Staphylococci Related to Farm Ostriches and Their Sensitivity to Enterocins

Abstract

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).

Introduction

Ostriches belong to the class Aves, the order Struthioniformes, the family Struthionidae and the genus Struthio. This not traditionally reared poultry in Slovakia is nowadays reared mainly for the meat. Their breeding has been ongoing here since 1992 (Sabolová et al., 2008). In December 1994 the Slovak Association of Ostrich Breeders was established. In 1995 ostriches were included in the list of domestic animals, having been transferred there from the category of exotic animals (§8 Section 1, Law No. 110/1972). There is still limited information related to microflora of ostriches, including staphylococci.

The genus Staphylococcus belongs to the phylum Firmicutes, the class Bacilli, the order Bacillales, and the family Staphylococcaceae. Their pathogenity correlates with the production of coagulase. In general, coagulase-positive species of staphylococci are supposed to be pathogenic and coagulase-negative staphylococci are nonpathogenic with the potential to be facultatively pathogenic (De Vos et al., 2009). Based on comparative 16S ribosomal RNA sequence studies, staphylococci are divided into 11 clusters: S. aureus cluster with the species S. aureus, S. simiae; cluster S. auricularis represented by the species S. auricularis; S. carnosus cluster with the species S. carnosus, S. condimenti, S. massiliensis, and S. piscifermentans; cluster S. epidermidis with the species S. capitis, S. caprae, S. epidermidis, and S. saccharolyticus; cluster S. haemolyticus possessing the species S. devriesei, S. haemolyticus, and S. hominis; cluster S. hyicus-intermedius with the species S. chromogenes, S. felis, S. delphini, S. hyicus, S. intermedius, S. lutrae, S. microti, S. muscae, S. pseudointermedius, S. rosteri, and S. schleiferi; cluster S. lugdunensis and its species S. lugdunensis; cluster S. saprophyticus with the species S. arlettae, S. cohnii, S. equorum, S. gallinarum, S. kloossi, S. leei, S. nepalensis, S. saprophyticus, S. succinus, and S. xylosus; cluster S. sciuri and its species S. fleuretii, S. lentus, S. sciuri, S, stepanovicii, and S. vitulinus; cluster S. simulans with the species S. simulans; and cluster S. warneri with the species S. pasteuri and S. warneri (Takashi et al., 1999).

Although adult ostriches are able to adapt to diverse climatic conditions and have a well-developed immune system, chicks are more predisposed to different diseases involving bacteria, so methicillin-resistant staphylococci in particular could also play a key role.

Knowing the composition of microflora is very important for the recognition of potential agents of disorders. Nowadays, bacteria resistant to antibiotics present a frequent problem in animals; as a result of a resistant bacteria, their elimination and disorders elimination, as well, is difficult. Resistance of bacteria to antibiotics can be natural or can be carried by DNA plasmids or DNA integrons from antibiotic resistant donor cell to sensitive recipient cell. For example, methicillin is and antibiotic active especially against penicillin-resistant strains of Staphylococcus aureus. With the acceptance of the gene mecA, which is responsible for methicillin resistance and is located on the chromosomal cassette mec (SCC mec), methicillin-resistant staphylococci arise from methicillin-sensitive strains (Hawkey, 2010). Farmers have made efforts to find natural substances to protect their husbandry and have mainly focused on probiotic bacteria. In our laboratory bacteriocins (enterocins) with broad inhibitory spectrum (produced by our isolates of Enterococcus faecium possessing probiotic properties) have been studied since 1985. In general, enterocins are small, thermo-stable, ribosomally synthesized antimicrobial proteinaceous substances with a wide spectrum of inhibitory activity against more or less related bacteria (Franz et al., 2007). Therefore, the aim of our study was not only to detect staphylococcal species in farm ostriches, but also to test their sensitivity to enterocins; however, their antibiotic profile was also tested. In our previous studies, staphylococci from different animals resistant to antibiotics were sensitive to enterocins (Lauková et al., 2003). Moreover, information concerning the distribution of staphylococcal species in farm ostriches has been very limited or not yet reported until now.

