The effect of enterocin A/P dipeptide on growth performance, glutathione-peroxidase activity, IgA secretion and jejunal morphology in rabbits after experimental methicillin-resistant Staphylococcus epidermidis P3Tr2a Infection.

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.

Microbiome Associated with Slovak Raw Goat Milk, Trace Minerals, and Vitamin E Content.

In Slovakia, goat milk production for direct consumption and cheese processing has attracted growing interest. However, there is a lack of information regarding the microbial consortium in Slovak raw goat milk analyzed by next-generation sequencing and trace elements and vitamin E as well. A randomly selected samples (G24-G50) of raw goat milk from different animals at farms in Slovakia were analyzed. The phylum Actinobacteria dominated (62.8%), followed by the phyla Firmicutes (20.5%), Proteobacteria (7.4%), and Bacteroidetes (6.4%). The family Microbacteriaceae was detected in the highest percentage (60.2%) followed by Staphylococcaceae, Bacteroidaceae, Streptococcaceae, Lactobacillaceae, Enterobacteriaceae, and others. Regarding the genera, the most prevalent was genus Curtobacterium (47.4%) followed by the genera such as Staphylococcus (8.3%) and Bifidobacterium (4%). The genera Streptococcus, Lactococcus, Enterococcus, Lactobacillus, and Lacticaseibacillus were evaluated in abundance percentage in range 1%-3.2%. The genus Veillonella reached abundance 3.2%. The genera Enterobacter, Pseudomonas (1.3% and 0.5%), and Bacteroides (6.4%) were evaluated in small percentage abundance too. Zinc was detected with the highest mean value (2.561 ± 0.6823 mg/L) in raw goat milk, followed by iron (1.383 ± 0.5087 mg/L). The mean value of copper and manganese was 0.1746 ± 0.0463 mg/L and 0.051 ± 0.0238 mg/L. The vitamin E reached the mean value 0.3783 ± 0.1976 mg/L. This study is an original contribution showing microbial consortium in raw goat milk from Slovak farms. It also contributes to trace elements and vitamin E status in raw goat milk showing it as a nutritionally healthy food.

Preventive Potential of Dipeptide Enterocin A/P on Rabbit Health and Its Effect on Growth, Microbiota, and Immune Response.

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 Strain Lactiplantibacillus plantarum LP17L/1 Isolated from Traditional Stored Ewe's Milk Cheese and Its Beneficial Potential.

Stored ewe's milk lump cheese is a local product that can be a source of autochthonous beneficial microbiota, especially lactic acid bacteria. The aim of this study was to show the antimicrobial potential of Lactiplantibacillus plantarum LP17L/1 isolated from stored ewe's milk lump cheese. Lpb. plantarum LP17L/1 is a non-hemolytic, non-biofilm-forming strain, susceptible to antibiotics. It contains genes for 10 bacteriocins-plantaricins and exerted active bacteriocin with in vitro anti-staphylococcal and anti-listerial effect. It does not produce damaging enzymes, but it produces ß-galactosidase. It also sufficiently survives in Balb/c mice without side effects which indicate its safety. Moreover, a reduction in coliforms in mice jejunum was noted. LP17L/1 is supposed to be a promising additive for Slovak local dairy products.

Susceptibility to Bacteriocins of Multiresistant, mecA Gene Possessing Staphylococcus pseudintermedius Strains from Lesions on Dogs in Vojvodina (Serbia).

