Anna Kandričáková

Use of bacteriocin-producing, probiotic strain Enterococcus faecium AL41 to control intestinal microbiota in farm ostriches

Probiotic enterococci can produce bacteriocins. Enterococcus faecium AL41 is an Enterocin M‐producing, probiotic strain which has previously shown beneficial effect in broiler chickens. In this study, it was used to control intestinal microbiota in farm ostriches in a 42‐day experiment with an experimental group (EG, 40 ostriches) and a control group (CG, 46). In addition to feed mixture, the ostriches in EG received Ent. faecium AL41 (109 CFU ml−1; by rifampicin‐marked variant) 400 μl per animal per day in their drinking water for 21 days. Sampling was carried out at the start of the experiment (at day 0/1), at day 21 (after 21 days of AL41 application) and at day 42 (21 days after AL41 cessation). Faeces (mixture, n = 6) were treated using the standard microbiological dilution method and cultivated on selective media (ISO). The highest count of AL41 was found at day 42. Its identity was confirmed with PCR and Maldi‐Tof. The ostriches were free of Salmonella and Campylobacter cells. At day 21, antimicrobial effect was demonstrated by significant reduction in coagulase‐positive and negative staphylococci in EG compared to CG (P < 0·001) and coliforms, Enterobacteria and Pseudomonas‐like bacteria (P < 0·001). We conclude that AL41 can be used to control intestinal microbiota in farm ostriches.

Staphylococcal Species Detected in Free-Living Trouts of East Slovakian Water Sources and their Relation to Antimicrobials

Abstract The aim of the study was to isolate and identify staphylococci from the intestinal samples of 24 trouts from East Slovakian waters. Moreover, their relation to antimicrobials was tested. The count of staphylococci in the trouts reached in average 4.0 x 101 colony forming units per gram. Twenty-two strains were identified by validated species-specific oligonucleotide array targeting the manganese-dependent superoxide dismutase-sodA gene. The identified strains were allotted to five species (Staphylococcus warneri, S. haemolyticus, S. epidermidis, S. hominis, S. pasteuri) clustered to three groups according to 16S rRNA sequences (S. epidermidis group, S. haemolyticus group, S. warneri group). These species belong to coagulase-negative staphylococci. All strains were sensitive to eight antibiotics out of 14 tested; the majority of strains were also sensitive to the remaining six antibiotics with the inhibitory zones from 13 to 41 mm. The strains were also sensitive at least to three enterocins of nine tested. Strains SW24/2, SHo 19/2, SHo20/1, SP19/1 were sensitive to eight of nine enterocins. All strains were sensitive to Ent A, P=EK13, and Ent EM41 with activity 100-6400 AU/mL. Strains SHo19/2 and SP19/1 were sensitive to Ent 2019 with activity up to 25600 AU/mL.

In vivo Model Experiment Using Laying Hens Treated with Enterococcus faecium EM41 from Ostrich Faeces and its Enterocin EM41

Abstract Enterococcus faecium EM41 is an isolate from ostrich faeces. It produces a thermo-stable proteinaceous substance, bacteriocin (enterocin) EM41 with the highest inhibition activity in late logarithmic phase of growth (25 600 AU/ml). This strain and its enterocin have not been previously tested in animals. Lohmann Brown laying hens (aged 45 weeks) were involved in this model/pilot experiment, divided into 3 groups 6 birds in each. E. faecium EM41 applied was a variant treated with rifampicin (109 cfu/ml, dose 400 μl/animal/day) to differentiate it from the other enterococci. Partially-purified enterocin EM41 (Ent EM41, dose 40 μl/animal/day) and its producer were applied to water for 21 days. The experiment lasted 35 days. Sampling was performed at days 0-1, 21 (3 weeks of additive application), 35 (2 weeks after cessation of additive application) from every bird. E. faecium EM41 sufficiently colonized the intestinal tract of laying hens from the initial concentration 109 cfu/g, its count reached 4.30 log cfu/g at day 21. PCR genotypization confirmed the identity of the EM41 strain with the species Enterococcus faecium. E. faecium EM41 and its enterocin EM41 showed antimicrobial effects demonstrated by reduction of coagulase-positive and coagulase-negative staphylococci, coliforms, Pseudomonas spp., Aeromonas spp. and Campylobacter spp. The hens were Salmonella spp. free. After administration of both additives, phagocytic activity was stimulated with a significant increase. The additives did not negatively influence biochemical and haematological parameters or weight gains.