Materials and Methods

Isolation of bacterial strains

Altogether, 140 ostriches from three age groups were sampled (n=18, faecal mixture samples from each group) over a half-year period in 2013 on a farm in Slovakia: 56 birds aged from hatching up to 3 weeks (group 1); 42 birds, 6-to 9-week-old (group 2); and 42 birds aged 12–16 months (group 3). Chicks were hatched indoors, and older birds (6–9 weeks or older) were transferred to an external field area with access to a stall in case of bad weather. The ostriches were administered feed mixture 1567 Pštros Mini Energys (De Heus a.s., Vyškov, Czech Republic) with access to drinking water ad libitum. Fifty-four fecal samples were collected with the farmer's agreement (approved by Slovak State Veterinary and Food Administration). Fresh feces were collected in the ostrich pen by hand using gloves, immediately after being voided by the birds to prevent other contamination; feces were put into sterile packs, placed into a transport fridge, and driven to our laboratory. Feces from each group (comprising 18 ostriches) were sampled into three packs per group (fecal mixture from six birds in each pack). Samples were treated according to the standard microbiological method of ISO (International Organization for Standardization) using appropriate dilutions [1 g of feces into 9 mL of Ringer's solution (Merck, Darmstadt, Germany)]; samples were stirred using the Stomacher–Masticator (IUL S.A. Barcelona, Spain) and diluted. Mannitol salt agar (ISO 6888; Difco, Detroit, MI) or Baird-Parker agar with supplement (ISO 6888-2; Becton and Dickinson, Cockeysville, MD) were used for cultivation. Plates were incubated at 37°C for 24–48 h. Bacterial richness was calculated as an average count of colonies grown in the highest dilution per sample and expressed in colony-forming units per gram of sample (log₁₀ CFU/g ± standard deviation). Eighty-one different colonies from 54 samples were picked, checked for purity, and prepared for species identification.

Identification of bacterial strains

Presumed colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Billerica, MA) based on the analysis of bacterial protein “fingerprints” (Alatoom et al., 2011). Protein profiles of bacteria are compared with databases and bacteria are taxonomically classified. Lysates of bacterial cells were prepared according to the producer's instructions prior to identification. Taxonomic identification was expressed on the basis of secure probable species identification/probable species identification (score value 2.000–2.299) of MALDI-TOF MS identification system (Bruker Daltonic) (Alatoom et al., 2011). Identitical colonies were excluded. Taxonomical allottation of the strains by MALDI-TOF MS was also validated by phenotypization: biochemical tests were completed using BBL Crystal Gram-positive kit (Becton and Dickinson). The parameters such as production of alkaline phosphatase, arginine dihydrolase, urease and fermentation of arabinose, fructose, lactose, mannitol, sucrose, and trehalose were compared as reported in Bergeys Manual by De Vos (2009) for type strains. Identified strains were stored using Microbank^™ system (Pro-Lab Diagnostics, Richmond, BC Canada).

Production of lactic acid and antibiotic profile of identified strains

Production of lactic acid (LA) by staphylococcal strains was tested by the quantitative spectrophotometric method (based on conversion of LA to acetaldehyde after heating by sulphuric acid), expressed in mmol/L. For testing, the strains were inoculated into Brain-heart infusion (BHI) broth (Becton and Dickinson) for 18 h at 37°C.

Antibiotic phenotypes of Staphylococci were tested by the agar disk method followed by antibiotics required in CLSI (2012): oxacilin (1 μg); clindamycin (2μg); novobiocin, neomycin (5μg); methicillin (Met), lincomycine, penicillin, gentamicin, and tobramycin (10μg); and erythromycin (15μg); phosphomycin (20μg); chloramphenicol vancomycin, kanamycin, rifampicin, tetracycline (30μg) (Becton and Dickinson; Lach-Ner, Czech Republic). The positive control strain was S. aureus ATCC 25923. The strains were cultivated in BHI broth (Becton and Dickinson) overnight at 37°C. The volume 100 μL was plated onto BHI agar enriched with defibrinated sheep blood (Becton and Dickinson) and aforementioned appropriate disks were applied. The evaluation was done according to the manufacturer's instructions; the inhibitory zones were expressed in millimeters. Antibiotic-free agar plates were included as a control for obligatory growth. The use of the antimicrobial agents was decided according to the manufacturers guides with the special attention for methicillin resistant strains.

