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

Abstract

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.

Introduction

C ampylobacter spp. are mainly recognized as a major cause of bacterial gastroenteritis in humans. However, these bacteria have been isolated from a range of animals, including poultry (EFSA, 2012). Campylobacter spp. infection is often asymptomatic, and, once in a flock, Campylobacter spp. are rapidly transmitted throughout broiler sheds by the fecal–oral route (Wassenaar, 2011). Thermophilic Campylobacter spp., particularly Campylobacter jejuni and Campylobacter coli, consist of Gram-negative spiral rods, oxidase and catalase positive (Vaishnavi et al., 2015). They can possess virulence factors such as flagella-mediated motility, adherence to intestinal epithelial cells, invasion and survival in the host cells, as well as the ability to produce toxins (Tam et al., 2003).

Most Campylobacter spp. infections are self-limiting and do not require antimicrobial therapy. However, in systemic infections, antibiotic therapy is usually necessary (Skirrow et al., 2012). Many studies have reported an increase in the resistance of Campylobacter spp. to various antibiotics (Rozynek et al., 2010). This is very alarming because some of the resistant isolates have been suspected of spreading from food animals to humans (Rozynek et al., 2010; Tareb et al., 2013).

A promising strategy to reduce Campylobacter spp. colonization in poultry and poultry products may be done due to the effect of the beneficial properties of probiotic bacteria. Lactobacillus spp. have been shown to be successful in reducing Campylobacter spp. colonization in broilers (Ghareeb et al., 1993). Recently, probiotic enterococci have been indicated as having reductive potential against Campylobacter spp.; for example, Enterococcus faecium AL41 = CCM8558 reduced Campylobacter spp. in chickens (Ščerbová et al., 2014). Its protective effect in the cecum of hens has also been assessed in relation to Toll-like receptor activation and production of luminal IgA challenged with C. jejuni CCM 6191 (Karaffová et al., 2016). In addition, some bacteria produce proteinaceous substances (bacteriocins) that could inhibit the growth of Campylobacter spp. in vitro (Svetoch et al., 2005). Bacteriocins also include enterocin-bacteriocins produced by some enterococci (Franz et al., 2007; Nes et al., 2014). Enterocins (Ents) are divided into four Classes: Class I—lantibiotic enterocins; Class II—bacteriocins with subgroups IIa, antilisterial/pediocin-like bacteriocins; IIb, two peptides bacteriocins; IIc, circular bacteriocins; IId, leaderless bacteriocins; Class III—other small heat-stable bacteriocins; and Class IV—bacteriolysins—an antibody that lyses bacterial cells (Nes et al., 2014). Enterocins have been studied in our Laboratory of Animal Microbiology since 1993. The enterocins used in this research are shown in Table 1. They showed antimicrobial/inhibition effect against different indicator bacteria under in vitro conditions (e.g., Lauková et al., 1993, 2008, Simonová and Lauková, 2007). Moreover, their beneficial effect (antimicrobial, stimulation of phagocytic activity, modulation of biochemical blood parameters, and so on) has also been monitored in vivo; for example, in rabbits (Pogány Simonová et al., 2013), Japanese quails (Lauková et al., 2003), ostriches (Lauková et al., 2015). In addition, bacteriocins (enterocins) showed antimicrobial effect also in situ in animal-derived food, for example, in cheese (Lauková and Vlaemynck, 2001) or in salami Púchov (Lauková and Turek, 2011). This study focuses on the sensitivity of Campylobacter spp. to enterocins and also indicates further practical use of enterocins. Feces from broiler chickens, laying hens, ducks, and ostriches were screened. Ostriches were previously not traditionally reared poultry in Slovakia. They have been included in the category of farm animals since 1995 (paragraph 8, section 1 of Law No. 110/1972 Statutes related to animal breeding).

Table 1.

