Isolation and Characterization of Enterococci Bacteriocinic Strains from Tunisian Fish Viscera

Migaw Sarra, Ghrairi Taoufik, Le Chevalier Patrick, Brillet Benjamin, Fleury Yannick, Hani Khaled

Laboratoire Universitaire de Biodiversité et d’Ecologie Microbienne (EA3882), IFR148 ScInBioS, Université de Brest, Université Européenne de Bretagne, IUT de Quimper, 6 Rue de l’Université, Quimper Cedex, France..
U12-ES03, Department of Biochemistry, Faculty of Medicine Ibn El Jazzar, University of Sousse, Sousse, Tunisia.

DOI: 10.4236/fns.2013.46089                

Abstract

A collection of lactic acid bacteria isolates from fish viscera was studied and investigated regarding to their functional properties and safety aspects. From these, three isolates GM1, GM2 and GM3 were identified as Enterococcus feacium species using molecular methods. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with restriction enzyme HaeIII separated these isolates into distinctive group which suggest genotypic variability within enterococci strains isolated from fish viscera. The three strains GM1, GM2 and GM3 exhibited antimicrobial activity. Indeed strains have been shown to produce bacteriocins with inhibitory effect against food spoilage bacteria and pathogenic fish including Carnobacterium maltaromaticum. The molecular mass of bacteriocin, as calculated by tricine-SDS-PAGE, was found to be 4.5 kDa. All isolates were tested positive upon PCR amplification of enterocin A structural gene. Investigations of antibiotic resistance show that the isolates were mostly sensitive to several antibiotics (ampicillin, penicillin, tetracycline, gentamycine) and resistance to rifampicin. All isolates grow in esculin azide agar as a selective medium for enumeration of probiotic enterococci. This study suggests that our strains can be employed as probiotic or to improve the safety of food products.

