Valentína Focková

Antibiotic-resistant, biofilm-forming Escherichia coli may constitute a reservoir of antibiotic resistance and other determinants that can be transmitted to pathogenic bacteria for animals and humans. Therefore, it is crucial to reduce the incidence of these types of E. coli. The aim of this study was to determine whether essential oils from oregano, thyme, sage, and coriander, as well as the postbiotic substance PS412, can inhibit virulence factor genes possessing pig-derived E. coli. It aimed to find a new tool for the prevention and/or elimination of virulent E. coli. Altogether, 16 pig-derived E. coli from a pig farm in the region of Vojvodina (Serbia) were taxonomically identified using MALDI-TOF mass spectrometry; 14 strains (87.5%) with secure genus identification/probable species identification and 2 with highly probable genus identification. The fimA gene was detected in 62.5% of E. coli strains, and the crl gene in 87.5% of the strains. Ec3419/2 contained five analyzed genes. Five E. coli were found to form biofilm, as indicated by their growth on Congo red agar. The strains were mostly multi-resistant to antibiotics. Each E. coli strain produced the damaging enzyme, such as β-glucuronidase and/or α-chymotrypsin. However, they were susceptible to herbal essential oils (HEOs) with average inhibitory zones from 15 to 27 mm in diameter. They were also (6) susceptible to the PS412 (activity to 6400 AU/mL). The results contribute to the practical effectiveness of postbiotic substances, HEOs, and their combination as a novel approach to combating the virulence factors of E. coli. This insight also contributes to the strategy behind the One Health Concept.

Altogether 16 pigs-derived Escherichia coli (isolated pig farms in Vojvodina region, Serbia) were taxonomically identified using MALDI-TOF mass spectrometry; 14 (87.5%) with secure genus identification/probable species identification and two with highly probable genus identification. Testing virulence factor genes, the gene fimA was detected in 62.5% of strains. Crl gene was detected in 14 strains (87.5 %). Ec3419/2 contained 5 analyzed genes. Using Congo red agar, five E. coli were biofilm-forming which was not confirmed using the quantitative plate assay. The strains were mostly multiresistnt to antibiotics. Each E. coli was found with production of non-useful enzymes in values from 5 up to 30 nmol. However, they were susceptible to herbal essential oils (HEO-oregano, thyme, sage, and coriander) with average inhibitory zone from 15 up to 27 mm in diameter. Tey were also (6) susceptible to postbiotic substance (Ent) 412 (activity up to 6400 AU/ml. Postbiotic substances represent one of novel approaches to fight with virulence factor possessing E. coli. Moreover, their combination with HEO increases their antimicrobial effect. These treatment conditions correspond with One Health Concept.

Background: Using bacterial (probiotic) strains can influence beneficial health statuses, e.g., through dairy products; however, they must be assessed as safe before consumption; broiler rabbits represent a suitable model for this purpose. This study evaluated the safety and effect (functionality) of the dairy-derived, plantaricin-producing strain Lactiplantibacillus plantarum LP17L/1 using broiler rabbits. The following parameters were assessed to evaluate safety and functionality: microbiota, phagocytic activity (non-specific immunity parameter), blood serum biochemistry, oxidative stress enzyme, growth performance, cecal hydrolytic activity, and jejunal morphometry. Methods: Previously validated methods were used for the analyses. The appropriate administrations approved the experiments. Results: Although only up to 1.0 colony forming unit (CFU)/g (log10) LP17L/1 reached the gastrointestinal tract of the rabbits, the total lactic acid bacteria and amylolytic streptococci were significantly increased (p < 0.001). The other microbiota were not influenced, meaning there was no negative influence on phagocytic activity, growth parameters, and biochemistry in the analyzed blood serum. Conversely, lower GPx values were measured in the experimental group than in the control group, meaning LP17L/1 did not induce oxidative stress. LP17L/1 caused an increase in villi length to crypt depth ratio, while hydrolytic activity was also significantly increased (p < 0.01, p < 0.001). Conclusions: L. plantarum LP17L/1 was confirmed as safe. Regarding the functionality, no negative influence on the microbiota was noted, and the tested parameters were not negatively influenced. Demonstrating that the LP17L/1 strain is safe provides further chances for its industrial application. Additional studies are being conducted.

