Rendiconti Lincei. Scienze Fisiche e Naturali https://doi.org/10.1007/s12210-018-0722-7

Distinctive antagonistic role of new Enterococcus faecium ER‑3M strain and its bacteriocin effect against Staphylococcus aureus Pneumonia Marwa S. Abdel‑Hamid1 · Anis Anis2   · Rania H. Elbawab3 · Abeer A. B. Mohammed1 · Sahar H. Orabi4 · Said I. Fathalla5 Received: 16 June 2017 / Accepted: 4 June 2018 © Accademia Nazionale dei Lincei 2018

Abstract Bacteriocins secreted by some bacteria such as Enterococcaceae has a protected role against Staph. aureus pneumonia. This study identified a new Enterococcus sp. that produces efficient bacteriocin which attenuates Staph. aureus pneumonia in rats. Identification of Enterococcus sp. was achieved according to Bergey’s manual of systematic bacteriology using 16S rRNA analysis. Bacteriocin was extracted, partially purified and characterized. Antagonistic activity of the bacterial cells or its bacteriocin was estimated in vitro against different pathogenic bacteria and in vivo with an animal experiment, where histopathological and hematological examinations were evaluated. The isolate was identified as Ent. faecium ER-3M, with accession number KY774316. It exhibits broad antimicrobial spectrum against Gram-positive and Gram-negative organisms in vitro. Treatment of rats with bacterial cells or bacteriocin (500 Au mg−1), previously infected with Staph. aureus, significantly decreased the symptoms, WBCs, neutrophils, and liver functions. Histopathological examination of rat lung tissues revealed that treatment with probiotic bacteria and/or its bacteriocin can attenuate the pneumonic lesions caused by Staph. aureus bacteria. Partially purified bacteriocin was characterized by a large molecular weight of approximately 40 kDa a proteinous nature according to both Fourier transform infrared spectrometry (FTIR) spectroscopy and 1H-Nuclear magnetic resonance (NMR) analysis and belongs to class III of bacteriocins termed enterolysin. Based on the results, the successful treatment of Ent. faecium ER-3M or its bacteriocin may be one of the promising treatments against Staph. aureus pneumonia. Keywords  Enterococcus faecium · Bacteriocin · Antagonistic activity · Staphylococcus aureus pneumonia

* Marwa S. Abdel‑Hamid [email protected]; [email protected]

Anis Anis [email protected]; [email protected]

Rania H. Elbawab [email protected]

2



Pathology Department, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Menoufyia Governorate, P.O. Box 79/22857, Egypt

3



Ministry of Health, Health Insurance Organization (HIO), Sadat City Clinic, Sadat City, Menoufyia Governorate, Egypt

4



Biochemistry and Chemistry of Nutrition Department, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Menoufyia Governorate, P.O. Box 79/22857, Egypt

5



Physiology Department, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Menoufyia Governorate, P.O. Box 79/22857, Egypt

Abeer A. B. Mohammed [email protected] Sahar H. Orabi [email protected] Said I. Fathalla [email protected] 1



Microbial Biotechnology Department, University of Sadat City, Genetic Engineering and Biotechnology Research Institute, Sadat City, Menoufyia Governorate, P.O. Box 79/22857, Egypt

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1 Introduction The nosocomial infections caused by multidrug-resistant microorganisms (MDR) are the most serious problems of clinical medicine and pose a major public health concern (Cecchini et al. 2015). Staph. aureus is the main bacteria associated with nosocomial infections (Fuzi 2016). MDR develops numerous strategies of resistance to antimicrobial agents and host defenses. Therefore, patients infected by these microorganisms have increased length of hospital stay and the rate of mortality compared to individuals infected with susceptible strains (Spicknall et  al. 2013). Staph. aureus pneumonia specifies 1–10% of cases of communityacquired pneumonia and 16% of cases of nosocomial pneumonia (Tong et al. 2015). Furthermore, Staph. aureus can induce hemorrhagic necrotizing pneumonia with massive polymorph nuclear leukocyte infiltration in lung parenchyma and the formation of micro-abscesses this result was later contradicted by Hamouda and Abou-El-Souod (2018). Microorganisms have secreted various biomaterials that support the limitation of infection of Staph. aureus and E. coli (El Bialy et al. 2017) and support our environment as reducing wastewater Abd El-Zaher et al. 2017); formation lipids that can be converted to biofuel (Hamouda et al. 2016; El-Sheekh and Hamouda 2016) and, meanwhile, could produce bio nanoparticles that inhibit Ehrlich ascites carcinoma tumor in mice (O’Connor et al. 2015). Otherwise, lactic acid bacteria produce ribosomal synthesized antimicrobial peptides or bacteriocins which attract considerable attenuation as natural and nontoxic food preservatives (Cotter et al. 2013) and as therapeutic agents for human and veterinary applications and in the animal production field (Jiménez et al. 2015). Bacteriocin-producing lactic acid bacteria are also promising vectors for delivery of therapeutic peptides and as probiotics (Shehata et al. 2016). Enterococcus faecium is potential probiotic that known to have desirable characteristics as resistance to gastric juice and bile salts and production of antimicrobial compounds such as bacteriocins (Ford et al. 2014). Bacteriocin family is the most abandoned and diverse group of bacterial defense system (Yang et al. 2014). Probiotic strains as Enterococcus sp. are not only secreting bacteriocin that significantly inhibited Staph. aureus infection, but also treating types of diseases at different sites of the human body-oral cavity, respiratory tract, gastrointestinal tract, and urogenital tract Girardin and Seidman (2011). Zacharof and Lovittb (2012) reported that bacteriocins have been classified on the basis of their molecular mass; biochemical characteristics and mode of action against related groups into three groups. Class I termed lantibiotics, which have molecular mass varies between 2 and 4 kDa and act through pore formation. Class II nonlantibiotics are containing small (4–6 kDa), heat-stable,

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Rendiconti Lincei. Scienze Fisiche e Naturali

non-modified peptides (Arnison et al. 2013). Class III bacteriocins: This class contains of heat labile proteins which has a large molecular weight (> 30 kDa) and classified into two subunits: helveticin I produced by Lactobacillus helveticus and enterolysin produced by Enterococcus faecium (Alvarez-Sieiro et al. 2016). Most of LAB bacteriocins are amphiphilic and membrane permeable peptides. The cell wall of Gram-positive (+) bacteria permits passage of relatively large molecules. Teichoic and lipoteichoic acids, as an important part of the cellular wall, play essential role in the initial interaction of anionic bacteriocins produced by Gram-positive (+) bacteria. Bacteriocin may be amino acid sequence homologies not only within the mature peptide, but also in the N-terminal leader region and the associated proteins in bacteriocin secretion and function (Garsin et al. 2014). Antimicrobial compounds or the chemical nature of bacteriocin can be identified by Fourier transform infrared spectrometry (FTIR) and nuclear magnetic resonance (NMR) spectroscopy as reported in Yamamoto et al. (1994) and Marshall and Rawson (1999), a powerful and rapid tool for identification structural studies of bacteriocin. The present search was focused on identification a new Enterococcus spp., which, able to produce effective medical products as bacteriocin. Moreover, the evaluation of antagonistic role the new Enterococcus faecium ER-3M strain as its bacteriocin against Staphylococcus aureus Pneumonia in rats was targeted.

