.e. in blood samples with low cholesterol values it increased to the physiological level and in those with high levels it decreased; cholesterol was not influenced in 3 dogs.

Characterization of Staphylococcus xylosus and Staphylococcus carnosus isolated from Slovak meat products

The aims of this study were to isolate, identify and characterize the population of coagulase-negative staphylococci in different types of Slovak traditional sausages and to determine the metabolic properties of selected Staphylococcus xylosus and S. carnosus strains for the selection of potential starter cultures to use in the processing of sausages. The strains were tested for lactic acid production, survival in the presence of bile and sensitivity to antibiotics. Bacteriocin production, adhesion ability as well as biogenic amine (BA) production by isolates were also analysed. Most of the isolates were identified as S. xylosus and S. carnosus. Lactic acid values ranged from 0.40 to 1.03mmol/l and strains survived in the presence of 1% bile. Most of the strains studied were sensitive to all antibiotics. Two strains, S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 inhibited Listeria innocua and Pseudomonas sp. S. xylosus strains did not produce any BA, while S. carnosus SO2/F/2/5 did. S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 appeared as the most adhesive strains. S. xylosus SO3/1M/1/2 with antimicrobial activity against Enterococcus avium EA5, L. innocua LMG13568 and Pseudomonas sp. SO1/1M/1/4, adhesion ability and free BA production could be used as starter culture in sausage manufacture.

Adhesion properties of enterococci to intestinal mucus of different hosts.

The adhesive capacity of selected enterococci to human, canine and porcine intestinal mucus was investigated in order to select for potential probiotic strains with good adhesive properties for human or animal use. The adhesion to the human intestinal mucus of the tested strains was found to range from log10 3.8 to log10 8.6 cfu per microtitre plate well. The highest adhesion to the human intestinal mucus was found among strains from horse faeces, dog faeces and dog feed. The adhesion to canine mucus was observed to range from log10 3.8 to log10 8.3 cfu/well, with the highest adhesive capacity among strains from dog faeces, horse faeces and dogs feed; on average log10 7.9, 7.3 and 7.0 cfu/well, respectively. Isolates from dogs did not bind at higher levels to canine mucus than to human mucus. A strong correlation was observed for the adhesion to human and canine intestinal mucus (p<0.0001) and also between porcine and canine or human mucus (p<0.05 for both). When comparing the adhesion ofEnterococcus faecium andE. faecalis, no statistical significant differences were observed for any of the tested mucus types. The testedEnterococcus strains were found to exhibit a strain dependent onin vitro adhesion to human, canine and porcine intestinal mucus and did not exhibit host specificity in their adhesion, though some sources appeared to contain more adhesive strains than others. To our knowledge, this is the first report on thein vivo adhesion to intestinal mucus of a large number of enterococci from different sources.

Lactobacilli and enterococci--potential probiotics for dogs.

Forty strains of enterococci and forty strains of lactobacilli isolated from feces of 10 healthy dogs were tested for the antimicrobial activity, tolerance to bile and adhesion activity. The total count of fecal enterococci reached 5.5 log CFU/g and of lactobacilli 7.6 log CFU/g. Screening for production of bacteriocin-like substances showed an to partly inhibit the growth ofEnterobacter sp. (hazy zones of inhibition). Ten strains ofEnterococcus sp. and nine strains ofLactobacillus sp. were found without any inhibitory activity against all indicators used. Seven enterococcal strains and six lactobacilli with the broadest antimicrobial spectrum were selected for further probiotic assays. In the presence of 1 % bile, the survival rate of selected enterococci (71.7–97.5 %) was higher than that of lactobacilli (66.7–75.4 %). The adhesion of strains to human intestinal mucus (5.1–8.2 % by enterococci, 2.7–8.3 % by lactobacilli) was found to be similar as adhesion to canine intestinal mucus (3.7–10.6 % by enterococci, 2.1–6.0 % by lactobacilli). Strain AD1, one lactobacillus isolate, reduced the higher level of serum cholesterol and alanine aminotransferase after oral administration to dogs suffering from diseases of the gastrointestinal tract.

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella+probiotic group (EFSE) received E. faecium EF55 (10(9) CFU - 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10(8) CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.

Potential of enterococci isolated from horses.

Faecal samples of 122 horses (from farms in Slovakia) were examined to select enterococci to study their probiotic potential for their further use as additives. Each gram of faeces contained 1.0-5.0 cfu (log 10) of enterococci. Of the 43 isolates, 25 (58.1%) were identified as Enterococcus faecium, 3 strains were (6.9%) Enterococcus mundtii and one strain was identified as E. faecalis. Fourteen isolates were not characterized further. A significant proportion of the isolates were resistant to kanamycin, vancomycin and gentamicin. Low urease activity of enterococci dominated. The values of lactic acid ranged from 0.98 to 1.91 mmol/L. Porcine fibronectectin and bovine lactoferrin were bound weakly by tested enterococci, while bovine fibrinogen was bound more strongly. Enterococci from horses did not bind bovine apotransferrin. The isolates adhered with the same ability to human as well as to canine mucus. At least one enterocin gene was detected among 16 analyzed isolates. Ent B gene was detected in all strains tested (16, 100%), followed by the genes ent A, ent P and ent L50B. Three suitable candidates-the strains of E. faecium EF 412, EF 462 and EF 491 were selected for further detail studies and possibilities to be used as additives.