Enterocins, Their Producer, and Sources

Producer	Enterocin	Source	References
Enterococcus faecium EM41	Ent EM41	Ostrich feces	Lauková et al. (2012a)
E. faecium AL41^a	Ent M	Environmental	Mareková et al. (2007)
E. faecium EK13^b	Ent A (P)	Environmental	Mareková et al. (2003)
E. faecium EF55	Ent 55	Chick feces	Strompfová and Lauková (2007)
E. faecium EF412	Ent 412	Horse feces	Lauková et al. (2008)
E. faecium EF9296	Ent 9296	Silage	Marciňáková et al. (2005)
E. faecium 4231	Ent 4231	Ruminants	Lauková et al. (1993)
E. faecium EF2019^c	Ent 2019	Rabbit feces	Simonová and Lauková (2007)
E. faecium EFHč131	Ent 131	Beaver colon	Unpublished

E. faecium AL41 = CCM8558.

E. faecium EK13 = CCM7419.

E. faecium EF2019 = CCM7420.

Materials and Methods

Isolation and identification of Campylobacter spp

Feces (102 samples) from a total of 237 different poultry birds were collected at different days/times. Ostriches (140) from three age groups were sampled, and 54 fecal mixtures (18 from each group) were sampled over a half-year period in 2013 on a farm in Slovakia: 56 birds aged from hatching up to 3 weeks (group 1); 42 ostriches aged 6 to 9 weeks (group 2); and 42 ostriches aged 12–16 months (group 3); 10 broiler chickens (6 fecal mixture samples twice from 3 birds and once from 4 birds), 47 laying hens (27 fecal mixture samples, 5 from 24 hens, 18 from individual birds, 2 from 5 birds, and 6 from 18 birds); and 40 ducks (15 fecal mixture samples). Sampling was performed with the farmer's agreement (approved by the Slovak Veterinary and Food Administration) or with the consent of individual owners. Fresh feces were collected in the poultry pen by hand using gloves, immediately after being voided by the birds to prevent other contamination; they were put into sterile packs, placed in a portable fridge, and driven to our laboratory. Samples were treated according to the standard microbiological method of ISO (International Organization for Standardization); 1 g of feces was added into 9 mL of Ringer solution (Merck); samples were stirred using the Stomacher–Masticator and diluted. Campylobacter agar base, Karmali (Oxoid Ltd.), was used as a selective medium to enumerate Campylobacter spp. colonies. The plates were placed in a jar enriched with microaerobic atmosphere—approximately 5% O₂, 10% CO₂, 85% N₂, and cultivated at 42°C for 48 h in an incubator. Bacterial richness was expressed in colony-forming units per gram of sample (log₁₀ CFU/g ± SD), calculated as an average count of colonies grown in the highest dilution per sample.

Presumptive Campylobacter spp. colonies were controlled for purity and identified using the MALDI-TOF MS (mass spectrometry) identification system based on protein “fingerprints” (Alatoom et al., 2011; MALDI-TOF MS, Bruker Daltonics), performed using a Microflex MALDI-TOF MS mass spectrophotometer. Briefly, a single fresh colony from Karmali agar was mixed with matrix (α-cyano-4-hydroxycinnamic acid and trifluoroacetic acid); the suspension was spotted onto a MALDI plate and ionized with a nitrogen laser (wavelength 337 nm, frequency 20 Hz). Results were evaluated using the MALDI Biotyper 3.0 (Bruker Daltonics) identification database. Taxonomic allotment was evaluated on the basis of highly probable species identification (value score 2.300–3.000) and secure probable species identification/probable species identification (2.000–2.299) as used in the database. The positive controls used were strains included in the MALDI-TOF MS identification system database. Identical colonies evaluated with the MALDI-TOF MS score value were excluded from the other analyses. Twenty-three identified strains were stored using the Microbank™ system (Pro-Lab Diagnostic).

Campylobacter spp. identified by means of MALDI-TOF MS spectrometry were then genotyped using PCR (Techne Thermocycler) with specific 16S rRNA gene (Luebeck et al., 2003). The bacterial colony was suspended in TE buffer (20 mM Tris, 10 mM EDTA, pH 8.0) and centrifuged at 6000 g for 5 min. Bacterial DNA was extracted using the commercial Bactozol™ Bacterial DNA Isolation Kit (Molecular Research Centre, Inc.) according to the manufacturer's instruction. The primers used were as follows: OT1559 5′–CTG CTT AAC ACA AGT TGA GTA AGG-3′ and 18–1 5′-TTC CTT AGG TAC CGT CAG AA-3′. The PCR mixture included 0.1 μL from each primer, 2.5 μL of 10 mM deoxynucleoside triphosphates (Invitrogen, Life Technologies), 2 μL of 50 mM MgCl₂, 2.5 μL 10 × PCR buffer, 0.2 μL/U Taq polymerase (Invitrogen), and water 16.6 μL. The final thermocycling program was as follows: initial denaturation 94°C at 2 min, then 35 cycles of denaturation at 94°C for 30 s; annealing at 58°C for 15 s, and extension at 72°C for 30 s; and finally an extension at 72°C for 4 min. PCR product was evaluated with agarose electrophoresis (1% gel; 287 bp; Sigma-Aldrich). Visualization of PCR product (bands) (UV transilluminator; Sigma-Aldrich) was done with ethidium bromide staining solution (1 μg/mL; Sigma-Aldrich). Marker (100 bp) was used as standard molecular mass DNA (Jena Bioscience). Positive control strain was C. jejuni CC M6191.