Aims: Staphylococci are considered commensal organisms; however, they can also cause diseases in animals. The species Staphylococcus pseudintermedius was originally isolated from the nares and anal mucosa of healthy dogs; nowadays, the incidence of methicillin-resistant S. pseudintermedius strains in dogs has increased, and this species has also become the most common cause of canine skin lesions. S. pseudintermedius were tested for their susceptibility to bacteriocins to show the potential of bacteriocins to eliminate/reduce S. pseudintermedius as a causative agent. Material and Methods: In this study, S. pseudintermedius were isolated from skin lesions on dogs of various breeds and ages in the Vojvodina region (Serbia) in the framework of our international co-operation. Results: S. pseudintermedius strains were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the species allocation was confirmed by genotypization and corroborated using phenotyping tests. The strains were multiresistant, involving methicillin-resistance. In addition, the mecA gene was detected in S. pseudintermedius strains, confirming the presence of a genetic feature for methicillin resistance. S. pseudintermedius strains produced large amounts of the enzymes alkaline phosphatase and acid phosphatase; they were low-grade biofilm forming (0.1 ≤ A570 < 1.0), except S. pseudintermedius SPs1. All strains were susceptible to gallidermin (inhibition activity up to 204,800 arbitrary unit [AU]/mL); however, they were mostly resistant to enterocins. However, biofilm-forming S. pseudintermedius SPs1 was susceptible to enterocins (200 AU/mL). Conclusion: Establishing the susceptibility of multiresistant and mecA gene-possessing S. pseudintermedius strains to gallidermin is an innovative, original result; it provides a new opportunity for preventing/eliminating infection caused by those agents.

Antimicrobial Susceptibility to Natural Substances of Campylobacter jejuni and Campylobacter coli Isolated from Italian Poultry.

Campylobacter spp. isolated from poultry are a major public health concern. Antimicrobial resistance is increasing in general, which is associated with limited treatment of diseases in animal husbandry and in human as well. The use of natural substances (enterocins and phytoadditives) seems to be a promising alternative, either in the prevention or treatment of the gastrointestinal tract infections. A total 28 broiler carcasses from Italian poultry were sampled and 39 Campylobacter strains were isolated. Twenty-four isolates were identified as Campylobacter jejuni and 15 strains were identified as Campylobacter coli by polymerase chain reaction. All Campylobacter strains were determined to be resistant to nalidixic acid, ciprofloxacin, tetracycline (E-test), cefotaxim, and ampicillin using the disk diffusion method. Interestingly, the study revealed multidrug resistance of the Campylobacter strains; however, the strains were susceptible to natural substances, such as enterocins, oregano, and sage. Eighty-five percent of the Campylobacter strains were susceptible to at least one enterocins, and 77% of the strains were susceptible to enterocin (Ent) 9296, 69% were susceptible to Ent EM41, and 69% were susceptible to Ent A(P). Sage inhibited the growth of 38% of C. jejuni strains and 53% C. coli strains, whereas oregano inhibited the growth of 50% of the C. jejuni strains and up to 67% C. coli strains. Owing to the One health concept, application of enterocins and herbal extracts is a more ecological approach for preventing bacterial contamination during production and processing of poultry, which also leads to the protection of human health.

Effect of enterocin M and durancin ED26E/7 supplementation on blood parameters, immune response and jejunal morphometry in rabbits.

Natural feed additives application in rabbit nutrition can help to control and prevent digestive disturbances and improve gut health and immunity around the critical weaning period. While probiotics are frequently used in rabbits, in vivo administration of bacteriocins is often limited. Therefore, the present study evaluates the effect of enterocin EntM, durancin EntED26E/7 and their combination on serum biochemistry, phagocytic activity and jejunal morphometry of rabbits. Eighty rabbits (aged 35 days, meat line M91, both sexes) were divided into experimental groups E (EntM; dose 50 µl/animal/day, activity 25,600 AU/ml), D (EntED26E/7; 50 µl/animal/day, 12,800 AU/ml), E + D (50 µl EntM + 50 µl EntED26E/7 /animal/day) and control group (C). Additives were administrated in drinking water for 21 days. Both enterocins positively influenced tested serum parameters, with emphasis on durancin EntED26E/7 administration, alone and/or in combination with EntM. Increased total proteins (E, D: p < 0.001), urea (D: p < 0.001), albumin (D: p < 0.05) and triglycerids (E, D, E+D: p < 0.001) were found. Hypocholesterolaemic effect of both additives was recorded (p < 0.001), with the lowest HDL concentration in E + D. The most of tested hepatic enzymes were positively influenced by enterocins combination (E + D; p < 0.001). The lowest AST was noted in group D (p < 0.001). Mineral profile was also improved (p < 0.001), with the highest values in D. Oxidative stress, was not evoked during enterocins application. Both additives showed a tendency to improve phagocytic activity (prolonged effect of EntED26D/7; D, E+D: p < 0.05) and jejunal morphometry parameters (increased villus cut surface; E, D, E+D; p < 0.001). Diet supplementation with EntM and mostly with EntED26E/7 can improve serum biochemistry, phagocytic activity and jejunal morphometry.