2 Experimental Present investigation was carried out in Microbial Biotechnology department, Genetic engineering and biotechnology institute in cooperation with physiology department, faculty of veterinary medicine university of Sadat city, Menoufyia Governorate, Egypt. All the microbial growth media constitutes were obtained from Thermo fisher (Oxide microbiology product, UK). Antibiotics discs were provided from Sigma-Aldrich™. ­Nucleospin ® Extract II kit (MACHEREY–NAGEL GmbH & Co. KG, Germany) was used to purify the PCR Products. The primers 27F (5′-GAG AGT TTG ATC CTG GCT CAG-3′) and 1495R (5′-CTA CGG CTA CCT TGT TAC GA-3′) were used for the amplification of the 16S rRNA gene, while primer Bact1098R used in sequencing (5′-AAG​GGT​TGC​GCT​CGT​TGC​G- 3′) was used for fragment sequencing. Pure double distilled water was used as solvent solution in all the experiment units. All other chemicals were of analytical grade. All rats’ experimental design and conditions were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, University of Sadat City, Egypt.

Rendiconti Lincei. Scienze Fisiche e Naturali

2.1 Enterococcus sp. isolation and identification Local Enterococcus sp. ER-3M was isolated from cattle raw milk in Sadat city, (30.446369N, 30.624044E), Menoufyia Governorate, Egypt. Cells were isolated on MRS medium (De Man et al. 1960) containing 10.0 g/l peptone, ‘8.0 g/l Lab-Lemco’ powder, 4.0 g/l yeast extract, 20.0 g/l glucose, 1 ml sorbitan mono-oleate, 2.0 g/l dipotassium hydrogen phosphate, 5.0 g/l sodium acetate 3­ H2O, 2.0 g/l triammonium citrate, 0.2 g/l magnesium sulphate ­7H2O, 0.05 g/l manganese sulphate ­4H2O, 10.0 g/l agar, and pH 6.2 ± 0.2 at 25 °C. The isolate was identified according to Bergey’s| manual of systematic bacteriology (Brenner et al. 2005). The morphology of cells was examined by Transmission Electron Microscope (TEM) to investigate the surface of cells using Joel 1200-EXII, Tokyo, Japan.

2.2 Safety assessment of isolated Enterococci Safety assessment tests were performed to confirm the absence of virulence characteristics of the isolated Enterococci and to determine potential pathogenic activity containing the following:

2.3 Antibiotic susceptibility The tested antibiotics comprised, namely, Vancomycin (VA30), Streptomycin (S10), Kanamycin (Kan30), Ciprofloxacin (CIP5), Oxy-tetracycline (T30), and Gentamycin (CN10). Inoculum of ER-3M (100 µl) containing 2.6 × 107 CFU ml−1 was plated and disc-diffusion assay was carried out in accordance with the clinical and laboratory standard institute guidelines (Arnison et al. 2013). After incubation at 37 °C for 24 h, the data were recorded for the growth parameters or not according to (De Kraker et al. 2011).

2.4 Coagulase test Two test tubes of citrated rabbit plasma (0.5 ml) were used in this test. The first tube was inoculated with enough Staph. aureus paste (50 µl) for 18–24 h broth culture to each tube to make a cloudy suspension. The other tube was inoculated with Ent. faecium ER-3M. Both tubes were incubated at 35 °C for 1–4 h in a water bath. Afterward, examine both tubes for the presence or absence of clouding and clots. Staph. aureus was used as a positive control in this experiment according to Clinical and Laboratory Standards Institute (2010).

2.5 Gelatinase test A heavy inoculum of Ent. faecium ER-3M (18–24 h) was inoculated on the tube containing nutrient gelatin medium

(peptone 5 g, beef extract 3 g, and gelatin 120 g/l. The inoculated tube and the uninoculated one were kept at 37 °C up to 2 weeks. The tubes were taken daily from the incubator and placed in the ice bath or refrigerator (4 °C) for 15–30 min (until control is gelled) to check for gelatin liquefaction. The tubes were immersed in an ice bath or kept at 4 °C to confirm that liquefaction was due to gelatinase activity. Staph. aureus was used as a positive control in this experiment (Prescott 2002).

2.6 Haemolytic activity Enterococcus sp. was tested for hemolytic activity, by the method described by Prescott (2002). Staph. aureus was used as a positive control in this experiment.

2.7 Molecular identification of isolated Ent. faecium ER‑3M Genomic DNA of the isolate was extracted from cells according to the protocol for bacterial DNA extraction with the Gene Jet Genomic DNA purification and visualization on 1% agarose gels (Vogelstein and Gillespie 1979). The 16srRNA gene of the new isolate was amplified by thermocycler (Biometra thermocycler, Germany) using universal primers; Bact 27f (5′-AGA​GTT​TGATC(A/C)-TGG​CTC​ AG-3′), Bact1492r (5′-TACGG(C/T)-ACC​TTG​TTA​CGA​ CTT-3′), and Bact 1098r (5′-AAG​GGT​TGC​GCT​CGT​TGC​ G- 3′) (Chang et al. 2000; Picard et al. 1999). DNA sequencing reactions were performed using Applied B ­ iosystems® 3500 Genetic Analyzers. Blasting of the sequences has been carried out from sequences into gene bank NCBI (National Center for Biotechnology Information) to search the similarities and characteristics. The determined 16S rRNA gene nucleotide sequences were entered for BLAST searching the Web site of NCBI (http://www.ncbi.nlm.nih.gov/blast​/).

2.8 Growth curve of the Ent. faecium ER‑3M and bacteriocin production Ent. faecium ER-3M was inoculated in MRS broth at 37 °C for 48 h with constant shaking at 150 rpm, and the aliquot of the culture medium turbidity was measured spectrophotometrically 280 nm at different time intervals. Bacteriocin production was determined by measured inhibition zone after 0, 2, 4, 6, 8, 10, 12, 24, and 48 h (Chang et al. 2000). The arbitrary unit (AU) was defined as the reciprocal of the highest dilution (1:1), (1:2), (1:4), (1:8), and (1:16) producing a clear zone of growth inhibition of the test isolate. AU was calculated as (1000/100) × D, where 1000: constant, 100: volume of supernatant in a well (μl) and D: the dilution factor (Yusuf and Abdul Hamid, 2012). The activity units of bacteriocin were examined by serially diluted twofold with

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phosphate buffer (10 mM, pH 7.0). About 100 µl from each dilution was added to the plates containing Staph. aureus as indicator strain to determine bacteriocin activity by welldiffusion assay.