Antibiotic resistance test

Identified Campylobacter spp. strains were tested for their antibiotic phenotype sensitivity/resistance. The disk diffusion method was used according to the Clinical Laboratory Standard Institute guidelines (CLSI, 2013). Briefly, tested strains were cultivated in brain heart infusion (BHI; Becton and Dickinson) at 42°C for 40–48 h. Bacterial culture (100 μL) was plated onto Mueller–Hinton agar (Bio-Rad) enriched with defibrinated horse blood (5%; Oxoid Ltd). Then, the following antibiotic discs supplied by Oxoid Ltd and/or Lach-Ner were applied: ciprofloxacin (5 μg), ampicillin (10 μg), erythromycin, azithromycin (15 μg), streptomycin (25 μg), nalidixic acid, cefotaxim, cephalothin, chloramphenicol, tetracycline (30 μg), and gentamicin (120 μg). Concentrations of antibiotics were decided according to the Oxoid Ltd commercial recommendations as well as those of the European Centre for Disease Prevention and Control. Inhibition zones (in mm) were evaluated according to the European Committee on Antimicrobial Susceptibility Testing guidelines for Campylobacter spp. or to the CLSI guidelines for Enterobacteriaceae (CLSI, 2013). C. jejuni CCM 6191 was used as a positive control. Antibiotic-free agar plates were included as a control for obligatory strain growth. Campylobacter spp. are naturally (chromozomally) resistant to cephalothin, therefore, this antibiotic was also included in the testing.

Sensitivity of Campylobacter spp. to enterocins

The strains resistant to antibiotics were used to test their sensitivity to enterocins. For this purpose, partially purified enterocins were used (produced by different E. faecium strains isolated and characterized at our Laboratory of Animal Microbiology, Institute of Animal Physiology Slovak Academy of Sciences, Košice, Slovakia; Table 1). Briefly, a 16-h culture (300 mL) of the producer strains in MRS broth (Merck) was centrifuged (30 min, 10,000 g). After removing cells, supernatants were adjusted to pH 5.0 or 5.5; ammonium sulfate was added to the supernatant (40% saturation) and stirred at 4°C for 2–4 h. After centrifugation, the pellet was resuspended in 10 mmoL of sodium phosphate buffer (pH 6.5). Inhibition activity was measured using the quantitative agar diffusion method (De Vuyst et al., 1996) with BHI agar (1.5%; 0.7%; Becton and Dickinson). Indicator strains were cultivated in BHI broth at 37/42°C, then 200 μL of culture was added to the volume of 4 mL in 0.7% BHI agar and applied on the plate agar surface (1.5% BHI agar). Partially purified enterocins (Ents) were diluted (1:1 in 10 mmoL of sodium phosphate buffer, pH 6.5) and dropped (10 μL) on the plate surface. Incubation was done at 42°C for 48 h in an incubator with microaerobic atmosphere. Inhibition activity was defined as the reciprocal of the highest dilution producing an inhibition zone against the indicator strain and evaluated in Arbitrary Units per mL (AU/mL). Control activity of Ents against EA5 strain (the principal indicator, strain from piglet, isolated in our laboratory) reached up to 25,600 AU/mL. Double testing was performed.

Results

Identified Campylobacter spp.

