Effect of Bacteriocin-Like Substance Produced by Enterococcus faecium EF 55 on the Composition of Avian Gastrointestinal Microflora

Strompfová V. , A. Lauková, D. MudroÀová: Effect of Bacteriocin-like Substance Produced by Enterococcus faecium EF55 on the Composition of Avian Gastrointestinal Microflora. Acta Vet. Brno 2003, 72: 559-564. The influence of daily orally administered crude extract of bacteriocin-like substance (at a concentration of 3200 AU/ml) produced by Enterococcus faecium EF55 on the total counts of lactobacilli, staphylococci, enterococci and E. coli in the faeces and caecum of 3-day-old Japanese quails was investigated. Enterococcus faecium EF55 was isolated from the crop content of a chicken (Gallus domesticus). The inhibitory activity of bacteriocin-like substance (BLIS) produced by the strain EF55 was assayed by the agar spot test using Gram-positive and Gramnegative indicator bacteria. A wide range of Gram-positive genera such as Enterococcus, Staphylococcus, Micrococcus, Lactobacillus, Lactococcus, Streptococcus and Aerococcus was susceptible to BLIS, but none of the Gram-negative bacteria. The antimicrobial substance produced by the strain EF55 was thermo-resistant (30 min at 100 °C), stable at pH 4.0 to 9.0 at -20 °C, 4 °C and 22 °C for 10 d tested, and inactivated by proteolytic enzymes indicating its proteinaceous nature. After the first administration of bacteriocin crude extract (BCE) of EF55 strain to Japanese quails, a reduction amounting to 0.83–1.3 log cycles of E. coli, enterococci, staphylococci and lactobacilli in faeces was observed within 24 h. This inhibitory effect was most visible after first extract addition, later this difference was diminished. By agar spot test, BLIS produced by the strain EF55 of Ent. faecium was active against Staphylococcus spp., Lactobacillus spp. and Enterococcus spp., isolates obtained from the experimental birds. However, no inhibition against E. coli was detected, despite of their decreased counts under in vivo conditions. Bacteriocin, Japanese quails, gastrointestinal microflora, effect The ability of bacteria to produce bacteriocins is well known (Klaenhammer et al. 1993). Bacteriocins are proteinaceous compounds with inhibitory activity against more or less related bacterial genera (Nes et al. 1996). Bacteriocin-producing bacteria have been isolated from a variety of habitats (Dykes 1995) and bacteriocins have been characterized from many different bacterial genera, especially from lactic acid bacteria (Jack et al. 1995). Nisin, produced by certain strains of Lactococcus lactis subsp. lactis, is the best characterized bacteriocin-lantibiotic widely used for decades as a food preservative (Delves-Broughton et al. 1996). More recently, also biomedical applications have been proposed which include its potential to treat mastitis infections in cows (Sears et al. 1995), gingivitis and gastrointestinal infections such as peptic ulcer disease caused by Helicobacter pylori (Blackburn and Projan 1994). Nisin has been proved to modulate the immune system of mice when included in the diet (De Pablo et al. 1999) or of turbot after its intraperitoneal injection (Vil lamil et al. 2002). Strains of enterococci, including Enterococcus faecium and Ent. faecalis, have also the ability to produce bacteriocins. Numerous studies concerning enterocins (bacteriocins produced by enterococci) have been published during recent ten years. Up to now the following enterocins have been characterized as to their homogenity: enterocin A (Aymerich et al. 1996), enterocin ACTA VET. BRNO 2003, 72: 559–564 Address for correspondence: MVDr. Viola Strompfová Institute of Animal Physiology, Slovak Academy of Sciences ·oltésovej 4-6, 04001 Ko‰ice, Slovakia Phone: +421-55-6330283 Fax: +421-55-7287842 E-mail: strompfv@saske.sk http://www.vfu.cz/acta-vet/actavet.htm B (Casaus et al. 1997), enterocin P (Cintas et al. 1997), enterocin I (Flor iano et al. 1998), enterocin M, a new variant of enterocin P (Mareková et al. 2002), and enterocin CCM 4231 (Lauková et al. 1997). Whereas the effectiveness of enterocins against food spoilage and pathogenic bacteria in various food systems is well demonstrated (Aymerich et al. 2000), little information is available on their possible antagonistic activity in the intestine in vivo. Therefore, the objective of this study was to