Antimicrobial activity of Enterococcus faecium ef 55 against Salmonella enteritidis in chicks.

The protective effect of Enterococcus faecium EF 55 against Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was studied in 1-day-old chicks. The EF 55 strain (isolated and characterised by the authors earlier) was applied daily (1.10(9) CFU/0.2 ml PBS) for 7 days. Oral inoculation of the SE PT4 strain was performed on day 8 in a single dose of 5.10(8) CFU/0.2 ml PBS. The experiment lasted for 21 days. Samples were collected on day 1 of the experiment to verify the absence of Salmonella, on day 8 to check colonisation of EF 55 and immunological status in experimental birds, and on days 2, 4, 6, 8 and 14 after SE PT4 infection of chicks. Strain EF 55 sufficiently colonised the digestive tract of chicks after 7 days of application. The highest numbers of EF 55 in the faeces of chicks were observed before SE infection and persisted to day 6 post infection (p.i.) in both the EF and EF+SE groups. PCR confirmed the identity of the EF 55 strain. The counts of SE PT4 strain in faeces of the EF+SE group were significantly reduced in comparison to those in the SE group on days 2 and 14 p.i. (P < 0.01). The significant reduction of salmonellae in the caecum was recorded at the end of the experiment (day 14 p.i.) in the EF+SE group in comparison to the SE group (P < 0.01). At day 4 p.i., colonies of S. Enteritidis PT4 were found in the liver of chicks of the SE group in a higher concentration than in chicks of the EF+SE group (P < 0.001). Salmonellae were isolated from the liver until days 8 and 6 p.i. in the SE and EF+SE groups, respectively. The mean values of actual lymphocyte subpopulations in the blood and the relative percentage of caecal intraepithelial lymphocyte subpopulations (CD4, CD8, CD44, TCR, MHC II and IgM) were not influenced at a statistically significant level by the application of the EF 55 and/or the SE PT4 strain. The results demonstrate the antimicrobial effect of E. faecium EF 55 against S. Enteritidis PT4.

Probiotic Potential of Enterococci Isolated from Canine Feed

Enterococci isolated from 28 different commercially available feeds (10–1000 CFU/mL) were identified and their probiotic potential was determined. Species identification of 22 selected strains was performed by intergenic length-polymorphism analysis (tRNA-PCR); PCR products were analyzed using capillary electrophoresis. Six strains were allotted to the species Enterococcus faecium, four to E. faecalis, one to E. hirae; the remaining strains were not classed. The strains were sensitive to vancomycin, ampicillin, tetracycline and rifampicin. They were able to adhere to human as well as canine intestinal mucus. They produced lactic acid (0.99–1.04 mmol/L) and most of them were urease-positive with sufficient survival in 5 % Oxgall-bile. They did not show any inhibitory activity due to antimicrobial substances. Plasmid DNA was detected in 8 strains, the bands responding to small molecular size (10 kbp). Considering all probiotically important properties, E. faecium strain EE3 was suggested as potential feed additive.

Bacteriocin-producing strain of Enterococcus faecium EK 13 with probiotic character and its application in the digestive tract of rabbits

Enterococcus faecium EK 13 is a bacteriocin-enterocin A producing strain with probiotic properties. In this study its colonization, stability and effect on microflora in rabbits was studied as well as its influence on zootechnical parameters. Fifty rabbits of both sexes (HYPLUS, 30-day old; after weaning) were divided into control (CG) and experimental (EG) groups. They were fed a standard diet. Moreover, 25 rabbits in EG were fed daily (for 4 weeks) 15 g (separate doses ∼1.6 g) of lyophilized EK13 strain (rifampicin resistant variant — rifR; 109 cfu/g) dissolved in drinking water. After cessation of EK13 (rifR) strain application, the rabbits in both groups were fed a standard diet for the next 2 weeks. Sampling was performed in double on day 0 (at the beginning of experiment), weekly during EK13 (rifR) strain application as well as on week 1 and 2 after cessation of EK13 (rifR) strain application. The counts of EK13 (rifR) strain reached 7.1 ± 2.6 log10 cfu/g after 4 weeks and even on week 2 after its cessation the counts 5.6 ± 2.3 log10 cfu/g were determined. The total counts of enterococci in the rabbits were already increased in EG comparing with CG (p < 0.05); even 2 weeks after EK13 (rifR) strain cessation, their counts in EG were 7.2 ± 2.6 log10 cfu/g (p < 0.001). Enterococci in CG reached at the same time the value 3.7 ± 2.6 log10 cfu/g. The counts of E. coli were significantly reduced in EG during 4 weeks (p < 0.05, p < 0.001). Even 2 weeks after EK13 (rifR) strain cessation significant difference in E. coli counts between CG and EG was detected (p < 0.001). Enterobacteria in EG were significantly reduced (p < 0.001). Average daily gain in EG was 41.0 ± 3.83 in comparison to CG (40.6 ± 3.72); it means almost the same; although rabbits in EG showed higher feed intake per kg of gain than rabbits in CG. Preliminary results demonstrated that EK13 is a perspective probiotic candidate for rabbits.