Altogether 23 Campylobacter spp. were allotted through the MALDI TOF MS system. Based on the identification score value, Campylobacter spp. were allotted to two taxonomical species, C. jejuni (19 strains) and C. coli (4 strains). Identified C. coli were isolated from the feces of ducks; C. jejuni were from broilers and laying hens. Campylobacter spp. were not detected in the feces of ostriches. The most strains were identified on the basis of secure probable species identification/probable species identification (Tables 2 and 3). Five strains (C. coli SZ4, C. jejuni 4/E/1, 3/E/1a, 3c, 1SL/1) were identified on the basis of highly probable species identification. The rest of the strains were identified in the score value range up to 1.999. Moreover, allotment of strains to the genus Campylobacter was also confirmed by means of PCR.

Table 2.

Identification Score Value of Campylobacters, Their Antibiotic Profile, and Sensitivity to Enterocins

Strain	S/V	EM41	131	M	55	A (P)	2019	9296	412	4231	Ctx	Ery	Amp	Str	Cip	Azm
Campylobacter jejuni
BK1/A	2.278	100	—	—	—	100	—	100	—	—	23	31	R	31	R	45
BK1	2.210	—	100	—	—	—	—	—	—	—	16	27	R	28	R	20
BK	22.290	—	100	—	—	100	—	—	—	—	20	26	18	33	R	30
5/E/2	2.254	—	—	—	100	—	100	—	—	—	d	26	R	28	R	37
2/E/2	2.139	—	—	100	100	100	100	100	100	—	20	30	d	30	R	33
4/E/1	2.303	—	100	—	—	100	—	100	—	—	22	22	R	36	R	33
1/A/1	1.893	—	100	—	100	—	—	—	100	100	20	30	R	35	R	R
6/P/1	2.010	—	100	—	—	100	—	—	—	—	d	28	R	33	R	32
3/E/1a	2.320	100	100	100	100	100	100	100	100	—	R	R	26	R	R	32
3c	2.328	100	—	100	100	100	100	100	—	—	d	34	R	33	R	23
1SL/1	2.328	100	100	100	100	100	—	100	—	100	25	R	R	37	R	25
NB1	2.100	—	100	100	—	100	—	100	100	—	d	28	d	31	R	23
2d/2	1.852	100	100	100	—	—	—	100	—	—	d	28	d	37	R	30

C. jejuni CCM6191: susceptible to all enterocins (100 AU/mL); R to Ent 4231; Ctx-R, Ery-S22, CnS27 Trim, Na, Amp, Chl, Sts R, Tct S23 CipR, AzmR, R-resistant, d-dubious reaction; S-sensitive, 100 AU/mL-Arbitrary unit per mL; Na-nalidixic acid, Amp-ampicillin, Chl-chloramphenicol, Tct-tetracycline, Ery-erythromycin, Cip-ciprofloxacin, Ctx-cefotaxim, S(no)-sensitive and size of inhibition zone in mm; Ent 55: 25,600 AU/mL; Ent EM41: 25,600 AU/mL; Ent 2019: 25,600 AU/mL; Ent 412: 25,600 AU/mL; Ent 9296: 51,200 AU/mL; Ent A (P): 51,200 AU/mL; Ent EF131: 25,600 AU/mL; Ent M: 12,800 AU/mL; Ent 4231: 800 AU/mL. BK1/A, BK1, BK2 from broilers, the rest from laying hens; —, not inhibited/not sensitive; S/V, score value; AU/mL, Arbitrary Units per milliliter.

Table 3.

Identification Score Value of Campylobacters, Their Antibiotic Profile, and Sensitivity to Enterocins

Strain	S/V	EM41	Hc131	M	55	A (P)	2019	9296	412	4231	Ctx	Ery	Amp	Str	Cip	Azm
3e/2	1.935	—	—	—	—	—	—	100	100	—	28	26	R	30	R	27
1e/2	1.803	—	100	—	—	—	—	—	—	—	d	28	R	32	R	40
2D/2	1.987	100	—	100	—	100	100	—	100	100	30	36	R	30	R	R
1/A	1.944	—	—	—	—	—	—	—	—	—	d	30	R	33	R	33
1/E	1.915	—	100	100	—	—	—	100	—	—	d	37	R	32	R	33
1/D	1.943	—	—	—	—	—	—	—	—	—	d	26	R	32	R	23
Campylobacter coli
Kc1	1.869	—	—	—	—	—	—	—	—	—	d	30	R	25	R	30
SZ3	2.218	—	—	—	—	—	—	—	—	—	R	R	R	27	d	35
SZ4	2.326	—	—	—	—	100	—	—	100	—	R	R	R	29	d	33
SZ5	2.248	100	—	100	100	100	—	100	100	—	d	R	R	26	d	35