determine the effect of orally administered bacteriocin-like substance (crude extract), produced by the isolate EF55 of Enterococcus faecium, on the composition of intestinal microflora of conventional Japanese quails. Additionally, antimicrobial activity of this strain against a number of Gram-positive and Gram-negative bacteria was tested. Materials and Methods Experimental animals A group of fourteen 3-day-old Japanese quails was divided into two groups of seven birds each. The experiment lasted for seven days. All birds were fed the commercial diet BR 1/FAT (Tatrafat s.r. Huncovce, Slovakia) and had access to water ad libitum. The experimental group (n = 7) was orally administered the crude bacteriocin extract of Ent. faecium EF55 at a concentration of 3200 AU/ml (one oral dose 0.10-0.25 ml) every 24 hours. The control group (n=7) was given placebo – Brain heart infusion (BHI) broth (Becton & Dickinson, Cockeysville, USA). Sampling of faeces was done from each quail after 1, 3 and 7 d to monitor the effect of the bacteriocin crude extract (BCE) on the microflora of their gastrointestinal tract. At the end of the experiment all animals were sacrificed and their caecum was separated. Its content was mixed using Stomacher (80I, England). The birds were weighed both at the beginning and at the end of the experiment. Bacter ia l counts The samples of faeces and caecum content were serially diluted in saline buffer (0.85%) according to the standard microbiological method and plated on the following media: Mac Conkey agar (Imuna, ·ari‰ské Michaoany, Slovakia) for enumeration of E.coli, Mannitol salt agar (Becton & Dickinson) for staphylococci, M-Enterococcus agar (Becton & Dickinson) for enterococci and Rogosa agar (prepared according to Oxoid manufacturer, pH 6.2) for lactobacilli. Enterococci, staphylococci and E. coli were cultivated at 37 oC for 24 and 48 h, respectively. Lactobacilli were cultivated in a 3% CO2 atmosphere at 37 oC for 48 h. Numbers of colony forming units (cfu) were expressed as log 10 cfu per gram. The results are given as arithmetical means ± S.E.M. Preparat ion of crude extract The bacteriocinogenic strain EF55 of Ent. faecium was grown in 300 ml of BHI broth (Becton & Dickinson) at 37 oC for 18 h (O.D.600 1.1). Culture supernatant was collected by centrifugation (10 000 g for 30 min), neutralized and concentrated in a rotary evaporator. The activity of crude extract was determined by the agar spot test (De Vuyst et al. 1996) on BHI agar plates (1.5 and 0.7%; Becton & Dickinson) and expressed in arbitratry units per ml of culture medium (AU/ml). Arbitrary unit is defined as the reciprocal of the highest dilution giving growth inhibition of the indicator organism. As the indicator was used EA5 strain of Enterococcus avium. Ant imicrobial act ivi ty assays The inhibitory activity of the cell-free supernatant of Ent. faecium EF55 was assayed by the agar spot test (De Vuyst et al. 1996) on BHI agar plates (1.5 and 0.7%; Becton & Dickinson) against the target of Gram-positive and Gram-negative bacteria. A complete list of indicator bacteria used in this study is given in Table 1. The bacteriocin activity of crude extract against strains isolated from the faeces of the experimental quails by using selective growth media mentioned above was also tested by the agar spot test. Effect of enzymes, pH and heat on bacter iocin act ivi ty Sensitivity to proteolytic enzymes was tested individually at a concetration of 1 mg/ml by adding of the following enzymes to cell-free supernatant of Ent. faecium EF55: protease (Sigma), trypsin (Sigma), α-chymotrypsin (Sigma). Reaction mixtures were incubated at 37 oC for 1 h. The thermostability of the antibacterial activity was determined by heating of cell-free neutralized supernatant fluids at 60 oC, 80 oC and 100 oC for 30 min. To determine the effect of pH on bacteriocin activity, cell-free supernatant fluid was adjusted to pH levels ranging from 4.0 to 9.0 (intervals of 1.0) with 1N HCl or 5N NaOH. The pH-adjusted supernatants were kept at -20 oC, 4 oC and room temperature (22 oC) for 10 d. Bacteriocin activity of all tests was determined using agar spot test method and Ent. avium EA5 as indicator. Stat is t ical analysis Statistical evaluation of the results was performed by the Students’ t-test with the level of significance set at P < 0.05. 560