2.9 Antibacterial spectrum The antibacterial spectrum of identified strains was performed by agar well-diffusion assays as described by De Kraker et al. (2011). Staphylococcus aureus ATCC6538, Proteus mirabilis ATCC 9240, and E. coli were provided from Microbial Biotechnology Department, GEBRI, University of Sadat City, Egypt. Salmonella typhimurium ATTC 25566, listeria monocytogenes ATCC19116, and Bacillus cereus DSN354 have been obtained from MIRCEN Fac. of Agriculture, Ain Shams University, Egypt for screening bacteriocin production. Initial inoculum 1­ 05 CFU ml−1 of the indicator bacteria was used according to Pilasombut et al. (2005).

2.10 Determination of total protein Total protein of bacterial supernatant was determined using BioMed-Total Protein kit (BIOMED diagnostic, Germany) using bovine albumin as standard to calculate specific and total protein activities measured by the method of Lowry et al. (1951).

2.11 Extraction and purification of bacteriocin Culture Free Supernatant (CFS) prepared from the culture of the Enterococcus sp. grown in MRS broth medium at 37 °C for 24 h. The cells were removed by centrifugation (10,000 rpm, 4 °C 20 min) supernatants containing bacteriocins were harvested, neutralized to pH 7 with 1 M NaOH and treated with catalase (1 mg/ml) to avoid inhibitory activity due to hydrogen peroxide and filter sterilized through 0.45 µm filters to remove cellular debris. This CFS was brought to a final ammonium sulphate concentration of 40, 50, 60, and 70% saturation by slow addition of the salt, and was stirred overnight at 4 °C. Then, the mixture was centrifuged at 6000 rpm on 4 °C for 30 min) and the formed pellicles were harvested and resuspended in 10 ml of 0.1 M sodium phosphate buffer (pH 6). This partially purified bacteriocin was stored at − 20 °C. Dialysis contain 500 ml of phosphate buffer (pH 6) in SPECTRA/POR1 Dialysis Tubing MWCO laboratories-SERVA) at 4 °C overnight (Aslam et al. 2011). Gel filtration chromatography Sephadex G-100 and 0.1 M phosphate buffer (pH 6) the sample fractions each of 2.0 ml were collected at a flow rate of 2 ml/min and read at 280 nm using spectrophotometer according to (Al-Saman et al. 2015). Antimicrobial assay against Staph. aureus and protein concentration were performed.

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2.12 Partial characterizations of extracted bacteriocin Partially purified bacteriocin was conducted by their chemical structure and molecular weight as: (1) determination of the molecular weight using Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) according to the method described by Laemmli (1970). (2) Fourier transform infrared spectrometry (FTIR) (JASCO, IR Affinity 1, Tokyo, Japan) determines its functional groups. The recorded spectrum was determined in the range of 4000–400 cm−1. (3) The atomic components of the extracted bacteriocins were identified by 1H-nuclear magnetic resonance (NMR) analyses. The NMR data were recorded on JEOL-ECA (MA, USA) at 400 and 100 MHz. Calibration was performed using the signal of the residual protons of the solvent as a secondary reference. Deuterium oxide was obtained from SDS (Vitry, France). (4). Amino acid analysis of bacteriocins was carried out by amino acid analyzer/ionexchange resin with ninhydrin post-column derivatization Biochrom 30 instruction manual. (5). Effect of temperature on bacteriocin activity was carried out at 50 and 60 °C, for 15, 30, and 60 min. The residual activity was then determined for each analysis by agar well-diffusion assay (Heng et al. 2007).

2.13 Animal experiment Thirty female Albino rats (body weight about 100 g) were used for the in vivo study. Animals were kept for 1 week to accommodate on laboratory conditions. Control and treated animals were fasted for 12 h with free access to water before each experiment (Swarnakar et al. 2005). The animals were divided into five groups of six animals each. Subcutaneous injection of Staph. aureus was used for pneumonia induction. Experimental design of the animal experiment of this study is presented in Table 1. Rats were killed under halothane inhalation anesthesia. Lung tissues were taken and fixed in 10% neutral buffered formalin (NBF) for histopathological investigation. After 72 h of fixation, samples were dehydrated, embedded in paraffin wax, and sectioned (3 µm) for hematoxylin and eosin (HE) staining. Histological photos were taken by using Leica EC3 digital camera. Hematological parameters such as red blood cell count (RBCs), hemoglobin concentration (Hb), packed cell volume (PCV), and total (TLC) and differential leukocytic counts were evaluated as described previously (Young 2001). Albumin, Serum glucose level, and CRP were estimated by the methods of Lowry et al. (1951), Young (2001), Swarnakar et al. (2005), respectively. In addition, the activities of serum AST and ALT were estimated as described previously (Bowman 1993).

Rendiconti Lincei. Scienze Fisiche e Naturali

2.14 Statistical analysis Data were expressed as mean ± SE and analyzed using a one-way analysis of variance (ANOVA) with Duncan’s post hoc test to determine the significant differences among data in this study. The differences between means were analyzed at the level (P ≤ 0.05) and were considered statistically significant (Duncan 1955).

3 Results 3.1 Biochemical, molecular and safety assessment characterizations of Enterococcus sp. The results confirmed that Ent. faecium ER-3M has a broad spectrum against Gram-negative and positive bacteria (Table 2). The promising isolate ER-3M showed the highest inhibition zone against Staph. aureus (15 mm). On the other hand, this isolate produced antibacterial activity with

10 and 5 mm in diameter against E. coli and Proteus mirabilis ATCC 9240, respectively. Moreover, our isolate did not produce any activity against Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus. Results clarify that ER-3M isolate is highly sensitive to ciprofloxacin, oxy-tetracycline, kanamycin, gentamycin, streptomycin, and vancomycin, respectively, as described in Table 3. Assessment of virulence factors by qualitative tests revealed that the isolate was not hemolytic in blood agar base media (Thermo scientific, Oxoid microbiology, UK) supplemented with 5–7% of sterile sheep blood. Both gelatinase and coagulase were negative with our isolate. Micromorphology of the isolate showed cocci to cocobacilli in shape that aggregates in different patterns (single, diplo or clusters). Colony morphology of our isolate on MRS agar was off-white, round, with smooth edges and raised up from a centre. The isolate was Gram-positive cocci with an arrangement of singles cells to clusters for Enterococcus sp. The isolate was fermented glucose without gas production and does not produce a catalase enzyme with

Table 1  Experimental design of animal experiment in this study Group no.