Campylobacter jejuni CCM6191: susceptible to all enterocins (100 AU/mL); R to Ent 4231; Ctx-R, Ery-S22, CnS27 Trim, Na, Amp, Chl, Sts R, Tct S23 CipR, AzmR, R-resistant, d-dubious reaction; S-sensitive, 100 AU/mL-Arbitrary unit per mL; Na-nalidixic acid, Amp-ampicillin, Chl-chloramphenicol, Tct-tetracycline, Ery-erythromycin, Cip-ciprofloxacin, Ctx-cefotaxim, S(no)-sensitive and size of inhibition zone in mm; Ent 55: 25,600 AU/mL; Ent EM41: 25,600 AU/mL; Ent 2019: 25,600 AU/mL; Ent 412: 25,600 AU/mL; Ent 9296: 51,200 AU/mL; Ent A (P): 51,200 AU/mL; Ent EF131: 25,600 AU/mL; Ent M: 12,800 AU/mL; Ent 4231: 800 AU/mL. Strains from laying hens and ducks; —, not inhibited/not sensitive; S/V, score value; AU/mL, Arbitrary Units per milliliter.

Antibiotic profile, in vitro testing of sensitivity to enterocins

C. jejuni and C. coli, 23 strains from poultry birds showed high resistance to antibiotics used. They were also resistant to nalidixic acid. On the contrary, they were susceptible to chloramphenicol, tetracycline, gentamicin, and to aminoglycoside streptomycin (inhibition zone size up to more than 30 mm). Only a few strains showed dubious reaction. Campylobacter spp. were mostly resistant to ciprofloxacin or showed dubious reaction. Ampicillin is a broad-spectrum antimicrobial antibiotic, to which most strains were resistant (except C. jejuni BK2 and C. jejuni 6/P/L, Table 2). On the contrary, the strains were mostly susceptible to macrolide antibiotics such as erythromycin (size of inhibition zone up to 40 mm, Tables 2 and 3) and they were also susceptible to azithromycin. The Campylobacter spp. tested were sensitive, resistant, or with dubious reaction to cefotaxim. In general, the strains showed multiresistance to antibiotics, including the control strain C. jejuni CCM 6191 (resistance to 7 out of the target of 11 antibiotics tested). However, these multiresistant strains were sensitive to at least one of the nine enterocins used (except C. coli Kc1, SZ3, and C. jejuni 1/D). These strains were most sensitive to Ent A (P) (12 strains, Tables 2 and 3), to Ent 131 (11 strains, Tables 2 and 3), and to Ent 9296 (11 strains, Tables 2 and 3). This means that among the 23 strains, the growth of almost 52% was inhibited by Ent A (P), while 48% of strains were inhibited by Ent 131 and Ent 9296. Ent M inhibited the growth of nine Campylobacter spp. (39%). Ent 412 inhibited the growth of eight strains (35%), and Ent 55 and Ent EM41 inhibited the growth of seven strains (30%). Five strains (22%) were sensitive to Ent 2019. Ent 4231 inhibited the growth of three Campylobacter spp. strains (13%). The most sensitive was strain C. jejuni 3/E/1a, sensitive to eight out of nine Ents, followed by C. jejuni 1SL/1, sensitive to seven Ents, and C. jejuni 2/E/2, SZ, and 2D/2, which were sensitive to six Ents. The rest of the strains were sensitive to at least one Ent as previously mentioned. Control strain C. jejuni CCM 6191 was sensitive to all Ents except Ent 4231. Seven strains were sensitive to EM41 (produced by E. faecium EM41 from ostriches). Campylobacter spp. sensitivity to enterocins reached inhibition activity of 100 AU/mL.