Sensitivity of methicillin-resistant strains to enterocins

The sensitivity of methicillin-resistant staphylococci to enterocins was tested using the quantitative agar spot test (De Vuyst et al., 1996) using Brain Heart agar (Becton and Dickinson). In this study, seven semipurified substances (enterocins) produced by Enterococcus faecium strains of ruminal, enviromental, and animal origin isolated and characterized in our laboratory were used as follows: Ent EM41 produced by E. faecium EM41 from ostrich (Lauková et al., 2012b); Ent EK13 [A (P)]; EntM produced by environmental strains E. faecium EK13 (CCM7419) and AL41 (CCM8558) (Mareková et al., 2003, 2007; Lauková et al., 2012a); Ent 55 produced by E. faecium EF55 from the chicken crop (Strompfová and Lauková, 2007); Ent 2019 produced by rabbits E. faecium 2019 (CCM7420) (Pogány Simonová et al., 2013); Ent M3a produced by E. faecium M3a from rabbit meat (Szabóová et al., 2013); and Ent 412 produced by E. faecium EF412 from horses feces (Lauková et al., 2008). They were prepared following the protocols indicated in previous references. Control activity of the used enterocins against the principal indicator strain E. avium EA5 (our isolate from piglets feces) ranged from 6400 to 51,200 Arbitrary units per mL (AU/mL). Briefly, a 16-h culture (300 mL) of the producer strains in MRS broth (Merck, Germany) was centrifuged (30 min, 10,000 × g). After removing cells, supernatants were adjusted to pH 5.0 or 5.5; ammonium sulphate 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). Indicator strain was cultivated 18 h in BHI broth at 37°C, then 200 μL of culture was added to the volume 4 mL in 0.7% BHI agar and applied to the plate agar surface (1.5% BHI agar). Semipurified enterocins were diluted (1:1 in 10 mmoL of sodium phosphate buffer, pH 6.5) and dropped (10 μL) on the plate surface. Plates were incubated at 37°C for 18 h. The inhibitory activity was defined as the reciprocal of the highest dilution producing an inhibitory zone against the indicator strain and evaluated in AU/mL.

Results

Bacterial counts and bacterial identification

Staphylococci in ostriches reached an average 4.3 ± 0.63 (log10) CFU/g. In feces of group 1, staphylococci reached 3.99 ± 0.99 CFU/g. The counts of staphylococci in ostrich's feces of groups 2 and 3 were 4.63 ± 0.66 CFU and 6.26 ± 0.71 CFU/g. Twenty-four strains (from 81 isolated; 29.6%) were taxonomically classified to eight species of the genus Staphylococcus (Table 1): six strains (25%) were identified as Staphylococcus equorum, five strains (20.8%) were allotted to the species S. xylosus, and four strains (16.7%) to the species S. epidermidis. To the species S. haemolyticus, three strains (12.5%) were allotted; two strains (8.3%) belonged to each species S. cohnii and S. succinus; and one strain each (4.2%) was allotted to the species S. warneri and S. hominis. Identified strains were evaluated by secure probable species identification/probable species identification (score value up to 2.299). Strains which did not reach requested score value were excluded.

Table 1.

Staphylococcal Species with Strains Identified In Ostrich Feces

Staphylococcus species	Number of strains
S. equorum	6
S. xylosus	5
S. epidermidis	4
S. haemolyticus	3
S. succinus	2
S. cohnii	2
S. hominis	1
S. warneri	1

Biochemical properties (phenotypic tests) were in accordance with those properties presented for the type strains: acid formation from trehalose, fructose, lactose, arabinose, mannitol, and sucrose and positive reaction related to alkaline phosphatase, arginine, dihydrolase, and urease.

Production of lactic acid and antibiotic profile of identified strains

The amounts of lactic acid (LA) produced by staphylococci from ostriches ranged from 0.93 to 2.23 mmol/L (Table 2). The average value was 1.35 ± 0.43 mmol/L. The strains of the species S. equorum produced an average concentration of LA 1.1 ± 0.14 mmol/L; the species of S. xylosus produced an average concentration of LA 1.63 ± 0.55 mmol/L; S. epidermidis 1.76 ± 0.32 mmol/L; S. haemolyticus 1.85 ± 0.14 mmol/L; S. succinus 1.22 ± 0.27 mmol/L; S. cohnii 1.00 ± 0.09 mmol/L; S. hominis 0.99 ± 0.17 mmol/L and production of LA by S. warneri was an average concentration of 1.13 ± 0.46 mmol/L.