Dose of subcutaneous injection

Dose of treatment via gastric tube

G1 (negative control) G2 G3

0.1 ml saline/10 days 100 µl of Staph. aureus (3.0 × 107 CFU ml−1) 2 days 100 µl of Staph. aureus (3.0 × 107 CFU ml−1) 2 days 100 µl of Staph. aureus (3.0 × 107 CFU ml−1) 2 days None

0.1 ml saline/10 days None Bacteriocin* of Ent. faecium ER-3 M (500 AU ­ml−1)/10 days

G4 G5

Ent. faecium ER-3 M cells 250 µl (2.6 × 106 ­CFUml−1)/10 days Ent. faecium ER-3 M cells 250 µl (2.6 × 106 ­CFUml−1)/10 days

*Bacteriocin was sterilized by filtration in 0.45 mm Millipore filters

Table 2  Antimicrobial activity of isolated Ent. faecium ER-3M against indicators’ bacteria Bacteriocin producer strain code

ER-3M

Diameter zone (mm) Indicator bacteria Staph. aureus ATCC6538

E. coli

Proteus mirabilis ATCC 9240

Listeria monocytogenes ATCC19116

Salmonella typhimurium ATTC 25566

Bacillus cereus DSN354

15

10

5

0

0

0

Table 3  Safety assessment of Enterococcus sp. in this study

Isolate

Virulence factors Haemolytic activity Coagulase test Gelatinase test

Antibiotic susceptibility K 30 T 30 CN 10 S 10 VA 30 CIP 5 Diameter of inhibition Zone (mm) ER-3M 25

39

23

15

15

40

−ve

−ve

−ve

CIP ciprofloxacin, T oxy-tetracycline, K kanamycin, CN gentamycin, S streptomycin, VA vancomycin

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13

− + + + + + + + + ER-3M +

10 45

7 8 9.6 2 3 4 6.5

+

+

+

+

+

+

+

−

−

−

−

Kanamycin escu- Sugars fermentation Gram stain Starch Urease test Citrate test Oxilin azide medium hydrolysis dase Ribose Mannitol Lactose Sorbitol Raffinose test NaCl  % pH value Growth temperature (°C)

Biochemical characterizations Isolate

Table 4  Biochemical characterizations of Ent. faecium ER-3M

Catalase test



hydrogen peroxide. It catabolized a variety of energy sources including ribose, mannitol, lactose, raffinose, and sorbitol. Meanwhile, the isolate can survive environmental stress including extremely alkaline pH (9.6) and salt concentrations until 6.5% NaCl, as shown in Table 4. ER-3M isolate can grow in the range of 10–45 °C and able to grow in 0.9 M NaCl, and hydrolyze esculin in the presence of 40% bile salts (Fig. 1). The bacterium can analyze esculin to glucose which combines with iron ions to produce a black complex visible as black zone around colonies. These colonies appear smooth and creamy with entire edges (Fig. 1a). TEM investigation revealed a coupled bacterial cell with high-resolution back-scattered electron imaging. Cells were ovoid (75 µm), and occur singly or in pairs (Fig. 1b).

3.2 16SrRNA gene sequencing Figure 2 shows agarose gel PCR amplification fragment of 16S rRNA gene ~ 1.5 Kbp from local isolated Enterococcus sp. 16S rRNA sequences of isolate ER-3M that have been deposited in the Gene Bank Database under accession number KY7743160. Figure 3 which revealed phylogenetic tree of the new strain Ent. faecium ER-3M indicating the genetic relationship with the references strains.

3.3 Growth curve Ent. faecium ER‑3M and bacteriocins activity The culture supernatant of Ent. faecium ER-3M was sampled at various times during growth phases and tested for bacteriocin activity (Fig. 4). The exponential phase of growth started at the 4th hour from the time of incubation and this was maintained up to the 8th hour of growth. During this stage, there was an increase in the optical density 600 nm (from 0.77 to 2.86 nm) and the production of antimicrobial peptides starts at 8th hour which happens to be the peak of the exponential phase. Bacteriocin production started early during logarithmic phase (8th hour) and the activity reached to the maximum in early stationary phase 500 Au ml−1 and then remained constant for certain period (about 5 h) during this stage of growth. The stationary phase starts at the 8th hour of growth, while decline phase in optical density was observed at the 18th hour from the time of incubation from the onset of incubation with declining in the level of activity to reach 100 Au ml−1 after 24 h incubation.

3.4 Extraction and characterization of bacteriocin The results revealed that 60% saturation level appeared to be the best saturation level and highest activity for Enterococcus bacteriocin in comparison with dialysis bag and Sephadex G-100 as gel column chromatography (Fig. 5). Seventy-five fractions were collected for measuring protein

Rendiconti Lincei. Scienze Fisiche e Naturali

Fig. 1  a Bacterial growth on Kanamycin esculin azide agar medium, b photograph of the bacteria by scanning electron microscope examination of Ent. faecium ER-3M isolate (0.5 µm)

Fig. 2  Agarose gel electrophoresis of PCR amplification fragment of 16S rRNA gene ~ 1500 Kbp from our local isolated Enterococcus sp. Lane M: DNA marker (1 Kbp)

concentration and bacteriocin activity the eluted fractions were measured at 280 nm for bacteriocin activity that equilibrated and eluted with 0.1  M sodium phosphate buffer (pH 6), flow rate was 2 ml/min, in which absorbance was dramatically increased to reach 0.05 at fraction no. 40 and then fall down. Results in Table 4 indicated that ammonium sulfate and Sephadex methods reduce the activity  % of bacteriocin (160 and 500 Au ml−1), respectively, with a

Fig. 3  Phylogenetic tree of the new strain Ent. faecium ER-3M indicating the genetic relationship with the references strains

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600

3.5

500

3 2.5

400

2

300

1.5

200

1

100

0.5

0

Absorbance at 600 nm

Rendiconti Lincei. Scienze Fisiche e Naturali

Resdual Activity (Au ml -1)



0 0

2

4

6

8

10 12 14 16 18 20 22 24 36 48 Incubaon me (h)

Acvity (Au/ml)

Absorbance at 600nm

0.06 0.05

400

0.04

300

0.03

200

0.02

100

0.01

Absorbance (at 280 nm)

600 500

0

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47

Resudai Ac vity (Au ml -1)

Fig. 4  Growth curve of Ent. faecium ER-3M with bacteriocins activity

Frac on no. Acvity (Au/ml)

Absorbance (at 280 nm)

Fig. 5  Purification steps of bacteriocin produced by Ent. faecium ER-3M using Gel filtration chromatography

Fig. 6  Molecular weight of the bacteriocin was determined by SDSPAGE gel electrophoresis