Can Enterocin M in Combination with Sage Extract Have Beneficial Effect on Microbiota, Blood Biochemistry, Phagocytic Activity and Jejunal Morphometry in Broiler Rabbits?

The present study investigated the effects of enterocin (Ent) M and sage extract alone and also in combination on the gut microflora, phagocytic activity, blood biochemistry, and morphometry of rabbits. Sixty-four rabbits (aged five weeks, M91 meatline, both sexes) were divided into three experimental groups: E (EntM; 50 µL/animal/day), S (sage; 10 µL/animal/day), and E + S (EntM + sage) groups and control group (C). The additives were administered in drinking water for a period of 21 days. Dietary supplementation of EntM and sage significantly reduced the coliforms (E: p < 0.001; S: p < 0.001; E + S: p < 0.001) in feces, while simultaneous addition of EntM and sage decreased enterococci (E + S: p < 0.0001), lactic acid bacteria (E + S: p < 0.01), and coagulase-positive staphylococci (E + S: p < 0.0001) in the appendix. Sage addition reduced HDL (S: p < 0.001) and LDL cholesterol (S: p < 0.001; E + S: p < 0.001), LDL/HDL ratio (S: p < 0.001; E + S: p < 0.01), and increased urea (S: p < 0.01; E + S: p < 0.001) and creatinine (S: p < 0.001; E + S: p < 0.001) in serum. EntM and sage application, alone or in combination, improve the jejunal morphometry (p < 0.0001) in rabbits.

Virulence Factor Genes Incidence among Enterococci from Sewage Sludge in Eastern Slovakia following Safety Aspect.

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylum Firmicutes. In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the species Enterococcus faecium with highly probable species identification and E. faecalis EEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesin E. faecium (efaAfm, in 22 E. faecium strains and in one E. faecalis). Adhesin efaAfs gene was detected in E. faecalis EEV20 and in two E. faecium. GelE gene was present in three strains. E. faecium EF/EC31 was absent of virulence factor genes.

Oral administration of bacteriocin-producing and non-producing strains of Enterococcus faecium in dogs.

The current effort to incorporate microbial cultures in canine nutrition and thus intake of them on daily base increases our interest in careful and more complex study of their effects in dogs. Many of the commercially used strains have not been tested in dogs and are incorporated only on the base of beneficial effects observed in humans with specific disorders. Moreover, no information on the effects of bacteriocin-producing strains in dogs is available. Therefore, we decided to test and to compare overall effect of bacteriocin non-producing Enterococcus faecium DSM 32820 and enterocin B-producing E. faecium LMG 30881 strain (both of canine origin). Dogs were divided into three treatment groups of ten dogs each: control; DSM 32820 group; and LMG 30881 group, dosing 109 CFU/day/dog. The experiment lasted 35 days with a 14-day treatment period (sample collection at days 0, 7, 14, 35). Despite bacteriocin production is believed that may provide a competitive advantage over neighbouring sensitive strains within shared environment, results indicated somewhat better survival for the DSM 32820 compared to the LMG 30881 group. Furthermore, dogs of DSM 32820 group had optimal faecal consistency throughout the experiment, significantly stimulated phagocytic activity (days 7 and 14) and metabolic burst activity of leukocytes (days 14 and 35) and lower serum glucose concentration (day 35). In contrast, dogs of LMG 30881 group showed higher faecal count of Gram-negative bacteria (day 35), lower haemoglobin and glucose concentration (day 35), and higher metabolic burst activity (days 14 and 35). These results are further evidence of the existence of inter-strain differences in efficacy despite the same origin.

Sensitivity to Enterocins of Biogenic Amine-Producing Faecal Enterococci from Ostriches and Pheasants.