Table 2.

Values for Lactic Acid Produced by Staphylococcal Species Detected in Ostrich Feces

Staphylococcus species/strains	Lactic acid concentration (mmol/L)
S. equorum
Sq31	0.94 ± 0.14
Sq342	1.09 ± 0.10
Sq352	1.34 ± 0.01
Sq2184	1.12 ± 0.12
Sq3163	1.00 ± 0.10
Sq3184	1.10 ± 0.28
S. xylosus
SX242	2.23 ± 0.17
SX272	2.08 ± 0.07
SX2121	1.71 ± 0.43
SX2133	1.08 ± 0.01
SX3172	1.03 ± 0.03
S. epidermidis
SE211	1.88 ± 0.29
SE273	2.14 ± 0.23
SE281	1.59 ± 0.32
SE3122	1.42 ± 0.42
S. haemolyticus
SHae111	1.91 ± 0.10
SHae214	1.69 ± 0.55
SHae371	1.96 ± 0.36
S. succinus
Su392	1.41 ± 0.04
Su3162	1.03 ± 0.30
S. cohnii
Sco210	0.93 ± 0.20
SCo2122	1.06 ± 0.20
S. hominis
SHo3112	0.99 ± 0.17
S. warneri
SW292	1.13 ± 0.46

Among 24 tested staphylococci, three strains of different species (S. epidermidis SE 273, S. hominis SHo 3112, and S. succinus SU 3162) were sensitive to 11 of 15 tested antibiotics reaching inhibitory zones up to 31 mm. Four strains (S. haemolyticus SHae 111, S. haemolyticus SHAe 371, S. xylosus SX 2133, and S. warneri SW 292) were resistant to methicillin. S. hominis SHo 3112 and S. succinus Su3162 were sensitive to all antibiotics—their growth was inhibited by antibiotic treatment. All strains were sensitive to vancomycin and chloramphenicol. Most strains were sensitive to oxacillin (54.1%), novobiocin (66.6%), neomycin (70.8%), lincomycin (70.8%), penicillin (75%), tobramycin (70.8%), erythromycin (91.6%), and phosphomycin (54.1%). The representants of the species S. haemolyticus, S. cohnii, and S. warneri were sensitive to penicillin. S. cohnii was sensitive to novobiocin. All strains of S. epidermidis were sensitive to tobramycin. The representants of the species S. xylosus, S. haemolyticus, S. cohnii, and S. warneri were sensitive to erythromycin. Eleven strains (three strains of S. xylosus, two strains of S. epidermidis, two strains of S. haemolyticus, two strains of S. equorum, one strain of S. warneri, and one strain of S. cohnii; 45.9%) were resistant to phosphomycin, and the rest strains were sensitive to phosphomycin.

Sensitivity of methicillin resistant strains to enterocins

The sensitivity of four methicillin-resistant staphylococci (S. haemolyticus SHAe 111 and SHAe 371, S. warneri SW 292, and S. xylosus SX 2133) was tested with the seven semipurified enterocins. S. haemolyticus SHAe 111, S. haemolyticus SHAe 371, and S. warneri SW 292 were sensitive to six of seven tested enterocins, with inhibitory activity in the range from 200 to 12,800 AU/mL (for the strains SHae 111 and SW292, Table 3) and from 200 to 1600/3200 for strains SHae371 and SX2133. S. xylosus SX 2133 was sensitive to five of seven enterocins (inhibitory activity range, 400–3200 AU/mL) (Table 3). The most sensitive was S. warneri SW 292; inhibitory activity reached by Ent 2019 was 12.800 AU/mL.

Table 3.

Sensitivity of Methicillin-Resistant Staphylococci to Enterocins

	Staphylococcus strains
Enterocins	SHae111	SHae371	SW292	SX2133
EntEM41	200	200	–	–
Ent A(P)	800	200	200	400
Ent55	6400	1600	3200	3200
EntM	800	200	400	400
Ent412	–	200	800	800
Ent2019	3200	800	12,800	800
EntM3a	200	–	100	–

Inhibitory activity is expressed in Arbitrary units per milliliter (AU/mL). The initial activity of enterocins tested against the principal indicator Enterococcus avium EA5 ranged up to 51,200 AU/mL.