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specific activity (83.33 and 333.33 Au/mg) in comparison with cell-free supernatant enhanced activity (500 Au ml−1). Meanwhile, the protein concentration was enhanced up to 1.92 mg/ml by ammonium sulfate, while it fell down with crude bacteriocin and Sephadex 1.52 and 0.24, respectively. Results showed that the specific activity was affected using ammonium sulfate gave 83.33 Au mg−1, while it gave higher value with crude bacteriocin and Sephadex 328.5 and 333.3 Au mg−1. The crude extract produced highest total activities (250,000 AU) with yield (100%) among the three steps of purification. The molecular weight of bacteriocin from Ent. faecium ER-3M was determined by SDS-PAGE. One protein band was observed when stained with coomassie blue was approximately appeared at 40 kDa. Results in Fig. 6 clearly indicated the purity of the protein. Evidence for the homogeneity of bacteriocin was provided by uniform activities for eluted fractions, further chromatography resulting in elution of a single peak of absorbance corresponding to activity, and absence of contaminating proteins on SDS-PAGE. In the present study, the FTIR spectroscopy was used to detect the functional group which it could be in partial purified bacteriocin. Bacteriocin absorbance in frequency at 3437.17 cm−1 was observed which it was referred to the large content of amine N–H stretch primary amines group. Absorbance band in the region of 2929.34 cm−1 indicated that alkyl C–H stretch group. In addition, formation of carboxylic acid OH stretch group which may include amino acids, protein, phosphodiester, and polysaccharide, as revealed by decreased in spectra in the region 2378.76 cm−1 Bacteriocins also showed frequency decrease in the range of amide C=O stretch 1639.2 which corresponding the carbonyl stretching groups. The FTIR spectrum of bacteriocin of Enterococcus reflects an increase in aromatic C=C bending 1408.75, C–O 1112.73 cm−1, and aromatic CH bending 610.36 cm−1, corresponding assignments of fatty acids (Fig. 7). Table 5 presents results of the proton NMR spectrum of the partial purified bacteriocin displayed, where the absence of aromatic protons and the presence of aliphatic chains (multipliers between 1 and 4 ppm). Signals in the anomeric region (about 4–3 and 1–2 ppm) of the spectrum and the coupling of the anomeric protons (57.40, 18.30, and 24.30) may provide useful information about the number of residues in a repeating unit and the anomeric configuration, respectively. Results given in Fig. 8 indicate that Ent. faecium ER-3M partially purified bacteriocin which contained 16 amino acids. Glycine, proline, and glutamic showed the higher content of almost 0.21, 0.17, and 0.15 g/100 ml, respectively. Cysteine, tyrosine, and histidine showed the lower content, reaching 0.01 g/100 ml. The concentration of the other amino acids was in the range 0.02–0.04 g/100 ml.

Rendiconti Lincei. Scienze Fisiche e Naturali Fig. 7  FTIR spectrum of bacteriocin of Ent. faecium ER-3M

Table 5  Purification steps of bacteriocin produced by Ent. faecium ER-3 M Purification steps

Volume (ml) 1 arbitrary unit (Au/ ml)

Cell-free supernatant (CFS) 500 Ammonium sulfate 60% precipitation 50 Sephadex G-100 8

500 160 80

2 specific Protein concentration (mg/ activity (Au/ mg) ml)

3 total activity (AU) 4 purification fold

5 yield (%)

1.52 1.92 0.24

250,000 8000 640

100 3.2 0.26

328.5 83.33 333.3

1 0.25 1.01

0.25 0.2

Residual acvity ( AU ml -1 )

Concentration (g/100ml)

Arbitrary unit AU: (1000/100) × D, where 1000: constant, 100: volume of supernatant in a well (μl), and D: the reciprocal highest dilution. Specific activity (Au/mg) represents bacteriocin activity divided by protein concentration. Total activity (Au) represents activity (AU/ml) × volume (ml). Purification fold represents the specific activity of purified fraction divided by specific activity of the crude extract. Yield (%) represents (a total activity of purified fraction divided by total activity of crude extract) × 100

0.15 0.1 0.05 0

Amino acids

500 450 400 350 300 250 200 150 100 50 0

37°C 50°C 60°C

Time (min)

Fig. 8  Amino acid analysis of partial purified bacteriocin produced by Ent. faecium ER-3M strain

Fig. 9  Effect of heat temperature on partial purified bacteriocin produced by Ent. faecium ER-3M strain

Results in Fig. 9 clarify that heat temperature affecting the sensitivity of the bacteriocin which started with 500 ­(AUml−1) at 37 °C. Residual activity was sharply fell down (200 and 100 ­AUml−1) after exposure at 50 °C for 15 and 30 min, respectively, while it lost its activity after 60 min. Residual activity was completely lost at 60 °C for 15 and 30 min and 1 hour.

3.5 Impact of Ent. faecium ER‑3M against Staph. aureus in vivo Results in Table 6 revealed a significant decrease in Staph. aureus count in blood (1.7 × 103 CFU ml−1). This decrease was observed on the 13th day post-infection in the group no (3) of infected rats that were treated with the bacteriocin

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Table 6  1H NMR spectral results of the extracted bacteriocin Functional group

1

1

H NMR(ppm) extracted Bacteriocin

Literature H NMR(ppm)

2 ­CH3 3,4 ­CH2 2,1 NH–CH2 1,1 ­NH2, NH –COOH CH2–C=O

2.51–2.52 3.4–3.9 1.14–1.13 and 1.8 4–4.3 – –

2.12 3.2 1.8 0 0 2.67

(500 Au ml−1). Ent. faecium ER-3M cells were used to treat group 4 post-infection which caused significant lower in the count of Staphylococci (1.1 × 102 C ­ FUml−1) in comparison with positive control, as shown in Table 6. Results in Table 7 indicated that the weight of all rats increased except in group no. 2 (91.83) g. A significant decrease in albumin value (P < 0.05) was obtained in group no. 2 which gave (2.22) g/ dl compared to control (1st group, 3.78 g/dl). An increase in ALT, AST, and CRP levels (21.67 and 13.50 μ/l) and (52.00 mg/l) was observed in G2 compared to control (1st group, 9.50 and 5.83 μ/l) and (3.00) mg/l, respectively, and there is no significant difference in glucose levels between groups 1 and 2 (71.67 and 77.67) g/dl. However, administration of bacteriocin and Ent. faecium ER-3M cell postinfection in groups 3 and 4 was normalized albumin (3.73 and 3.40 g/dl), ALT (10.83 and 9.67 μ/l) and AST (10.17 and 8.33 μ/l), and CRP (4.50 and 5.00 mg/l) levels, respectively. In addition, results in Table 7 in group no. 5 illustrate that there is no significant difference in ALT, AST, albumin, glucose, and CRP levels in group 2 (9 μ/l, 7.83 μ/l, 3.98 g/dl, Table 7  Effect of Ent. faecium ER-3 M cells and its bacteriocin on Staph. aureus count in blood culture

