Probiotic Potential and Antimicrobial Activity of Enterococcus faecium Isolated from Chicken Caecal and Fecal Samples
Enterococci are important inhabitants of the animal
intestine and are widely used in probiotic products. A probiotic strain
is expected to possess several desirable properties in order to exert
beneficial effects. Therefore, the objective of this study was to
isolate, characterize and identify Enterococcus sp. from chicken cecal
and fecal samples to determine potential probiotic properties.
Enterococci were isolated from chicken ceca and feces of thirty three
clinically healthy chickens from a local farm. In vitro studies were
performed to assess antibacterial activity of the isolated LAB (using
agar well diffusion and cell free supernatant broth technique against
Salmonella enterica serotype Enteritidis), survival in acidic
conditions, resistance to bile salts, and their survival during simulated
gastric juice conditions at pH 2.5. Isolates were identified by
biochemical carbohydrate fermentation patterns using an API 50
CHL kit and API ZYM kits and by sequenced 16S rDNA. An isolate
belonging to E. faecium species exhibited inhibitory effect against S.
enteritidis. This isolate producing a clear zone as large as 10.30 mm
or greater and was able to grow in the coculture medium and at the
same time, inhibited the growth S. enteritidis. In addition, E. faecium
exhibited significant resistance under highly acidic conditions at pH
2.5 for 8 h and survived well in bile salt at 0.2% for 24 h and showing
ability to survive in the presence of simulated gastric juice at pH 2.5.
Based on these results, E. faecium isolate fulfills some of the criteria
to be considered as a probiotic strain and therefore, could be used as a
feed additive with good potential for controlling S. Enteritidis in
chickens. However, in vivo studies are needed to determine the safety
of the strain.
[1] H. A. Abdel-Rahman., S. M. Shawky, H. Ouda, A. A. Nafeaa, and S. H.
Orabi, "Effect of Two Probiotics and Bioflavonoids Supplementation to
the Broilers Diet and Drinking Water on the Growth Performance and
Hepatic Antioxidant Parameters". Global Veterinaria 10 (6): 734-741,
2013.
[2] R. Herich, and M. Levkut, "Lactic acid bacteria, probiotics and immune
system". Veterinární medicína, 47, 169-180, 2002.
[3] P. Hlivak, J. Odraska, M. Ferencik, L. Ebringer, E. Jahnova, and A.
Mikes, "One-year application of probiotic strain Enterococcus faecium
M-74 decreases serum cholesterol levels". Bratisl. Lek. Listy 106, 67–
72, 2005.
[4] Y. Huang, and Y. Zheng, "The probiotic Lactobacillus acidophilus
reduces cholesterol absorption through the down-regulation of
Niemann–Pick C1-like 1 in Caco-2 cells". Br. J. Nutr. 9, 1–6, 2009.
[5] L. Pascual, F. Ruiz, W. Giordano, and I. L. Barberis, "Vaginal
colonization and activity of the probiotic bacterium Lactobacillus
fermentum L23 in a murine model of vaginal tract infection". Journal of
Medical Microbiology 59, 360–364, 2010.
[6] O. Ashayerizadeh, B. Dastar, M. Shams Shargh, E. Rahmatnejad, and A.
Ashayerizadeh, "Influence of prebiotic and two herbal additives on
interior organs and hematological indices of broilers". Journal of Animal
and Veterinary Advances. 8(9), p. 1851-1855, 2009.
[7] A. Bhardwaj, G. Kaur, H. Gupta, S. Vij, and R. K. Malik, "Interspecies
diversity, safety and probiotic potential of bacteriocinogenic Enterococcus faecium isolated from dairy food and human faeces".
World Journal of Microbiology and Biotechnology, 27, 591-602, 2011.
[8] J. A. Patterson, and K. M. Burkholder, "Application of prebiotics and
probiotics in poultry production. Poultry Sci., 82: 627-631, 2003.