Ent2019, enterocin produced by E. faecium EF2019 (CCM7420) from rabbits feces; Ent412, produced by E. faecium strain from horses feces; Ent55, produced by E. faecium EF55 from chicken crop; Ent A(P), enterocin produced by E. faecium EK13 (CCM7419) (environmental origin); EntEM41, enterocin EM41 produced by Enterococcus faecium strain from ostriches feces; EntM, produced by E. faecium AL41 (CCM8558) isolated from environment; EntM3a, E. faecium isolated from rabbit meat; SHae, S. haemolyticus; SW, S. warneri; SX, S. xylosus.

Discussion

Staphylococcal species variability was noted in the feces of ostriches. Strains of eight different species were determined and clustered to four different groups/clusters following the taxonomy reported by Takashi et al. (1999). The most frequently detected were the species of the S. saprophyticus cluster—the species S. equorum, S. xylosus, S. succinus, and S. cohnii—and then the representants of the S. haemolyticus cluster, such as S. haemolyticus and S. hominis. The representant species of S. epidermidis cluster was also detected, the strains of S. epidermidis (SE211, SE273, SE281, SE3122), as was the representant of the S. warneri cluster—S. warneri (SW292). All species detected belong to coagulase-negative staphylococci. Coagulase-positive staphylococci were not detected. The identification of staphylococci to the species level was in accordance with secure genus and probable genus/species identification (value score up to 2.299) confirmed by phenotypic properties. Phenotypic properties were in accordance with the properties for formerly mentioned species reported by De Vos et al. (2009) for type strains. S. cohnii, S. succinus, and S. hominis were the species detected also in the feces of common pheasants (Lauková and Kandričáková, 2015). The representatives of coagulase-negative staphylococci (CoNS) are dominant staphylococci also in ruminants (Lauková and Mareková, 1993), horses (Lauková et al., 2011), and small wild mammals (Hauschild, 2001). The count of staphylococci in the feces of ostriches correlates with the counts enumerated in another poultry. For example, staphylococci in the count 4.78 ± 0.17 log10 CFU/g were enumerated in the feces of turkeys (Marciňáková et al., 2005); in feces of common pheasants, staphylococci were detected in the count 3.79 ± 0.9 log10CFU/g (Lauková and Kandričáková, 2015). In cecum of Japanese quails, staphylococci were detected in the average count 2.50 ± 013 log 10 CFU/g (Lauková et al., 1991).

Staphylococci belong to lactic acid bacteria, but compared with lactobacilli or enterococci they produce lower values of LA. The LA values detected in this study were similar to those in ruminal CoNS reported by Lauková (1994) or in CoNS isolated from common pheasants (Cohn and Middleton, 2010). Our strains did not show bacteriocin activity; however, there are some bacteriocin-producing CoNS, for example, S. xylosus SO3/1M/1/2 (Lauková et al., 2010) is a bacteriocin-producing strain and its inhibitory effect could by potentiated also by LA effect.

Frequent use of antibiotics can cause the problem with antibiotic-resistant bacteria. As previously mentioned, genes of antibiotic resistance can be carried by DNA plasmids or DNA integrons from antibiotic-resistant donor cell to antibiotic-sensitive recipient cell (Hawkey, 2010). Our strains were mostly sensitive to antibiotics. Similarly, Mauriello et al. (2000) described, for example, some S. cohnii, S. xylosus, S. saprophyticus, and S. hominis isolates from food to be sensitive to vancomycin, chloramphenicol, lincomycin, and oxacillin. In spite of increasing occurrence of methicillin resistant CoNS in general and/or among different animals such as dogs, cats, horses, and fishes (Couto et al., 2011; Lauková et al., 2011; Ruzauskas et al., 2014; Sargelidis et al., 2014), in our study only four methicillin-resistant strains were found (S. haemolyticus SHAe 111, S. haemolyticus SHAe 371, S. xylosus SX 2133, and S. warneri SW 292) among ostrich's staphylococci. It has been speculated that CoNS may be the original source of the mecA gene (Cohn and Middleton, 2010). Although CoNS are traditionally not considered as pathogenic, they were detected in some systemic infections in dairy or dogs (Gilespie et al., 2009). Especially because of methicillin resistance, researchers have been looking for innovative methods to eliminate methicillin-resistant strains. The use of bacteriocins–antimicrobial substances is very promising. Enterocins (bacteriocins produced mostly by enterococci) are known to possess broad antimicrobial activity (Franz et al., 2007). Our methicillin-resistant strains (S. haemolyticus SHAe 111 and, SHAe 371, S. xylosus SX 2133, and S. warneri SW 292) were sensitive to enterocins (inhibitory activity in concentration range 200–12 800 AU/mL). Moreover, staphylococci are Gram-positive bacteria that are in general more sensitive to enterocins or bacteriocins compared with Gram-negative bacteria. Also in our previous studies, staphylococci including CoNS (even Met^R) were found sensitive to enterocins or the other bacteriocin under both in vitro (Lauková, 1994; Lauková et al., 2011; Lauková et al., 2015) and in vivo conditions, meaning a decrease of staphylococci in, for example, rabbits treated with Ent M (Lauková et al., 2012a) or the anti-staphylococcal effect of enterocin in Sunar and yogurt (Lauková et al., 1999).