The ability of bacteria to produce bacteriocins is well known (Klaenhammer et al. 1993).Bacteriocins are proteinaceous compounds with inhibitory activity against more or less related bacterial genera (Nes et al. 1996).Bacteriocin-producing bacteria have been isolated from a variety of habitats (Dykes 1995) and bacteriocins have been characterized from many different bacterial genera, especially from lactic acid bacteria (Jack et al. 1995).Nisin, produced by certain strains of Lactococcus lactis subsp.lactis, is the best characterized bacteriocin-lantibiotic widely used for decades as a food preservative (Delves-Broughton et al. 1996).More recently, also biomedical applications have been proposed which include its potential to treat mastitis infections in cows (Sears et al. 1995), gingivitis and gastrointestinal infections such as peptic ulcer disease caused by Helicobacter pylori (Blackburn and Projan 1994).Nisin has been proved to modulate the immune system of mice when included in the diet (De Pablo et al. 1999) or of turbot after its intraperitoneal injection (Villamil et al. 2002).Strains of enterococci, including Enterococcus faecium and Ent.faecalis, have also the ability to produce bacteriocins.Numerous studies concerning enterocins (bacteriocins produced by enterococci) have been published during recent ten years.Up to now the following enterocins have been characterized as to their homogenity: enterocin A (Aymerich et al. 1996), enterocin B (Casaus et al. 1997), enterocin P (Cintas et al. 1997), enterocin I (Floriano et al. 1998), enterocin M, a new variant of enterocin P (Mareková et al. 2002), and enterocin CCM 4231 (Lauková et al. 1997).Whereas the effectiveness of enterocins against food spoilage and pathogenic bacteria in various food systems is well demonstrated (Aymerich et al. 2000), little information is available on their possible antagonistic activity in the intestine in vivo.Therefore, the objective of this study was to determine the effect of orally administered bacteriocin-like substance (crude extract), produced by the isolate EF55 of Enterococcus faecium, on the composition of intestinal microflora of conventional Japanese quails.Additionally, antimicrobial activity of this strain against a number of Gram-positive and Gram-negative bacteria was tested.

Experimental animals
A group of fourteen 3-day-old Japanese quails was divided into two groups of seven birds each.The experiment lasted for seven days.All birds were fed the commercial diet BR 1/FAT (Tatrafat s.r.Huncovce, Slovakia) and had access to water ad libitum.The experimental group (n = 7) was orally administered the crude bacteriocin extract of Ent.faecium EF55 at a concentration of 3200 AU/ml (one oral dose 0.10-0.25 ml) every 24 hours.The control group (n=7) was given placebo -Brain heart infusion (BHI) broth (Becton & Dickinson, Cockeysville, USA).Sampling of faeces was done from each quail after 1, 3 and 7 d to monitor the effect of the bacteriocin crude extract (BCE) on the microflora of their gastrointestinal tract.At the end of the experiment all animals were sacrificed and their caecum was separated.Its content was mixed using Stomacher (80I, England).The birds were weighed both at the beginning and at the end of the experiment.