Discussion

The rapid and reliable identification of microorganisms is a crucial requirement in many fields of microbiology. The MALDI-TOF MS identification system was originally developed and applied for rapid identification of clinically important bacteria and is still mainly used for this purpose. Campylobacter spp. belong among the bacteria, which are important from the clinical aspect. For this reason, we followed their detection and confirmation in poultry in terms of their sensitivity to enterocins. MALDI TOF MS is an identification method based on the rapid and precise assessment of the mass of molecules in a range varying from 0.1 to 100 kDa (Cobo, 2013). Kopčáková et al. (2014) reported sufficient species detection, for example, from environmental samples using MALDI TOF MS. However, in our previous studies, additional identification methods such as phenotypization confirmed MALDI-TOF MS identification (Ščerbová et al., 2014). Campylobacter spp. are commensal in the gastrointestinal tract of many domestic and wild animals, especially birds (Takamyia et al., 2011). Fraqueza et al. (2016) reported dominance of C. jejuni in broilers confirmed by PCR, but in Japanese quails and turkeys the species C. coli was dominant (Kashoma et al., 2014; Fraqueza et al., 2016). In our case, four C. coli from ducks were identified.

The identified Campylobacter spp. showed high resistance to antibiotics on one hand, while on the other they showed high susceptibility to Ents. Fraqueza et al. (2014, 2016) reported 100% resistance to nalidixic acid of C. jejuni strains from Japanese quails and high resistance to ciprofloxacin. Our strains were also resistant to nalidixic acid. Some authors reported that nalidixic acid resistance in Campylobacter spp. is naturally (chromozomally) coded (Siegrist, 2014). In contrast to our findings, Vaishnavi et al. (2015) detected strains of Campylobacter spp. sensitive to ciprofloxacin and nalidixic acid. However, in accordance with our findings, they reported campylobacters sensitive to gentamicin and erythromycin. Susceptibility or resistance could vary depending on the animal source. Similarly, as in our study, Carbonero et al. (2012) detected low resistance to erythromycin in strains of Campylobacter spp. isolated from dogs. Of course, it could also vary depending on the species of Campylobacter. However, because the antibiotic resistance rate is rising worldwide, it is necessary to seek out “replacements”for antibiotics; in this case, natural substances are promising. For this reason, enterocins with a broad antimicrobial spectrum have been used in this research (Lauková et al., 1993, 2003, 2008, 2012a; Mareková et al., 2003, 2007; Marciňáková et al., 2005; Strompfová and Lauková, 2007;). They belong among the Class II enterocins as previously mentioned, and they are commonly indicated as being thermostable small peptides (molecular mass 3–10 kDa). As shown by Cintas et al. (2000), enterocins can possess a broad antimicrobial spectrum. This also indicates the effectiveness of our Ents. We have not studied the mode of action of our Ents on a molecular basis (but it is similar to Class II enterocins); however, in in vivo experiments a reduction of Gram-negative species (to which also Campylobacter spp. belong) has been shown through enterocins (Ščerbová et al., 2014). On the contrary, for example, Enterocin S37, for instance, produced by Enterococcus faecalis S37 strain from poultry was tested in vitro against C. jejuni strain; however, its growth was not inhibited (Belguesmia et al., 2010). In contrast, our enterocins inhibited the growth of Campylobacter spp. This could be explained by the different sensitivity of Campylobacter spp. to the permeabilization ability of Ents. In further experiments, therefore, we would like to apply for example Ent EM41 produced by E. faecium EM41 from ostriches in an animal model. Ent EM41 has not been tested in this manner. Based on our previous results with bacteriocin-producing strains, they show a tendency to reduce, for example, coliforms, Salmonella Enteritidis, and also Campylobacter spp. (Lauková et al., 2012b, 2015, Ščerbová et al., 2014). In addition, they also stimulate unspecific immunity parameter (phagocytic activity, PA) by increasing PA (Lauková et al., 2012b; Pogány Simonová et al., 2013).

In conclusion, the species C. jejuni and C. coli were identified in the feces of broilers, laying hens, and ducks. In ostriches, Campylobacter spp. were not detected. The identified Campylobacter spp. showed high resistance to antibiotics on one hand, while on the other, they were susceptible to enterocins. This indicates a broad inhibition spectrum of enterocins and also their application potential.

Footnotes

Acknowledgments

This study was supported by the project VEGA 2/0004/14 of the Slovak Scientific Agency. The authors thank Mrs. Margita Bodnárová for her laboratory assisstance. They are also grateful to professor Vladimír Kmeť (from our Institute) for his kindness, advice, and help with the PCR method. Detailed description of sampling regarding ostriches has been already reported by Kandričáková et al. (2016) in Foodborne Path Dis 13, doi:10.1089/fpd.2015.2069. Finally, they thank Mrs. Andrew Billingham for her kindness to check English language.

Disclosure Statement

No competing financial interests exist.

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