Results presented here highlight and expand our knowledge concerning the staphylococcal microflora in ostriches and also enterocins produced by the strains E. faecium isolated and characterized in our laboratory; these results contribute mainly to the basic research. In addition, it is information regarding the application possibilities of enterocins: they can probably be used to prevent staphylococcal bacteriosis.

Footnotes

Acknowledgments

This work was supported by the projects Vega 2/0002/11 and 2/0004/14 by Slovak Scienific Agency. We would like to thank Mrs. Margita Bodnárová for her excellent laboratory work and the farm supervisor for his kindness and permission to perform sampling.

Disclosure Statement

There is no conflict of interest to declare. No competing financial interests exist.

References

Alatoom

, Cunningham

, Ihde

, Mandrekar

, Patel

. Comparison of direct colony method versus extraction method for identification of Gram-positive cocci by use of Bruker Biotypermatrix-assissted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol, 2011; 49:2868–2873.

[CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard, 11 ^th ed. CLSI document M02-A11. Wayne, PA: Clinical and Laboratory Standards Institute, 2012.

Cohn

, Middleton

. A veterinary perspective on methicillin-resistant staphylococci. J Vet Emerg Clin Care, 2010; 20:31–45.

Couto

, Pomba

, Moodley

, Giardabassi

. Prevalence of meticillin-resistant staphylococci among dogs and cats at a veterinary teaching hospital in Portugal. Vet Rec, 2011; 169:72.

De Vos

, Garrity

, Jones

, Krieg

, Ludwig

, Rainey

, Schleifer

K-H

, Whitman

(eds.). Bergey's Manual of Systematic Bacteriology, 2 ^nd ed., vol. 3. New York: Springer, 2009.

De Vuyst

, Callewaert

, Pot

. Characterization of antagonistic activity of Lactobacillus amylovorus DCE471 and large scale isolation of its bacteriocin amylovorin L471. Syst Appl Microbiol, 1996; 19:9–20.

Franz

, van Belkum

, Holzapfel

, Abriouel

, Gálvez

. Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev, 2007; 31:293–310.

Gilespie

, Headrick

, Boonyayatra

, Oliver

. Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds. Vet Microbiol, 2009; 134:65–72.

Hauschild

. Phenotypic and genotypic identification of staphylococci isolated from wild small mammals. System Appl Microbiol, 2001; 24:411–416.

10.

Hawkey

. Methicillin resistant S. aureus . J Antimicrob Chemother, 2010; 65, Suppl 1:13–17.

11.

Lauková

. Antimicrobial susceptibility of ruminal coagulase-negative staphylococci. Microbiologica, 1994; 17:123–132.

12.

Lauková

, Chrastinová

, Pogány Simonová

, Strompfová

, Plachá

, Čobanová

, Formelová

, Chrenková

, Ondruška

. Enterococcus faecium AL41, its Enterocin M and their beneficial use in rabbits husbandry. Prob Antimicrob Prot, 2012a;4:243–249.

13.

Lauková

, Czikková

, Burdová

. Anti-staphylococcal effect of enterocin in Sunar and Yogurt. Folia Microbiol, 1999; 44:707–711.

14.

Lauková

, Hádryová

, Imrichová

, Strompfová

, Kandričáková

. Enterococus faecium EM41 isolate from ostrichs and its enterocin. Folia Vet, 2012b;56 Suppl. 2:34–36.

15.