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).

Beneficial effect of bacteriocin-producing strain Enterococcus durans ED 26E/7 in model experiment using broiler rabbits

Lauková A., Pogány Simonová M., Chrastinová Ľ., Kandričáková A., Ščerbová J., Plachá I., Čobanová K., Formelová Z., Ondruška Ľ., Štrkolcová G., Strompfová V. (2017): Beneficial effect of bacteriocin-producing strain Enterococcus durans ED 26E/7 in model experiment using broiler rabbits. Czech J. Anim. Sci., 62, 168–177. From the aspect of probiotic properties and bacteriocins, Enterococcus faecium belongs to the most frequently studied species among enterococci. This study deals with testing the strain of the species Enterococcus durans ED 26E/7 in broiler rabbits. The strain ED 26E/7 isolated from ewes lump cheese produces an antimicrobial substance durancin. Forty-eight post-weaned rabbits (aged 5 weeks) of both sexes were divided into experimental group (EG) and control group (CG) per 24 animals each, and kept in standard cages, two animals per cage. EG group rabbits were additionally administered the ED 26E/7 strain (500 μl/animal/day) into water for 21 days. CG group rabbits were fed a commercial feed. The experiment lasted 42 days. Faeces and blood samples were taken on days 0–1 (experiment onset), 21 (after a 3-week application), and 42 (3 weeks after ED 26E/7 strain cessation). On days 21 and 42, rabbits were slaughtered and caeca and appendix were sampled. The rabbits’ digestive tract was found to be sufficiently colonized by the strain ED 26E/7; the antimicrobial effect was demonstrated in caecum and appendix (e.g. decrease in coliforms). Reduction of Eimeria sp. oocysts in EG compared to CG rabbits was detected on day 21, when also a significant (P < 0.05) increase of phagocytic activity in EG was registered. Values of glutathioneperoxidase were lower in EG than in CG rabbits on day 21 implying that the ED 26E/7 application had not evoked oxidative stress. Biochemical blood parameters and quality of meat were not negatively influenced. First time tested in animals, E. durans ED 26E/7 seems to be a new candidate for use in rabbits husbandry.

Clinical streptococci and their sensitivity to enterocins produced by different strains of the species Enterococcus faecium (short communication).

In children, acute otitis media (AOM) is one of the most frequently occurring infections caused by Streptococcus pneumoniae and Str. pyogenes. The standard treatment of AOMis provided by antibiotics; however, an increased resistance of the causative agents to antibiotics requires the need to search for innovations. This study was focused on in vitro testing sensitivity of streptococci isolated from AOM to enterocins produced by 9 different origin strains of E. faecium. Enterocins (Ent) represent ribosomally synthesized proteinaceous substances with antimicrobial activity against Gram-positive and/or Gram-negative bacteria which are produced mostly by strains of the species Enterococcus faecium. Str. pneumoniae were sensitive at least to 1 Ent. Str. pneumoniae SPn 754 was sensitive to 5 Ent. Five Str. pyogenes were sensitive to enterocins. Ent A (P) inhibited the growth of 3 Str. pneumoniae, and 4 Str. pyogenes (activity between 100 and 3,200 AU/ml). Most of Ent inhibited the growth of streptococci tested (100-3,200 AU/ml). Str. pyogenes were more sensitive to Ent than Str. pneumoniae. Although more detailed further studies are required, our results indicate a new possibility for enterocin use.