Table 8  Effect of Ent faecium ER-3M and its bacteriocin on serum biochemical tests of Staph. aureus infected rats

70.83 g/dl, and 3 mg/l, respectively) comparing to group no. 1(9.5 μ/l, 5.83 μ/l, 3.78 g/dl, 71.67 g/dl, and 3 mg/l, respectively). Moreover, there is an increase in body weight of group no. 5 (129.8 g) comparing to control group (120 g). Table 8 reveals significant decrease between the groups in Hb concentration, RBCs, hematocrit, and lymphocyte (11.78  g/dl, 5.43 × 106/mm 3, and 35.27%, 64.20 × 103/ mm3), respectively (p < 0.05) in group no. 2 compared to control (1st group) (12.57 g/dl, 6.80 × 106/mm3, 38.67%, 85.85 × 103/mm3). However, treatment with bacteriocin and Ent. faecium ER-3M postinfection in rats normalized Hb concentration, RBCs count, hematocrit%, and lymphocyte count. On the other hand, results in Table 9 indicated a significant increase in total leukocytic count (23.33 × 103/mm3) and neutrophils (35.80 × 103/mm3) in G2 compared to control (1st group) in which WBCS (12.82 × 103/mm3) and neutrophils (14.15 × 103/mm3). Supplementation of rats with bacteriocin and Enterococcus cells in groups 3 and 4 postinfection reduced WBCS (13.63, 14.18) × 103/mm3 and neutrophils (17.47, 17.38) × 103/mm3 compared to G2. There is increase in platelets count (679.83 × 103/mL) in G2 compared with group 1 (549.83 × 103/mL). At the other side, platelets counts were normalized in groups 3, 4, and 5. Hemorrhagic pneumonia was detected in lung tissues of Staph. aureus infected rat (G 2) and was characterized by extravasation of red blood cells in the alveolar and bronchiolar lumen, periarterial infiltration of inflammatory cells, periarterial edema, degenerative changes in Tunica muscular of the blood vessel, infiltration of mononuclear cells, and accumulation of eosinophilic material in the interstitial tissue and in the alveoli (Fig. 10b–d). Mild interstitial pneumonia

Group no. treatment

G3 Bacteriocin of Ent. faecium ER-3M

G4 Ent. faecium ER-3M cells

Prior infection (negative control) Post-infection (positive control) Post-treatment

No growth 3.3 × 107 CFUml−1 1.7 10 × 3 CFUml−1

No growth 2.8 × 107 CFUml−1 10 × 1.12 CFUml−1

Group no. parameter

G1

G2

G3

G4

G5

Body weight (g) ALT (μ/l) AST (μ/l) Albumin (g/dl) Glucose (g/dl) CRP (mg/l)

120 ± 2.2b 9.50 ± 0.76b 5.83 ± 0.95b 3.78 ± 0.29a 71.67 ± 5.74a 3.00 ± 0.00b

91.83 ± 0.79d 21.67 ± 1.84a 13.50 ± 2.77a 2.22 ± 0.27b 77.67 ± 5.16a 52.00 ± 9.63a

110 ± 2.9c 10.83 ± 1.01b 10.17 ± 0.87ab 3.73 ± 0.17a 78.00 ± 4.03a 4.50 ± 0.67b

107 ± 2.3c 9.67 ± 1.87b 8.33 ± 0.88b 3.40 ± 0.17a 76.00 ± 3.41a 5.00 ± 1.48b

129.8 ± 2.4a 9 ± 0.52b 7.83 ± 0.70bc 3.98 ± 0.22a 70.83 ± 1.25a 3.000 ± 0.00b

Mean value ± SE. The mean difference is significant at P < 0.05. The values in the same raw carrying different letters were significantly different

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Fig. 10  Rat’s lung, a control group, G 1: showing normal histological architectures. Alv alveoli. b–d Staph. aureus infected group G 2, hemorrhagic pneumonia which characterized by: b extravasation of red blood cells in the alveolar lumen (arrows), and in the bronchiolar lumen (Br). Alv alveoli. c Periarterial infiltration of inflammatory cells, periarterial edema (asterisk), degenerative changes in Tunica

muscular of the blood vessel (arrows). Alv alveoli. d Extravasation of red blood cells in the alveolar lumen (thick arrow), infiltration of mononuclear cells (thin arrow), accumulation of eosinophilic material in the interstitial tissue and in the alveoli (arrowhead). HE stains. a, b ×10; c ×20 & d ×40

Fig. 11  Rat’s lung. a, b Staph. aureus plus bacteriocin treated group (G 3). a Showing mild interstitial pneumonia with thickening of interalveolar septa (arrows). b Showing mild interstitial pneumonia with thickening of interalveolar septa (arrows) and slight intra-alveolar hemorrhage (arrowhead). c Staph. aureus plus Ent faecium ER-3M

cells treated group (G 4): showing mild interstitial pneumonia with thickening of interalveolar septa (arrows) and slight intra-alveolar hemorrhage (arrowhead). Alv alveoli. d Ent. faecium ER-3M treated group (G 5): showing normal histological architectures. HE stains. a ×10; b, d ×20; c ×40

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Table 9  Effect of bacteriocin and Ent. faecium ER-3M supplementation on hematological parameters of Staph. aureus infected rats Groups no. parameter

G1

G2

G3

G4

G5

Hb (g/dL) RBCs (× 106/mm3) HCT (%) MCV (Fl) MCH (pg) MCHC (%) WBCs (× 103/mm3) Lymphocyte (× 103/mm3) Neutrophil (× 103/mm3) Platelets (× 103/mL)

12.57 ± 0.18ab 6.80 ± 0.21ab 38.67 ± 0.80ab 57.13 ± 2.25b 18.55 ± 0.73b 32.48 ± 0.47a 12.82 ± 0.20c 85.85 ± 1.24a 14.15 ± 1.24c 549.83 ± 14.03b

11.78 ± 0.45b 5.43 ± 0.25c 35.27 ± 1.44b 64.85 ± 0.55a 21.72 ± 0.48a 33.45 ± 0.51a 23.33 ± 0.62a 64.20 ± 1.11c 35.80 ± 1.11a 679.83 ± 22.91a

13.22 ± 0.36a 7.10 ± 0.19a 39.50 ± 1.48a 55.67 ± 2.08b 18.62 ± 0.50b 33.28 ± 0.39a 13.63 ± 0.38bc 82.53 ± 1.02b 17.47 ± 1.02b 572.50 ± 20.34b

13.58 ± 0.37a 6.37 ± 0.24b 41.33 ± 0.92a 65.32 ± 2.65a 21.62 ± 1.00a 32.82 ± 0.32a 14.18 ± 0.38b 82.62 ± 0.60b 17.38 ± 0.61b 568.00 ± 24.71b

13.87 ± 0.19a 6.82 ± 0.07ab 38.67 ± 0.88a 57.03 ± 0.88b 20.90 ± 0.34a 36.40 ± 0.79a 12.42 ± 0.22c 84.55 ± 1.4ab 14.37 ± 1.3b c 572.2 ± 4.6b

Mean value ± SE. The mean difference is significant at P < 0.05. The values in the same raw carrying different letters were significantly different

which was characterized by thickening of interalveolar septa and slight intra-alveolar hemorrhage was detected in lung tissues of Staph. aureus plus probiotic-treated group (G 3) (Fig. 11a, b), and Staph. aureus plus Ent. faecium ER-3M treated group (G 4) (Fig. 10c). Lung tissues of a control group (G 1) and Ent. faecium ER-3M-treated group (G 5) showed normal histological architectures (Figs. 10a, 11d), respectively.