The increasing frequency of methicillin-resistant (MR) staphylococci in humans and animals need special attention for their difficult treatment and zoonotic character, therefore novel antimicrobial compounds on a natural base against antibiotic-resistant bacteria are requested. Currently, bacteriocins/enterocins present a new promising way to overcome this problem, both in prevention and treatment. Therefore, the preventive and medicinal effect of dipeptide enterocin EntA/P was evaluated against MR Staphylococcus epidermidis SEP3/Tr2a strain in a rabbit model, testing their influence on growth performance, glutathione-peroxidase (GPx) enzyme activity, phagocytic activity (PA), secretory (s)IgA, and jejunal morphometry (JM). Eighty-eight rabbits (aged 35 days, meat line M91, both sexes) were divided into experimental groups S (SEP3/Tr2a strain; 1.0 × 10⁵ CFU/mL; dose 500µL/animal/day for 7 days, between days 14 and 21 to simulate the pathogen attack), E (EntA/P; 50 µL/animal/day, 25,600 AU/mL in two intervals, for preventive effect between days 0 and 14; for medicinal effect between days 28 and 42), E + S (EntA/P + SEP3/Tr2a; preventive effect; SEP3/Tr2a + EntA/P; medicinal effect) and control group (C; without additives). Higher body weight was recorded in all experimental groups (p < 0.001) compared to control data. The negative influence/attack of the SEP3Tra2 strain on the intestinal immunity and environment was reflected as decreased GPx activity, worse JM parameters and higher sIgA concentration in infected rabbits. These results suggest the promising preventive use of EntA/P to improve the immunity and growth of rabbits, as well as its therapeutic potential and protective role against staphylococcal infections in rabbit breeding.

Goat milk is gradually becoming the preferred milk by consumers worldwide, including Slovakia. It is also demanded as a functional and/or nutraceutical drink as it is rich in bioactive components. However, another new development is the research into the beneficial autochthonous strains used to enrich goat dairy products. Among individual species, representatives in raw goat milk are also involved in lacticaseibacilli. Bacteriocin-like inhibitory substance-producing Lacticaseibacillus paracasei LPa 12/1 was isolated from raw goat milk. This study focuses on its potential as a new additive in dairy products. No mortality was found when checking the safety of the LPa 12/1 strain using Balb/c mice. The strain reached higher counts in ewe–goat milk yoghurt (up to 6.1 cfu/g log 10) than in cow milk yoghurt (almost 5.0 cfu/g log 10). Cow milk yoghurts remained in more stable consistency after LPa 12/1 supplementation compared with ewe–goat milk yoghurts, although specific organoleptic tests were not performed. However, LPa 12/1 has survived sufficiently in both types of yoghurts. This bacteriocin-like inhibitory substance-producing strain LPa 12/1 in its encapsulated form applied in yoghurts seems suitable to supplement dairy products.

Goat milk starts to be gradually the preferred milk by consumers worldwide and also in Slovakia. It is also demanded as functional and/or nutraceutical drink as it is rich in bioactive components. Goat milk contains complex of microbiota among which the phylum Firmicutes have been occurred with abundance 20.5 %. Among individual representatives of this phylum are involved also lacticaseibacilli. This study has been focused on bacteriocin-producing, beneficial strain Lacticaseibacillus paracasei LPa 12/1 isolated from raw goat milk and its potential to be additive in dairy products. Based on safety testing of bacteriocin-producing LPa 12/1 strain using BALB/c mice, it looks as safe. Even increase tendency in phagocytic activity in blood of mice was noted after LPa 12/1 strain application. Encapsulated form of LPa 12/1 strain producing thermo-stable bacteriocin seems to be a suitable to supplement dairy products. The strain colonized better ewe-goat milk yoghurt (up to 6.1 cfu/g log 10) than cow milk yoghurt (almost 5.0 cfu/g log 10). Although specific organoleptic tests were not involved, cow milk yoghurts remained in better consistency after LPa 12/1 strain supplementation in comparison with ewe-goat milk yoghurts. LPa 12/1 strain may be supposed as a new potential additive in dairy products/yoghurts.