Bacterial counts
The samples of faeces and caecum content were serially diluted in saline buffer (0.85%) according to the standard microbiological method and plated on the following media: Mac Conkey agar (Imuna, •ari‰ské Michaºany, Slovakia) for enumeration of E.coli, Mannitol salt agar (Becton & Dickinson) for staphylococci, M-Enterococcus agar (Becton & Dickinson) for enterococci and Rogosa agar (prepared according to Oxoid manufacturer, pH 6.2) for lactobacilli.Enterococci, staphylococci and E. coli were cultivated at 37 ºC for 24 and 48 h, respectively.Lactobacilli were cultivated in a 3% CO 2 atmosphere at 37 ºC for 48 h.Numbers of colony forming units (cfu) were expressed as log 10 cfu per gram.The results are given as arithmetical means ± S.E.M.

Preparation of crude extract
The bacteriocinogenic strain EF55 of Ent.faecium was grown in 300 ml of BHI broth (Becton & Dickinson) at 37 ºC for 18 h (O.D. 600 1.1).Culture supernatant was collected by centrifugation (10 000 g for 30 min), neutralized and concentrated in a rotary evaporator.The activity of crude extract was determined by the agar spot test (De Vuyst et al. 1996) on BHI agar plates (1.5 and 0.7%; Becton & Dickinson) and expressed in arbitratry units per ml of culture medium (AU/ml).Arbitrary unit is defined as the reciprocal of the highest dilution giving growth inhibition of the indicator organism.As the indicator was used EA5 strain of Enterococcus avium.

Antimicrobial activity assays
The inhibitory activity of the cell-free supernatant of Ent.faecium EF55 was assayed by the agar spot test (De Vuyst et al. 1996) on BHI agar plates (1.5 and 0.7%; Becton & Dickinson) against the target of Gram-positive and Gram-negative bacteria.A complete list of indicator bacteria used in this study is given in Table 1.The bacteriocin activity of crude extract against strains isolated from the faeces of the experimental quails by using selective growth media mentioned above was also tested by the agar spot test.

Effect of enzymes, pH and heat on bacteriocin activity
Sensitivity to proteolytic enzymes was tested individually at a concetration of 1 mg/ml by adding of the following enzymes to cell-free supernatant of Ent.faecium EF55: protease (Sigma), trypsin (Sigma), α-chymotrypsin (Sigma).Reaction mixtures were incubated at 37 ºC for 1 h.
The thermostability of the antibacterial activity was determined by heating of cell-free neutralized supernatant fluids at 60 ºC, 80 ºC and 100 ºC for 30 min.To determine the effect of pH on bacteriocin activity, cell-free supernatant fluid was adjusted to pH levels ranging from 4.0 to 9.0 (intervals of 1.0) with 1N HCl or 5N NaOH.The pH-adjusted supernatants were kept at -20 ºC, 4 ºC and room temperature (22 ºC) for 10 d.Bacteriocin activity of all tests was determined using agar spot test method and Ent.avium EA5 as indicator.

Statistical analysis
Statistical evaluation of the results was performed by the Students' t-test with the level of significance set at P < 0.05.