Keywords

Bacteriocins; Fish Viscera; Enterococcus; Listeria; Carnobacterium; Probiotic

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	T. J. Eaton and M. J. Gasson, “Molecular Screening of Enterococcus Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates,” Applied and Environmental Microbiology, Vol. 67, No. 4, 2001, pp. 1628-1635. doi:10.1128/AEM.67.4.1628-1635.2001
[2]	G. Giraffa, “Enterococci from Foods,” FEMS Microbiolo- gy Reviews, Vol. 26, No. 2, 2002, pp. 163-171. doi:10.1111/j.1574-6976.2002.tb00608.x
[3]	W. Chingwaru, S. F. Mpuchane and B. A. Gashe, “Ente- rococcus faecalis and Enterococcus faecium Isolates from Milk, Beef and Chicken and Their Antibiotic Resistance,” Journal of Food Protection, Vol. 66, No. 6, 2003, pp. 931-936.
[4]	A. S. Valenzuela, N. Ben Omar, H. Abriouel, M. M. Cana- mero and A. Gálvez, “Isolation and Identification of En- terococcus faecium from Seafoods, Antimicrobial Resis- tance and Production of Bacteriocin-Like Substances,” Food Microbiology, Vol. 27, No. 7, 2010, pp. 955-961. doi:10.1016/j.fm.2010.05.033
[5]	M. R. Foulquié-Moreno, P. Sarantinopoulos, E. Tsakali- dou and L. De Vuyst, “The Role and Application of En- terococci in Food and Health,” International Journal of Food Microbiology, Vol. 106, No. 1, 2006, pp. 1-24. doi:10.1016/j.ijfoodmicro.2005.06.026
[6]	T. Hagi, D. Tanaka, Y. Iwamura and T. Hoshino, “Diver- sity and Seasonal Changes in Lactic Acid Bacteria in the Intestinal Tract of Cultured Freshwater Fish,” Aquacul- ture, Vol. 234, No. 1-4, 2004, pp. 335-346. doi:10.1016/j.aquaculture.2004.01.018
[7]	A. Petersen and A. Dalsgaard, “Species Composition and Antimicrobial Resistance Genes of Enterococcus spp., Isolated from Integrated and Traditional Fish Farms in Thailand,” Applied and Environmental Microbiology, Vol. 5, No. 5, 2003, pp. 395-402. doi:10.1046/j.1462-2920.2003.00430.x
[8]	I. G. Wilson and G. G. McAfee, “Vancomycin-Resistant Enterococci in Shellfish Unchlorinated Waters, and Chic- ken,” International Journal of Food Microbiology, Vol. 79, No. 3, 2002, pp. 143-151. doi:10.1016/S0168-1605(02)00063-6
[9]	V. J. Harwood, J. Whitlock and V. Withington, “Classifi- cation of Antibiotic Resistance Patterns of Indicator Bac- teria Bydiscriminant Analysis, Use in Predicting the Source of Fecal Contamination in Subtropical Waters,” Applied and Environmental Microbiology, Vol. 66, No. 9, 2000, pp. 3698-3704. doi:10.1128/AEM.66.9.3698-3704.2000
[10]	C. M. A. P. Franz, M. E. Stiles, K. H. Schleiferc and W.H. Holzapfel, “Enterococci in Foods a Conundrum for Food Safety,” International Journal of Food Microbiology, Vol. 88, No. 2-3, 2003, pp. 105-112. doi:10.1016/S0168-1605(03)00174-0
[11]	F. Leroi, M. R. Foulquié-Moreno and L. De Vuyst, “Ente- rococcus faecium RZS C5, an Interesting Bacteriocin Producer to Be Used as a Coculture in Food Fermenta- tion,” International Journal of Food Microbiology, Vol. 88, No. 2-3, 2003, pp. 235-240. doi:10.1016/S0168-1605(03)00185-5
[12]	T. R. Klaenhammer, “Genetics of Bacteriocins Produced by Lactic Acid Bacteria,” FEMS Microbiology Reviews, Vol. 12, No. 1-3, 1993, pp. 39-85.
[13]	H. Chen and D. G. Hoover, “Bacteriocins and Their Food Applications,” Comprehensive Reviews in Food Science and Food Safety, Vol. 2, No. 3, 2003, pp. 82-100. doi:10.1111/j.1541-4337.2003.tb00016.x
[14]	L. L. Agerholm, M. L. Bell, G. K. Grunwald and A. As- trup, “The Effect of a Probiotic Milk Product on Plasma Cholesterol, a Meta-Analysis of Short-Term Intervention Studies,” European Journal of Clinical Nutrition, Vol. 54, No. 11, 2000, pp. 856-860. doi:10.1038/sj.ejcn.1601104
[15]	A. Weiss, K. J. Domig and W. Kneifel, “Comparison of Se- lective Media for the Enumeration of Probiotic Entero- cocci from Animal Feed,” Food Technology and Bio- technology, Vol. 43, No. 2, 2005, pp. 147-155.
[16]	C. M. A. P. Franz, W. H. Holzapfel, and M. E. Stiles, Enterococci at the Crossroads of Food Safety. International Journal of Food Microbiology, vol. 47, 1999, pp. 1–24. doi:10.1016/S0168-1605(99)00007-0
[17]	M. T. Aymerich, M. Garriga, J. Ylla, J. Vallier, J. M. Mon- fort and M. Hugas, “Application of Enterocins as Biopre- servatives against Listeria innocua in Meat Products,” Journal of Food Protection, Vol. 63, No. 6, 2000, pp. 721-726.
[18]	W. H. Holzapfel, “Appropriate Starter Culture Technolo- gies for Small-Scale Fermentation in Developing Coun- tries,” International Journal of Food Microbiology, Vol. 75, No. 3, 2002, pp. 197-210. doi:10.1016/S0168-1605(01)00707-3