The present study investigated the effect of the dipeptide enterocin (Ent) A/P on growth, immune response, and intestinal microbiota in rabbits. Eighty-eight rabbits (aged five weeks, M91 meat line, both sexes) were divided into three experimental groups: E (Ent A/P; 50 µL/animal/day for 14 days; between 0–14 days); S (methicillin-resistant Staphylococcus epidermidis SE P3/Tr2a strain; 500 µL/animal/day for 7 days starting at day 14 to day 21); and E + S (Ent A/P between 0–14 days and SE P3/Tr2a strain between 14–21 days) groups, and the control group (C). The additives were administered in drinking water. Administration of Ent A/P lead to an increase in weight gain, reduction of feed conversion; phagocytic activity was stimulated and gut microbiota were optimized due to reduction of coliforms, total bacterial count, and methicillin-resistant staphylococci. Good health and increased weight gain also showed that methicillin-resistant S. epidermidis SE P3/Tr2a strain did not have any pathogenic effect on rabbits’ health status.

Bacteriocin-producing bacteria with probiotic character are known as nutritional supplements mainly for livestock. Among those beneficial bacteria we also found enterococci. Because the species strains Enterococcus mundtii also can produce bacteriocins, this study was focused on fecal strains E. mundtii from horses and their bioactivity with a view to their possible future use in breeding. Rectal removal from 47 horses (40 mares and 7 stallions), the Norik breed from Muráň were sampled in eastern Slovakia during November 2019 year. Horses age ranged from five months up to 23 years. Using MALDI-TOF mass spectrometry and 16S rRNA sequences analysis, 14 strains were allotted to the species E. mundtii. Bacteriocin substances produced by the strains EMKD 38/1, EMKD 40/2, EMKD 34/2 and EMKD 41/3 showed inhibitory activity against the most susceptible (principal) indicator strain Enterococcus avium EA5 and against listeriae as well (inhibitory activity from 100 up to 1 600 AU/mL). Only strain EMKD 41/3 possess Ent P and Mundticin KS genes and showed the broadest inhibitory activity. Ent B gene possessing strain EMKD 24/1 inhibited a growth of only indicator strain EA5. Identified E. mundtii tolerate low pH 3 and oxgall/bile. They were hemolysis, gelatinase and DNase negative and mostly susceptible to clinical antibiotics which are properties requested for application potential of strain. Substance from the strain with the broadest antimicrobial spectrum showed its practical/application potential, e.g. for optimizing the host microbiota which is important regarding the maintenance of animal`s health status.

Background: Goat milk and products from it are popular with the public in Slovakia. Consumers demand safe food. Therefore, methods to eliminate spoilage bacteria are necessary. This study focuses on susceptibility to lantibiotic bacteriocins of variable staphylococci, which were isolated from raw goat milk. A total of 53 raw goat milk samples collected from 283 goats in the regions of central and eastern Slovakia were analyzed.Results: In total, 37 strains were identified to use a combination of matrix‐assisted laser desorption ionization time‐of‐flight spectrometry and phenotypization. They were allotted to 14 species, which can be involved in 7 different clusters. Staphylococci showed mostly low‐grade biofilm formation ability; six different strains were high‐grade biofilm‐forming. Staphylococcus xylosus SX 17/1 showed the highest biofilm formation ability value (3.16 ± 1.77). Staphylococci were deoxyribonuclease and mostly hemolysis negative (γ‐hemolysis). They all were susceptible to bacteriocins (inhibition activity up to 204,800 AU/ml).Conclusions: Variable staphylococcal species were detected in raw goat milk, mostly coagulase‐negative species, involving Staphylococcus arlettae. The species Staphylococcus hominis dominated. However, also the coagulase‐positive species Staphylococcus delphini and Staphylococcus schleiferi were detected. This is the first study involving detailed testing of staphylococci from Slovak raw goat milk and indication of a promising practical tool and prophylaxis using bacteriocins.