4 Discussion The importance of genus Enterococcus is mainly related to its antibiotic resistance, which contributes to the risk of colonization and infection. Enterococcus species can be classified as intrinsic resistance, acquired resistance, and tolerance. Relative to the Streptococci, Enterococci are intrinsically resistant to many used antimicrobial agents (Zacharof and Lovittb 2012). Bacteriocin is extracellularly secreted into the medium during the growth of the producer bacteria (Sharma et al. 2014). However, many enterococcal strains acquire various pathogenic traits, such as hemolytic activity, that have been reported to be involved in nosocomial infections (Kayser 2003). Bacterial strains, especially those resistant to vancomycin, are of particular concern, since vancomycin is often the last resort for the treatment of enterococcal infections. Therefore, only the strains which are non-hemolytic and sensitive to commonly used antibiotics, especially vancomycin, can be regarded as safe (De Vuyst et al. 2003). Safety assessment tests were carried out to estimate the presence of virulence factors or not. The strain was found to be non-hemolytic and also sensitive to commonly used antibiotics. Identification of the isolated bacterium was performed according to Bergey᾿s manual of systematic bacteriology, where cells were appeared in pairs or in short chains and they were non-spore forming, as reported in Kenzaka and Tani (2012). Meanwhile, the strain survives

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environmental stress, including extremely alkaline pH (9.6) and salt concentrations up to 6.5% NaCl. Isolate ER-3M can grow in the range of 10–45 °C; it was able to hydrolyze esculin to glucose and combines with ferric ions to form a black complex visible as black zones around colonies, as earlier reported by Kristich et al. (2014). LAB produces a number of substances that have antimicrobial properties. These include acids, hydrogen peroxide, and bacteriocins (Nilsen et al. 2003). Therefore, it was considered necessary to do the preliminary characterization of the antimicrobial substance produced by Ent. faecium ER-3M before detailed studies on its production and identification could be carried out. The most important property of bacteriocins is their proteinaceous nature, which distinguishes them from other antimicrobials. The loss of antimicrobial activity after treatment with proteolytic enzyme confirmed this proteinaceous nature and gave a strong indication that Ent. faecium ER-3M produces a bacteriocin. The expression of antimicrobial activity under neutral pH conditions and in the presence of catalase also eliminated the possibility of any antimicrobial effect due to the presence of any acid or hydrogen peroxide. Similar results have been reported by various researchers, where the loss in activity was observed only after treatment with proteolytic enzymes (Todorov and Dicks 2006; DeVuyst and Leroy 2007). In this study, bacteriocin excretion started during the logarithmic phase (eighth hour); the activity reached the maximum in the stationary phase (eleventh hour) of 500 Au ml−1. This is in partial agreement with the results obtained by Ben et al. (2008), in which the production of enterocin RZC5 from E. faecium was noticed in the early growth phase and the maximum activity was noticed during the exponential phase of growth. The result is also in partial agreement with the findings of 30 in the production of bacteriocin of Ent. faecium HJ35, enterocin HJ35, which was produced at the mid-log growth phase, reaching a maximum rate up to 2300 AU/mL during the late stationary

Rendiconti Lincei. Scienze Fisiche e Naturali

phase. Yoon et al. (2005) noticed that the highest bacteriocin production occurs at the end of the exponential and early stationary phases. The maximum antimicrobial activity was recorded during the exponential phase of growth, which reflects its nature as secondary metabolites and as antibiotics. These results are in agreement with some of the previously reported data (Ghrairi et al. 2008; Hadji-Sfaxi et al. 2011). The results clarify the agreement with Nagwa et al. (2015), who reported that bacteriocins from enterococci species can be used as bio-preservatives in food or as probiotics, since they can inhibit the growth of pathogenic bacteria. Bacteriocin RM6 is active against Gram-positive bacteria, including L. monocytogenes, Bacillus cereus, and methicillin-resistant Staphylococcus aureus (Huang et al. 2013). Bacteriocins produced by Gram-positive LAB are inhibitory mainly to other Gram-positive bacteria and these properties allow for the development of human applications and the protection of diseases (Macwana and Muriana 2012). As before, the maximum antimicrobial activity was recorded during the exponential phase of growth, and these results are in agreement with some of the previously reported data (Ghrairi et al. 2008; Hadji-Sfaxi et al. 2011). According to Yusuf and Abdul Hamid (2012) clarified that the degradation of the bacteriocin by proteolytic enzymes is the main cause of bacteriocin reduction, where it may affecting on their production in small quantities. On the other hand, all of the bacteriocin activity may be lost after final purification or after a decrease in their activity, as mentioned in Sharma et al. (2014); Thomas et al. (2000). The results indicate that the bacteriocin produced by Ent. faecium ER-3M appeared as an active band around 40 kDa in the SDS-PAGE gel. Karpinski and Szkaradkiewicz (2013) reported that bacteriocin produced by Ent. faecalis belonged to class III with molecular weight 34,525 Da which was a heat-labile protein. On an SDS–polyacrylamide gel, a single major band was observed (Khan et al. 2012). These results may be reflecting that partially purified bacteriocins belong to Class III of bacteriocins. Class III termed enterolysin are large molecular weight (> 30 kDa), heat-unstable proteins, and contain the bacteriolytic enzymes which killing the sensitive strains by lysis of the cell wall (Hu et al. 2010; Perez et al. 2014). FTIR and H-NMR spectroscopy provides information about the secondary structure content of proteins, and for identifying the types of chemical bonds (functional groups) present in compounds (Ashokkumar and Ramaswamy 2014). FTIR is able to detect those functional groups to indicate protein presence. Compared to other specific functional groups of protein, an amide is one of the specific functional groups that can be easily detected (Sjahfirdi and Nasikin 2012). Results confirmed the proteinaceous nature of extracted bacteriocins from Ent. faecium ER-3M. Knowledge of antibacterial properties interacting with biological cell membranes is an essential requirement for the understanding