Lauková

, Kandričáková

. Staphylococci detected in faecal samples of common pheasants and their relation to enterocins. Int J Curr Microbiol Appl Sci, 2015; 4:788–797.

16.

Lauková

, Kmeť

, Boďa

. Microflora of Japanese quails under space flight conditions. Curr Trends in Cosm Biol, 1991; 2:225–232.

17.

Lauková

, Mareková

. Antimicrobial spectrum of bacteriocin-like substances produced by rumen staphylococci. Folia Microbiol, 1993; 38:74–76.

18.

Lauková

, Mareková

, Štyriak

. Inhibitory effect of different enterocins against fecal bacterial isolates. Berl Muench Tierarztl Wschr, 2003; 116:37–40.

19.

Lauková

, Simonová

, Strompfová

. Staphylococcus xylosus SO3/1M/1/2, bacteriocin- producing meat starter culture or additive. Food Control, 2010; 21:970–973.

20.

Lauková

, Simonová

, Strompfová

, Štyriak

, Ouwehand

, Várady

. Potential of enterococci isolated from horses. Anaerobe, 2008; 14:234–236.

21.

Lauková

, Strompfová

, Kubašová

, Pogány Simonová

. In vitro inhibitory activity of lantibiotic gallidermin against methicillin resistant staphylococci from dogs. In: Book of Contributions—International Conference “Structure and Stability of Biomacromolecules SSB 2015,” Košice, Slovakia, June 30–July 3, 2015. Bágeľová

, Fedunová

, Gažová

, Šipošová

(eds.). Košice, Slovakia: SSB, pp. 126–127.

22.

Lauková

, Strompfová

, PogánySimonová

, Szabóová

. Methicillin-resistant Staphylococcus xylosus isolated from horses and their sensitivity to enterocins and herbal substances. Slovak J Anim Sci, 2011; 44:167–171.

23.

Marciňáková

, Strompfová

, Boldižárová

, Simonová

, Lauková

, Naď

. Effect of potential probiotic Enterococcus faecium strains on selected microflora in turkeys. Czech J Anim Sci, 2005; 50:341–346.

24.

Mareková

, Lauková

, De Vuyst

, Skaugen

, Nes

. Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13. J Appl Microbiol, 2003; 94:523–530.

25.

Mareková

, Lauková

, Skaugen

, Nes

. Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41. J Ind Microbiol Biotechnol, 2007; 34:533–537.

26.

Mauriello

, Moschetti

, Villani

, Blaiotta

, Coppola

. Antibiotic resistance of coagulase-negative staphylococci isolated from artisanal baples-type salami. Int J Food Sci Nutr, 2000; 51:19–24.

27.

Pogány Simonová

, Lauková

, Chrastinová

, Plachá

, Strompfová

, Čobanová

, Formelová

, Chrenková

. Combined administration of bacteriocin-producing, probiotic strain Enterococcus faecium CCM7420 with Eleutherococcus senticosus and their effect in rabbits. Pol J Vet Sci, 2013; 16:619–627.

28.

Ruzauskas

, Siugzdinie

, Klimenie

, Virgalis

, Mockeliunas

, Vaskeviciute

, Zienius

. Prevalence of methicillin-resistant Staphylococcus haemolyticus in companion animals: A cross-sectional study. Ann Clin Microbiol Antimicrob, 2014; 13:56–62.

29.

Sabolová

, Egyedová

, Gulovič

. Ostrich as a farming animal. Slovak Vet J, 2008; 32:21–27. (In Slovak.)

30.

Sargeliis

, Abrahim

, papadopoulos

, Soultos

, Martyiou

, Koulourida

, Govaris

, Pexara

, Zdragas

, Papa

. Isolation of methicillin-resistant Staphylococcus spp. from ready-to-eat fish products. Lett Appl Microbiol, 2014; 59:500–506.

31.

Strompová

, Lauková

. In vitro study on bacteriocin production of Enterococci associated with chickens. Anaerobe, 2007; 13:228–237.

32.

Szabóová

, Lauková

, Chrastinová

. Antibacterial activity of oregano and sage plant extracts against decarboxylase-positive enterococci isolated from rabbit meat. Potravinárstvo, 2013; 7:18–21.

33.

Tkashi

, Satoh

, Kikuchi

. Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis. Int J Syst Bacteriol, 1999; 49:725–728.