[9] A. Ahmadova, S. D. Todorov, Y. Choiset, H. Rabesona, T. M. Zadi,
Kuliyev, A., B.D.G.M. Franco, J. M. Chobert, and T. Haertlé,
"Evaluation of antimicrobial activity, probiotic properties and safety of
wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal
cheese". Food Control 30, 631–64, 2013.
[10] B. B. Matijašic´, T. Obermajer, and I. Rogelj, "Quantification of
Lactobacillus gasseri, Enterococcus faecium and Bifidobacterium
infantis in a probiotic OTC drug by real-time PCR". Food Control 21,
419–425, 2010.
[11] L. Saavedra, M. P. Taranto, F. Sesma, and G. F. de Valdez, "Homemade
traditional cheeses for the isolation of probiotic Enterococcus faecium
strains". Int. J. Food Microbiol. 88, 241–245, 2003.
[12] J. Benyacoub, P. F. Perez, F. Rochat, K. Y. Saudan, G. Reuteler, N.
Antille, M. Humen, G. L. De Antoni, C. Cavadini, S. Blum, and E. J.
Schiffrin, "Enterococcus faecium SF68 enhances the immune response
to Giardia intestinalisin mice". J. Nutr. 135: 1171–1176, 2005.
[13] D. G. V. Emmanuel, A. Jafari, K. A. Beauchemin, J. A. Leedle, and B.
N. Ametaj, "Feeding live cultures of Enterococcus faecium and
Saccharomyces cerevisiae induces an inflammatory response in feedlot
steers". J. Anim. Sci. 85:233–239, 2007.
[14] U. Lodemann, K. Hubener, N. Jansen, and H. Martens, "Effects of
Enterococcus faecium NCIMB 10415 as probiotic supplement on
intestinal transport and barrier function of piglets". Arch. Anim. Nutr.
60:35–48, 2006.
[15] L. Scharek, J. Guth, K. Reiter, K. D. Weyrauch, D. Taras, P. Schwerk, P.
Schierack, M. F. Schmidt, L. H. Wieler, and K. Tedin, "Influence of a
probiotic Enterococcus faecium strain on development of the immune
system of sows and piglets". Vet. Immunol. Immunopathol. 105:151–
161, 2005.
[16] W. Theppangna, K. Otsuki, and T. Murase, "Inhibitory effects of
Enterococcus strains obtained from a probiotic product on in vitro
growth of Salmonella enteric serovar Enteritidis strain IFO3313". J.
Food Prot. 69: 2258–2262, 2006.
[17] D. Taras, W. Vahjen, M.Macha, and O. Simon, "Performance, diarrhea
incidence, and occurrence of Escherichia coli virulence genes during
long-term administration of a probiotic Enterococcus faecium strain to
sows and piglets". J. Anim. Sci. 84:608–617, 2006.
[18] M. Tokumaru, H. Konuma, M. Umesako, S.Konno, and K. Shinagawa,
"Rates of detection of Salmonella and Campyobacterin meats in
response to the sample size and the infection level of each species". Int.
J. Food Microbiol. 13:41–46, 1991.
[19] M. A. Ehrmann, P. Kurzak, J. Bauer, and R. F. Vogel, "Characterization
of lactobacilli towards their use as probiotic adjuncts in poultry". J.
Appl. Microbiol. 92:966–975, 2002.
[20] L. M. Shin, R. J. McNally, S. M. Kosslyn, W. L. Thompson, S. L.
Rauch, and N. M. Alpert, "Regional cerebral blood flow duringscriptdriven
imagery in childhood sexual abuse-related posttraumatic stress
disorder: A positron emission tomographic investigation". Am J
Psychiatry 156:575–584, 1999.
[21] K. Perelmuter, M. Fraga, and P. Zunino, "In vitro activity of potential
probiotic Lactobacillus murinus isolated from the dog". Journal of
Applied Microbiology, 104, 1718-1725, 2008.
[22] Y. Huang, and M. C. Adams, "In vitro assessment of the upper
gastrointestinal tolerance of potential probiotic dairy propionibacteria".