Results and Discussion
Enterococcus faecium EF55 (own isolate) was isolated from the crop content of a chicken.It produces a bacteriocin-like substance with the inhibitory activity against Gram-positive bacteria including enterococci, staphylococci, lactococci, streptococci, lactobacilli, micrococci, but no inhibition against Gram-negative bacteria tested in vitro was detected (Table 1).This observation correlated with numbers of enterocins studied (Giraffa 1995).The antimicrobial substance was thermo-stable (30 min at 60 ºC, 80 ºC and 100 ºC) and it was stable over a pH range of 4.0-9.0 at -20 ºC, 4 ºC and room temperature (22 ºC) for 10 d tested.The treatment with proteolytic enzymes (trypsin, α-chymotrypsin, protease) resulted in the loss of antibacterial activity, which confirmed the proteinaceous character of this substance.On the basis of these results, it is thermo-stable proteinaceous substance with a stability over a wide range of pH values such as most of enterocins (Giraffa 1995).Activity of bacteriocins over a wide pH range is an important asset, allowing their use under acidic conditions (e.g.small intestine, fermented milks) as well as in more neutral enviroments (large intestine, low-acid foods).
The crude bacteriocin extract of strain EF55 (at a concentration of 3200 AU/ml) was used in the experiment in vivo.It was daily orally administered to each quail during seven consecutive days.The total counts of selected bacterial groups in the faeces of birds with developing intestinal microflora are summarized in Table 2.After 24 h of crude extract addition, the total counts of all bacterial groups under study (enterococci, staphylococci, lactobacilli and E. coli) were lower compared with control group.The greatest cell count reduction (significant difference) was detected after the first administration of BCE except for the greatest reduction of total counts of staphylococci (difference of 1.01 log; P < 0.05) detected 72 h after first BCE administration.Lactobacilli were significantly (P < 0.01) reduced 24 h after first BCE application (difference of 1.30 log), and this reduction was relatively stable during the experiment.The total count of enterococci was significantly (P < 0.05) lower only after the first BCE application (difference of 1.16 log).The difference in the counts of E. coli between experimental and control groups was 1.03 log (P < 0.05) also on d 1, later it was diminished.Concerning the total counts of cells, staphylococci were the most inhibited bacterial group (reduction of 22.9%), followed by lactobacilli (15.6%), enterococci (14.9%) and E. coli (12.7%).In the contents of caecum, no significant difference in the total counts of bacterial genera was detected.No differences in body mass between the control and experimental groups were noted.By the testing of BCE of EF55 in vitro against the isolates from the faeces of quails obtained before experimental treatment (Table 3), a similar, most pronounced inhibitory effect was noted against Staphylococcus spp., followed Lactobacillus spp.and Enterococcus spp.Surprisingly, no inhibition against the isolates of E.coli was detected, despite of their reduction under in vivo conditions.This could be due to secondary interactions between intestinal microflora groups.Moreover, activity of enterocins towards Gram-negative bacteria like E. coli (Tomita et al. 1997) and Vibrio cholerae has been shown (Simonetta et al. 1997).
Many bacterial species inhabitating the animal gut produce compounds that are inhibitory, in vitro, to the growth of other bacteria (Du Toit et al. 2000).Many investigators have considered the possibility that these compounds influence the survival of a bacterium in an ecological niche in the gastrointestinal tract of animals.In our experiment, the bacteriocin-like substance produced by the strain EF55 of Ent.faecium was able to reduce the populations of present intestinal microflora in experimental birds, although this effect was especially remarkable after its first oral administration.Similarly, this type of inhibition kinetics has been observed before with Listeria as indicator and nisin, pediocin AcH and enterococcin EFS2 as antimicrobial substances (Song and Richard 1997), and may indicate that sensitive bacteria become resistant to the bacteriocin.It is generally admitted that each bacteriocin-sensitive bacterial population includes potentially tolerant and/or resistant cells with structural modifications or at least with a high predisposition to such modifications, which would allow them to spontaneously emerge in case of exposure to the bacteriocin (Hanlin et al. 1993).In particular, modifications in the cytoplasmic membrane composition are often investigated to explain bacteriocin resistance, considering the key role of the membrane in the activity of bacteriocins (Mazzotta and Montville 1997).Because the occurrence of bacteriocin tolerance and/or resistance is the main limiting factor for bacteriocin effectiveness, the use of a combination of several bacteriocins could prevent or delay the apparition of bacteriocin-resistant mutants (Morency et al. 2001).
In conclusion, bacteriocins produced by intestinal Enterococcus isolates may help to control the autochthonous microflora and may be advantageous to the producing strain for its establishment and competition in the gastrointestinal tract.Further studies on bacteriocins behaviour in the intestinal environment are required to determine their suitability for potential biomedical applications where they may provide valuable alternatives to antibiotics for the treatment of animal infections without any side effects and leaving residues in the animal products behind.

Table 1
Inhibitory spectrum of bacteriocin-like substance produced by strain EF55 against indicator strains

Table 2
In vivo effect of bacteriocin crude extract produced by the strain EF55 of Ent.faecium on bacteria in the gastrointestinal tracts of Japanese quails

Table 3
In vitro inhibition of bacteriocin crude extract of EF55 strain against isolates from the faeces of experimental J. quails