of their mode of action at the molecular level. Yamamoto et al. 1994 recorded that 300 MHz 1H-NMR spectrum of crude bacteriocin produced by Enterococcaceae in DMSO at 30 °C, and found three signals in the anomeric region with almost equal integrated intensities, suggesting that there was amino acid repeating unit for crude or partially purified bacteriocin (Robijn et al. 1995). Amino acid analysis showed that the Ent. faecium ER-3 M bacteriocins did not contain lanthionine acid in its active molecule. According to Seham et al. (2014) who revealed that bacteriocins group which contain lantibiotics are belonged to class I that has small molecular weight < 10 kDa and heat stable; class II which divided in the pediocin-like and anti-Listeria (class IIa); the other subclass II b and c. Class III contains large molecular weight > 30 KDa, heat labile, and non-lantibiotics. This indicated that Ent. faecium ER-3 M bacteriocins did not belong to class I and are similar to class III. On the other hand, Berg et al. (2012) were classified amino acids according to its polarities, so our results can indicate that six amino acids were nonpolar and seven were polar and three were charged as this finding clarify the role of antibacterial peptides in biocontrol of pathogens, since bacteriocins are heterogeneous in nature and exploit the charge and hydrophobic properties of bacteriocins, which are necessary for their activity against pathogenic strains (Cleveland et al. 2001). Polar amino acids carry either a (+) or a (−) charge. Polar amino acids are hydrophilic and non-polar amino acids are hydrophobic. Hydrophobic R-groups stay adjacent together in water. Substances are cationic peptides that produce hydrophobic and amphiphilic properties, where the bacterial membrane is the target for their activity in general (Floch 2014). Heat sensitivity of bacteriocin is an important criterion and can help to categorized whether the bacteriocin belongs to the class of heat-labile large proteins or is a heat-stable peptide (Heng et al. 2007). The current study was observed that the bacteriocin was heat-labile being inactivated when heated at 60 °C for 60 min. This was a positive indication that Ent. faecium ER-3M is most probably producing a large antimicrobial protein (> 10 kDa) belonging or similar to class III of bacteriocins. This group consists of heat labile proteins which has a large molecular weight (> 30 kDa) Zacharof and Lovittb (2012). It is important to evaluate the impact of Ent. faecium ER-3M and its bacteriocin as antibacterial and to assess the development of anti-staphylococcus activity in an experimental animal. The strain and its bacteriocin can reduce the bacterial growth of Staph. aureus as blood was inhabited by highly diverse and abundant bacteria that form a complex ecosystem, which is difficult to be simulated in vitro. In addition, bacteriocins derived from lactic acid bacteria strongly reduced the growth and biofilm formation of Staph. aureus as reported previously (Wang and Liu 2016). In this

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study, there was significant decrease in Staph. aureus counts in blood samples of groups 3 and 4 which indicate that both the Enterococcus faecium ER-3M isolate and its bacteriocin can suppress the growth of Staph. aureus bacteria. This result suggests a protective effect of enterolysin against Staph. aureus infection in vivo. White blood cells (WBCs) are important in defending the body against infection (Gibson and Marcel 1995). The results revealed a significant increase in a total leukocytic count in the infected group with S. aureus which could be explained by neutrophilia. The neutrophil is the most important phagocytic cell which participates in the innate immune response, also known as the nonspecific immune response (Kumar and Sharma 2010). Neutrophilia could be also attributed to the stress response as a result of the endogenous release of cortisol which has major role in regulating circulating concentration of leukocytes (Duncan et al. 1994). The present study indicated that supplementation of rats with bacteriocin post infection improved hematological parameter. This improvement effect of bacteriocin on hematological parameter can be explained by its antioxidant potential. These observations were in line with those of (Pieniz et al. 2011) that were reported that some isolates of Enterococcus showed antioxidant properties. Monitoring the serum values of some biochemical parameters revealed a significant increase in serum activity of AST, ALT, and CRP in rats infected with S. aureus. The increase in serum activity of AST and ALT may suggest some degree of hepatic dysfunction due to increase in releasing of tissue specific enzymes and other intracellular proteins due to oxidative stress response to Staph. aureus infection (Horsburgh et al. 2001). A high level of CRP in the blood is a marker of any condition that causes inflammation, from an upper respiratory infection to cancer. The increase in serum CRP may suggest some degree of inflammation in respiratory tract caused by infection with Staph. aureus which is an important pathogen in upper and lower respiratory tract infections (Micek et al. 2007; Quirasco et al. 2007; García-Cano et al. 2014), while supplementation of rats with bacteriocin post-infection normalized ALT, AST, and CRP levels. Histopathologically, lung tissues of rats infected with Staph. aureus show a severe hemorrhagic bronchopneumonia which characterized by extensive hemorrhages, lymphoid cell infiltration, and edema. Treatment of rats with Ent. faecium ER-3M isolate or its bacteriocin inhibit the progress of hemorrhagic pneumonia in lung tissues of Staph. aureus infected rats in groups 3 and 4. Only mild interstitial pneumonia was detected in thesis groups. Enterococci are often used for the preparation of traditional cheeses manufactured in Mediterranean countries from raw or pasteurized milk. Levels of enterococci (E. faecium and E. faecalis) in different cheeses range from ­104 to ­106 CFU/g (Foulquié Moreno et al. 2006). The vast majority of enterococci, including species that are major agents of nosocomial

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infection, are peaceful inhabitants of the gastrointestinal tracts of animals that range from insects to humans (Garsin et al. 2014).

5 Conclusion In this study, Ent. faecium ER-3M was isolated from Egyptian cattle raw milk. Biochemical and molecular characterizations was studied and the isolate was registered in GenBank with accession number KY774316. The new strain can produce bacteriocin, which belongs to class III of bacteriocins termed enterolysin with large molecular weight and consist of heat labile proteins. Moreover, it has a broad spectrum against Gram positive and negative pathogenic bacteria in vitro. The promising novel strain and its bacteriocin can inhibit the growth of Staph aureus in the respiratory tract and reduce the symptoms of pneumonia in vivo. On the other hand, it can enhance the immune responses and prolong rat survival time post Staph aureus infection. As the clinical influence of Staphylococcus aureus is rising as it considered a multidrug-resistant strains and dispersal of surgical infections. There is an urgent requirement to identify new effective bacterial strain that can be safe, economic and secrete efficient biological bacteriocin as protection as treatment for hemorrhagic pneumonia. Therefore, Ent. faecium ER-3M may offer a new strategy in combating pathogens infection. Acknowledgements  Special thanks go to Dr. Ahmed M. Hammad Associative professor, Department of Food Hygiene and Control, Faculty of Veterinary Medicine, University of Sadat City, Egypt, whose comments was very helpful during bacterial isolation stage though this study.

Compliance with ethical standards  Conflict of interest  The authors declared that the present study was performed in absence of any conflict of interest.

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