[19]	N. Ben Omar, A. Castro, R. Lucas, H. Abriouel, N. M. K. Yousif, C. M. A. P. Franz, W. H. Holzapfel, R. Pérez-Pu- lido, M. Martínez-Canamero and A. Gálvez, “Functional and Safety Aspects of Enterococci Isolated from Different Spanish Foods,” Systematic and Applied Microbiology, Vol. 27, No. 1, 2004, pp. 118-113. doi:10.1078/0723-2020-00248
[20]	T. Ghrairi, M. Manai, J. M. Berjaud and J. Frère, “Occur- rence of Anti-Listeria Activity in Lactic Acid Bacteria Strains Isolated from Rigouta, a Traditional Tunisian Cheese,” Journal of Applied Microbiology, Vol. 97, No. 3, 2004, pp. 621-628. doi:10.1111/j.1365-2672.2004.02347.x
[21]	T. Ghrairi, J. Frère, J. M. Berjaud and M. Manai, “Purifi- cation and Characterisation of Bacteriocins Produced by Enterococcus faecium from Tunisian Rigouta Cheese,” Food Control, Vol. 19, No. 2, 2008, pp. 162-169. doi:10.1016/j.foodcont.2007.03.003
[22]	K. Schleifer, and R. Kilpper-Balz, “Transfer of Streptoco- ccus faecalis and Streptococcus faecium to the Genus En- terococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecalis comb. nov.,” International Journal of Systematic Bacteriology, Vol. 34, No. 1, 1984, pp. 31-34. doi:10.1099/00207713-34-1-31
[23]	Y. Fleury, M. A. Dayem, J. J. Montagne, E. Chaboisseau, J. P. Le Caer, P. Nicolas and A. Delfour, “Covalent Struc- ture, Synthesis, and Structure-Function Studies of Mesen- tericin Y 105(37), a Defensive Peptide from Gram-Posi- tive Bacteria Leuconostoc Mesenteroides,” Journal of Biological Chemistry, Vol. 271, No. 24, 1996, pp. 14421- 14429. doi:10.1074/jbc.271.24.14421
[24]	D. G. Anderson and L. L. Mc Kay, “Simple and Rapid Method for Isolating Large Plasmid DNA from Lactis Streptococci,” Applied and Environmental Microbiology, Vol. 46, No. 3, 1983, pp. 549-552.
[25]	W. G. Weisburg, S. M. Barns, D. A. Pelletier and D. J. Lane, “16S Ribosomal DNA Amplification for Phyloge- netic Study,” Journal of Bacteriology, Vol. 173, No. 2, 1991, pp. 697-703.
[26]	J. Andrews, J. Ashby, G. Jevons, T. Marshall, N. Lines and R. Wise, “A Comparison of Antimicrobial Resistance Rates in Gram-Positive Pathogens Isolated in the UK from October 1996 to January 1997 and October 1997 to January 1998,” Journal of Antimicrobial Chemotherapy, Vol. 45, No. 3, 2000, pp. 285-293. doi:10.1093/jac/45.3.285
[27]	E. Ringo and F. J. Gatesoupe, “Lactic Acid Bacteria in Fish, a Review,” Aquaculture, Vol. 160, No. 3-4, 1998, pp. 177-203. doi:10.1016/S0044-8486(97)00299-8
[28]	G. Tiecco, “Microbiologia dei vari Prodotti Alimentary di Origine Animale,” In: C. Edagricole, Ed., Igiene e Tec- nologia alimentaire, Bologna, 2001, pp. 102-103.
[29]	J. J. Leisner, B. G. Laursen, H. Prevost, D. Djamel and P. Dalgaard, “Carnobacterium, Positive and Negative Ef- fects in the Environment and in Foods,” FEMS Microbi- ology Reviews, Vol. 31, No. 5, 2007, pp. 592-613. doi:10.1111/j.1574-6976.2007.00080.x
[30]	A. M. Baya, A. E. Toranzo, B. Lupiani, T. Li, B. S. Ro- berson and F. M. Hetrick, “Biochemical and Serological Characterization of Carnobacterium spp. Isolated from Farmed and Natural Populations of Striped Bass and Cat- fish,” Applied and Environmental Microbiology, Vol. 57, No. 11, 1991, pp. 3114-3120.
[31]	I. Kuhn, A. Iversen, M. Finn, C. Greko, L. G. Burman, A. R. Blanch and X. Vilanova, A. Manero, H. Taylor, J. Cap- lin, L. Domínguez, I. A. Herrero, M. A. Moreno and R. M?llby, “Occurrence and Relatedness of Vancomycin- Resistant Enterococci in Animals, Humans, and the En- vironment in Different European Regions,” Applied and Environmental Microbiology, Vol. 71, No. 9, 2005, pp. 5383-5539. doi:10.1128/AEM.71.9.5383-5390.2005
[32]	H. Blom, T. Katla, H. Nissen and H. Holo, “Characteriza- tion, Production, and Purification of Carnocin H, a Bacte- riocin Produced by Carnobacterium 377,” Current Mi- cryobiology, Vol. 43, No. 4, 2001, pp. 227-231. doi:10.1007/s002840010292
[33]	S. D. Todorov and L. M. T. Dicks, “Screening for Bacte- riocin-Producing Lactic Acid Bacteria from Boza, a Tra- ditional Cereal Beverage from Bulgaria Comparison of the Bacteriocins,” Process Biochemistry, Vol. 41, No. 1, 2002, pp. 11-19. doi:10.1016/j.procbio.2005.01.026
[34]	C. M. A. P. Franz, M. J. van Belkum, W. H. Holzapfel, H. Abriouel and A. Gálvez, “Diversity of Enterococcal Bac- teriocins and Their Grouping in a New Classification Scheme,” FEMS Microbiology Reviews, Vol. 31, No. 3, 2007, pp. 293-310. doi:10.1111/j.1574-6976.2007.00064.x
[35]	C. Herranz, P. Casaus, S. Mukhopadhyay, J. M. Martinez, J. M. Rodriguez, I. F. Nes, E. Herrandez and L. M. Cintas, “Enterococcus faecium P21, a Strain Occurring Naturally in Dry-Fermented Sausages Producing the Class II Bacte- riocins Enterocin A and Enterocin B,” Food Microbiology, Vol. 18, No. 2, 2001, pp. 115-131. doi:10.1006/fmic.2000.0382