Enterococci are widespread bacteria forming the third largest genus among lactic acid bacteria. Some possess probiotic properties or they can produce beneficial proteinaceous antimicrobial substances called enterocins. On the other hand, some enterococci produce biogenic amines (BAs), so this study is focused on the sensitivity to enterocins of biogenic amine-producing faecal enterococci from ostriches and pheasants. Altogether, 60 enterococci isolated from faeces of ostriches and pheasants were tested for production of BAs. This target of the identified enterococci involved 46 strains selected from 140 ostriches and 17 from 60 pheasants involving the species Enterococcus hirae, E. faecium, E. faecalis, and E. mundtii. Although BAs histamine, cadaverine, putrescine, and tryptamine were not detected in the enterococci tested, in general high BA production by the tested enterococci was noted. The species E. hirae formed the majority of the enterococcal strains from ostrichs faeces (34 strains). High production of tyramine (TYM) was measured with an average amount of 958.16 ± 28.18 mg/ml. Among the enterococci from pheasants, the highest was production of TYM compared to phenylethylamine, spermidine, and spermine. Enterococci featured high BA production; however, they were sensitive to seven enterocins with inhibition activity ranging from 100 up to 25,600 AU/ml.

Effect of lantibiotic gallidermin against biogenic amine-producing faecal staphylococci from ostriches and pheasants.

In ostriches and pheasants, there is still limited information relating to staphylococci and their properties. Biogenic amines (BAs) are nitrogenous low-molecular-weight substances with biological functions in animals, plants and microorganisms. In this study, we focused on BA production by targeted faecal staphylococci from ostriches and pheasants and their sensitivity to lantibiotic bacteriocin gallidermin. Gallidermin belongs in a group of polycyclic proteinaceous antimicrobial substances. Thirty-six faecal staphylococci (24 strains from 140 ostriches, 12 from 60 pheasants) comprising different species were tested. Staphylococci from ostriches and pheasants did not produce tryptamine-TRYP, putrescine-PUT, cadaverine-CAD or histamine-HIS. Production of tyramine-TYM, phenylethylamine-PEA was high or very high (100-1000 mg/L). Production of spermine-SPM and spermidine-SPD by staphylococci was very low or low although in the case of staphylococci from pheasants medium production of SPM was found. Because of the risk posed by BAs for consumers, the control of BA-producing bacteria is important from the points of view not only of safety assessment of food-producing animals but also of human health safety. The sensitivity to gallidermin in biogenic amine-producing staphylococci from ostriches and pheasants detected here is the most promising indication for further application of gallidermin for veterinary purposes. The novelty of our study lies in testing the ability of faecal staphylococci from ostriches and pheasants to produce BAs and in their treatment with gallidermin which has so far not been tested in this way.

Sensitivity to Enterocins of Thermophilic Campylobacter spp. from Different Poultry Species.

Campylobacter spp. have been isolated from various animals, including poultry. They are rapidly transmitted throughout broiler sheds by the fecal-oral route. A promising strategy to reduce Campylobacter spp. in poultry may be done due to the beneficial properties of probiotic bacteria and their bacteriocins. In this study, inhibition spectrum/activity of different enterocins was evaluated against Campylobacter spp. (isolated from different poultry) to indicate further practical use of enterocins. Enterocins are antimicrobial proteinaceous substances produced mostly by enterococci. Feces from broiler chickens (10), laying hens (47), ostriches (140), and ducks (40) were screened. Altogether, 23 strains were allotted to the species Campylobacter jejuni and Campylobacter coli using MALDI TOF mass spectrometry and confirmed by genotyping (PCR method). In the feces of ostriches, Campylobacter spp. were not confirmed. Campylobacter spp. isolated from different poultry showed resistance to nalidixic acid, ciprofloxacin, and ampicillin. Interestingly, strains demonstrating antibiotic resistance revealed sensitivity to at least one of the nine enterocins used (except C. coli Kc1, SZ3, and C. jejuni 1/D). Almost 52% strains were inhibited by Ent A (P). Enterocins can therefore be used to prevent or reduce Campylobacter spp.; it is a basis for practical use.

Staphylococci Related to Farm Ostriches and Their Sensitivity to Enterocins.