International Journal of Food Microbiology, 91, 253 -260, 2004.
[23] A. R. Madureira, M. S. Gião, M. E. Pintado, A. M. P. Gomes, C. Freitas,
and F. X. Malcata, "Incorporation and survival of probiotic bacteria in
whey cheese matrices". J. Food Sci. 70(3): M161-M165, 2005.
[24] M. Succi, P. Tremonte, A. Reale, E. Sorrentino, L. Grazia, and S.
Pacifico, "Bile salt and acid tolerance of Lactobacillus rhamnosus
strains isolated from Parmigiano Reggiano cheese". FEMS
Microbiology Letters, 244, 129-137, 2005.
[25] S. Pieniz, R. Andreazza, T. Anghinoni, F. Camargo, and A. Brandelli,
"Probiotic potential, antimicrobial and antioxidant activities of
Enterococcus durans strain LAB18s. Food Control 37, 251-256, 2014.
[26] P. Sun, J. Wang, and Y. Jiang, "Effects of Enterococcus faecium (SF68)
on immune function in mice". Food Chemistry, 123, 63-68, 2010.
[27] B. Hyronimus, C. L. Marrec, S. A. Hadj, and A. Deschamps, "Acid and
bile tolerance of spore-forming lactic acid bacteria". International
Journal of Food Microbiology, 61, 193-197, 2000.
[28] J. Gong, R., J. Forster, and H. Yu, "Diversity and phylogenetic analysis
of bacteria in the mucosa of chicken ceca and comparison with bacteria
in the cecal lumen". FEMS Microbiol Lett 208:1–7, 2002.
[29] J. B. Payne, J. A. Osborne, P. K. Jenkins, and B. W. Sheldon, "Modeling
the growth and death kinetics of Salmonella in poultry litter as a
function of pH and water activity". Poult. Sci., 86: 191-201, 2007.
[30] EFSA, The community summary report on trends and sources of
zoonoses and zoonotic agents in the European Union in 2007. EFSA J
223:1–320, 2009.
[31] H. R. Taheri, H. Moravej, F. Tabandeh, M. Zaghari, and M. Shivazad,
"Screening of lactic acid bacteria toward their selection as a source of
chicken probiotic". Poult. Sci. 88:1586-1593, 2009.
[32] M. Chichlowski, J. Croom, B. W. McBride, G. Davis, L. Daniel, and M.
Koci, "Direct-fed microbial and salinomycin modulate whole body and
intestinal oxygen consumption and intestinal enterocytes cytokine
production in the broiler chick". Poult. Sci., 86: 1100-1106, 2007.
[33] M. A. Riley, and J. E. Wertz, "Bacteriocins: evolution, ecology, and
application". Annual review of microbiology, Vol.56, pp. 117-137, ISSN
0066-4227, 2002.
[34] P. Audisio, A.De Biase, G. Antonini, M. Belfiore, and M. Oliverio,
"Morphological, molecular and ecological evidence of a new Euro-
Anatolian species of the Meligethes coracinus complex (Coleoptera:
Nitidulidae). Insect Systematics and Evolution, Volume 31, Issue
4, p. 361 – 385, 2000.
[35] R. Herich, T. Kokinčáková, A. Lauková, and M. Levkutová, "Effect of
preventive application of Enterococcus faecium EF55 on intestinal
mucosa during salmonellosis in Chickens". Czech J. Anim. Sci., 55, (1):
42–47, 2010.
[36] V. Strompfová, and A. Lauková, "In vitro study on bacteriocin
production of enterococci associated with chickens". Anaerobe 13: 228-
237, 2007.
[37] M. Ashraf, and N. A. Akram, "Improving salinity tolerance of plants
through conventional breeding and genetic engineering: an analytical
comparison". Biotechnol. Adv., 27: 744-52, 2009.
[1] H. A. Abdel-Rahman., S. M. Shawky, H. Ouda, A. A. Nafeaa, and S. H.