In Slovakia, ostriches are reared mainly for their meat. There is still limited information related to microflora of ostriches, including staphylococci. Knowing the composition of microflora is very important for the recognition of potential pathogenic agents. Recently, a frequent problem in animals is the occurrence of bacteria resistant to antibiotics. The aim of this study was to detect staphylococcal species in feces of farm ostriches and to test their sensitivity to antibiotics and enterocins. Altogether 140 ostriches from three age groups were sampled (n = 18, faecal mixture samples from each group) on a farm in Slovakia or on Slovak farm. From 54 fecal samples, the staphylococcal count reached an average 4. 3 ± 0. 63 (log10) CFU/g. Twenty-four lactic acid producing strains were taxonomically classified to eight species of the genus Staphylococcus: Staphylococcus equorum, S. xylosus, S. epidermidis, S. haemolyticus, S. cohnii, S. succinus, S. warneri, and S. hominis. Strains were evaluated by secure probable species identification/probable species identification (score value up to 2.299) confirmed also by phenotypization. Most strains were sensitive to antibiotics. Four strains (S. haemolyticus SHae 111, S. haemolyticus SHAe 371, S. xylosus SX 2133, and S. warneri SW 292) were resistant to methicillin but sensitive to six or five of the seven enterocins tested (inhibitory activity 200-12,800 AU/mL). S. warneri SW 292 was sensitive to all enterocins (activity up to 12,800 AU/mL).

Enterococci isolated from farm ostriches and their relation to enterocins.

The present study focuses on the detection of enterococci in ostrich faeces. Forty-six bacterial colonies from 140 ostriches were identified at the species level using the MALDI-TOF MS identification system. According to the score value evaluation, they were allotted to the species Enterococcus hirae, Enterococcus faecium and Enterococcus mundtii confirmed also by phenotypic testing. Dominated species E. hirae (34 strains) were submitted to more detailed testing. Those strains E. hirae produced either no or only slight amount of the enzymes related to disorders (N-acetyl-ß-glucosaminidase, ß-glucuronidase, α-chymotrypsin, trypsin). Most of the strains were not hemolytic. They did not harbour the hiracin-producing gene. Five E. hirae strains harboured virulence factor gene gelE; however, they were phenotypically gelatinase negative. They also harboured other virulence factor genes such as esp, efaAfm and ccf. E. hirae strains were mostly sensitive to antibiotics and those resistant at least to one antibiotic were sensitive to enterocins (200-25,600 AU/mL). This study represents original and novel results concerning the enterococcal microflora in ostriches; enterococci in ostriches have not been described in detail up to now; sensitivity to enterocins of E. hirae strains harbouring virulence factor genes to enterocins is also new.

Virulence factors genes in enterococci isolated from beavers (Castor fiber).

Only limited information exists concerning the microbiota in beaver (Castor fiber). This study has been focused on the virulence factors genes detection in enterococci from beavers. In general, animals are not affected by enterococcal infections, but they can be a reservoir of, e.g. pathogenic strains. Moreover, detection of virulence factors genes in enterococci from beavers was never tested before. Free-living beavers (12), male and female (age 4-5 years) were caught in the north-east part of Poland. Sampling of lower gut and faeces was provided according to all ethical rules for animal handling. Samples were treated using a standard microbiological method. Pure bacterial colonies were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system. Virulence factors genes-gelE (gelatinase), agg (aggregation), cylA (cytolysin A), efaAfs (adhesin Enterococcus faecalis), efaAfm (adhesin Enterococcus faecium) and esp (surface protein) were tested by PCR. Moreover, gelatinase and antibiotic phenotypes were tested. Species detected were Enterococcus thailandicus, E. faecium, E. faecalis and Enterococcus durans. In literature, enterococcal species distribution was never reported yet up to now. Strains were mostly sensitive to antibiotics. Vancomycin-resistant E. faecalis EE9Tr1 possess cylA, efaAfs, esp and gelE genes. Strains were aggregation substance genes absent. Adhesin E. faecium (efaAfm) gene was detected in two of three E. faecium strains, but it was present also in E. thailandicus. Esp gene was present in EE9Tr1 and E. durans EDTr92. The most detected were gelE, efaAfm genes; in EF 4Hc1 also gelatinase phenotype was found. Strains with virulence factors genes will be tested for their sensitivity to antimicrobial enterocins.