Orabi, "Effect of Two Probiotics and Bioflavonoids Supplementation to
the Broilers Diet and Drinking Water on the Growth Performance and
Hepatic Antioxidant Parameters". Global Veterinaria 10 (6): 734-741,
2013.
[2] R. Herich, and M. Levkut, "Lactic acid bacteria, probiotics and immune
system". Veterinární medicína, 47, 169-180, 2002.
[3] P. Hlivak, J. Odraska, M. Ferencik, L. Ebringer, E. Jahnova, and A.
Mikes, "One-year application of probiotic strain Enterococcus faecium
M-74 decreases serum cholesterol levels". Bratisl. Lek. Listy 106, 67–
72, 2005.
[4] Y. Huang, and Y. Zheng, "The probiotic Lactobacillus acidophilus
reduces cholesterol absorption through the down-regulation of
Niemann–Pick C1-like 1 in Caco-2 cells". Br. J. Nutr. 9, 1–6, 2009.
[5] L. Pascual, F. Ruiz, W. Giordano, and I. L. Barberis, "Vaginal
colonization and activity of the probiotic bacterium Lactobacillus
fermentum L23 in a murine model of vaginal tract infection". Journal of
Medical Microbiology 59, 360–364, 2010.
[6] O. Ashayerizadeh, B. Dastar, M. Shams Shargh, E. Rahmatnejad, and A.
Ashayerizadeh, "Influence of prebiotic and two herbal additives on
interior organs and hematological indices of broilers". Journal of Animal
and Veterinary Advances. 8(9), p. 1851-1855, 2009.
[7] A. Bhardwaj, G. Kaur, H. Gupta, S. Vij, and R. K. Malik, "Interspecies
diversity, safety and probiotic potential of bacteriocinogenic Enterococcus faecium isolated from dairy food and human faeces".
World Journal of Microbiology and Biotechnology, 27, 591-602, 2011.
[8] J. A. Patterson, and K. M. Burkholder, "Application of prebiotics and
probiotics in poultry production. Poultry Sci., 82: 627-631, 2003.
[9] A. Ahmadova, S. D. Todorov, Y. Choiset, H. Rabesona, T. M. Zadi,
Kuliyev, A., B.D.G.M. Franco, J. M. Chobert, and T. Haertlé,
"Evaluation of antimicrobial activity, probiotic properties and safety of
wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal
cheese". Food Control 30, 631–64, 2013.
[10] B. B. Matijašic´, T. Obermajer, and I. Rogelj, "Quantification of
Lactobacillus gasseri, Enterococcus faecium and Bifidobacterium
infantis in a probiotic OTC drug by real-time PCR". Food Control 21,
419–425, 2010.
[11] L. Saavedra, M. P. Taranto, F. Sesma, and G. F. de Valdez, "Homemade
traditional cheeses for the isolation of probiotic Enterococcus faecium
strains". Int. J. Food Microbiol. 88, 241–245, 2003.
[12] J. Benyacoub, P. F. Perez, F. Rochat, K. Y. Saudan, G. Reuteler, N.
Antille, M. Humen, G. L. De Antoni, C. Cavadini, S. Blum, and E. J.
Schiffrin, "Enterococcus faecium SF68 enhances the immune response
to Giardia intestinalisin mice". J. Nutr. 135: 1171–1176, 2005.
[13] D. G. V. Emmanuel, A. Jafari, K. A. Beauchemin, J. A. Leedle, and B.
N. Ametaj, "Feeding live cultures of Enterococcus faecium and
Saccharomyces cerevisiae induces an inflammatory response in feedlot
steers". J. Anim. Sci. 85:233–239, 2007.
[14] U. Lodemann, K. Hubener, N. Jansen, and H. Martens, "Effects of
Enterococcus faecium NCIMB 10415 as probiotic supplement on
intestinal transport and barrier function of piglets". Arch. Anim. Nutr.
60:35–48, 2006.
[15] L. Scharek, J. Guth, K. Reiter, K. D. Weyrauch, D. Taras, P. Schwerk, P.
Schierack, M. F. Schmidt, L. H. Wieler, and K. Tedin, "Influence of a
probiotic Enterococcus faecium strain on development of the immune
system of sows and piglets". Vet. Immunol. Immunopathol. 105:151–
161, 2005.
[16] W. Theppangna, K. Otsuki, and T. Murase, "Inhibitory effects of
Enterococcus strains obtained from a probiotic product on in vitro
growth of Salmonella enteric serovar Enteritidis strain IFO3313". J.
Food Prot. 69: 2258–2262, 2006.
[17] D. Taras, W. Vahjen, M.Macha, and O. Simon, "Performance, diarrhea
incidence, and occurrence of Escherichia coli virulence genes during
long-term administration of a probiotic Enterococcus faecium strain to
sows and piglets". J. Anim. Sci. 84:608–617, 2006.
[18] M. Tokumaru, H. Konuma, M. Umesako, S.Konno, and K. Shinagawa,
"Rates of detection of Salmonella and Campyobacterin meats in
response to the sample size and the infection level of each species". Int.
J. Food Microbiol. 13:41–46, 1991.
[19] M. A. Ehrmann, P. Kurzak, J. Bauer, and R. F. Vogel, "Characterization
of lactobacilli towards their use as probiotic adjuncts in poultry". J.
Appl. Microbiol. 92:966–975, 2002.
[20] L. M. Shin, R. J. McNally, S. M. Kosslyn, W. L. Thompson, S. L.
Rauch, and N. M. Alpert, "Regional cerebral blood flow duringscriptdriven
imagery in childhood sexual abuse-related posttraumatic stress
disorder: A positron emission tomographic investigation". Am J
Psychiatry 156:575–584, 1999.
[21] K. Perelmuter, M. Fraga, and P. Zunino, "In vitro activity of potential
probiotic Lactobacillus murinus isolated from the dog". Journal of
Applied Microbiology, 104, 1718-1725, 2008.
[22] Y. Huang, and M. C. Adams, "In vitro assessment of the upper
gastrointestinal tolerance of potential probiotic dairy propionibacteria".
International Journal of Food Microbiology, 91, 253 -260, 2004.
[23] A. R. Madureira, M. S. Gião, M. E. Pintado, A. M. P. Gomes, C. Freitas,
and F. X. Malcata, "Incorporation and survival of probiotic bacteria in
whey cheese matrices". J. Food Sci. 70(3): M161-M165, 2005.
[24] M. Succi, P. Tremonte, A. Reale, E. Sorrentino, L. Grazia, and S.
Pacifico, "Bile salt and acid tolerance of Lactobacillus rhamnosus
strains isolated from Parmigiano Reggiano cheese". FEMS
Microbiology Letters, 244, 129-137, 2005.
[25] S. Pieniz, R. Andreazza, T. Anghinoni, F. Camargo, and A. Brandelli,
"Probiotic potential, antimicrobial and antioxidant activities of
Enterococcus durans strain LAB18s. Food Control 37, 251-256, 2014.
[26] P. Sun, J. Wang, and Y. Jiang, "Effects of Enterococcus faecium (SF68)
on immune function in mice". Food Chemistry, 123, 63-68, 2010.
[27] B. Hyronimus, C. L. Marrec, S. A. Hadj, and A. Deschamps, "Acid and
bile tolerance of spore-forming lactic acid bacteria". International
Journal of Food Microbiology, 61, 193-197, 2000.
[28] J. Gong, R., J. Forster, and H. Yu, "Diversity and phylogenetic analysis
of bacteria in the mucosa of chicken ceca and comparison with bacteria
in the cecal lumen". FEMS Microbiol Lett 208:1–7, 2002.
[29] J. B. Payne, J. A. Osborne, P. K. Jenkins, and B. W. Sheldon, "Modeling
the growth and death kinetics of Salmonella in poultry litter as a
function of pH and water activity". Poult. Sci., 86: 191-201, 2007.
[30] EFSA, The community summary report on trends and sources of
zoonoses and zoonotic agents in the European Union in 2007. EFSA J
223:1–320, 2009.
[31] H. R. Taheri, H. Moravej, F. Tabandeh, M. Zaghari, and M. Shivazad,
"Screening of lactic acid bacteria toward their selection as a source of
chicken probiotic". Poult. Sci. 88:1586-1593, 2009.
[32] M. Chichlowski, J. Croom, B. W. McBride, G. Davis, L. Daniel, and M.
Koci, "Direct-fed microbial and salinomycin modulate whole body and
intestinal oxygen consumption and intestinal enterocytes cytokine
production in the broiler chick". Poult. Sci., 86: 1100-1106, 2007.
[33] M. A. Riley, and J. E. Wertz, "Bacteriocins: evolution, ecology, and
application". Annual review of microbiology, Vol.56, pp. 117-137, ISSN
0066-4227, 2002.
[34] P. Audisio, A.De Biase, G. Antonini, M. Belfiore, and M. Oliverio,
"Morphological, molecular and ecological evidence of a new Euro-
Anatolian species of the Meligethes coracinus complex (Coleoptera:
Nitidulidae). Insect Systematics and Evolution, Volume 31, Issue
4, p. 361 – 385, 2000.
[35] R. Herich, T. Kokinčáková, A. Lauková, and M. Levkutová, "Effect of
preventive application of Enterococcus faecium EF55 on intestinal
mucosa during salmonellosis in Chickens". Czech J. Anim. Sci., 55, (1):
42–47, 2010.
[36] V. Strompfová, and A. Lauková, "In vitro study on bacteriocin
production of enterococci associated with chickens". Anaerobe 13: 228-
237, 2007.
[37] M. Ashraf, and N. A. Akram, "Improving salinity tolerance of plants
through conventional breeding and genetic engineering: an analytical
comparison". Biotechnol. Adv., 27: 744-52, 2009.
@article{"International Journal of Biological, Life and Agricultural Sciences:69484", author = "Salma H. Abu Hafsa and A. Mendonca and B. Brehm-Stecher and A. A. Hassan and S. A. Ibrahim", title = "Probiotic Potential and Antimicrobial Activity of Enterococcus faecium Isolated from Chicken Caecal and Fecal Samples", abstract = "Enterococci are important inhabitants of the animal
intestine and are widely used in probiotic products. A probiotic strain
is expected to possess several desirable properties in order to exert
beneficial effects. Therefore, the objective of this study was to
isolate, characterize and identify Enterococcus sp. from chicken cecal
and fecal samples to determine potential probiotic properties.
Enterococci were isolated from chicken ceca and feces of thirty three
clinically healthy chickens from a local farm. In vitro studies were
performed to assess antibacterial activity of the isolated LAB (using
agar well diffusion and cell free supernatant broth technique against
Salmonella enterica serotype Enteritidis), survival in acidic
conditions, resistance to bile salts, and their survival during simulated
gastric juice conditions at pH 2.5. Isolates were identified by
biochemical carbohydrate fermentation patterns using an API 50
CHL kit and API ZYM kits and by sequenced 16S rDNA. An isolate
belonging to E. faecium species exhibited inhibitory effect against S.
enteritidis. This isolate producing a clear zone as large as 10.30 mm
or greater and was able to grow in the coculture medium and at the
same time, inhibited the growth S. enteritidis. In addition, E. faecium
exhibited significant resistance under highly acidic conditions at pH
2.5 for 8 h and survived well in bile salt at 0.2% for 24 h and showing
ability to survive in the presence of simulated gastric juice at pH 2.5.
Based on these results, E. faecium isolate fulfills some of the criteria
to be considered as a probiotic strain and therefore, could be used as a
feed additive with good potential for controlling S. Enteritidis in
chickens. However, in vivo studies are needed to determine the safety
of the strain.
", keywords = "Acid tolerance, antimicrobial activity, Enterococcus
faecium, probiotic.", volume = "9", number = "4", pages = "378-5", }
