Probiotic potential of Enterococcus strains with multiple enterocin-encoding genes

Zahia BENMOUNA; Eva VALDIVIA; Manuel MONTALBÁN-LÓPEZ; Fatiha DALACHE; Halima ZADI-KARAM; Nour-Eddine KARAM; Manuel MARTÍNEZ-BUENO

doi:10.55779/nsb16111792

Authors

Zahia BENMOUNA University of Sciences and Technology of Oran Mohamed Boudiaf, USTOMB, Faculty of Natural and Life Sciences, Department of Living and Environment, PO Box 1505, El Mnaouar, Oran 31000; University of Science and Technology Houari Boumediene, USTHB, Faculty of Biological Sciences, Laboratory of Research in Arid Areas, PO Box 32, El Alia, Bab Ezzouar, Algiers 16111 (DZ)
Eva VALDIVIA Universidad de Granada, Departamento de Microbiología, Avda. Fuentenueva, s/n, Granada 18071 (ES)
Manuel MONTALBÁN-LÓPEZ Universidad de Granada, Departamento de Microbiología, Avda. Fuentenueva, s/n, Granada 18071 (ES)
Fatiha DALACHE University of Mostaganem Abdelhamid Ibn Badis, Faculty of Natural and Life Sciences, Department of Biology, PO Box 188, Mostaganem 27000; University of Oran1, Laboratory of Microorganisms Biology and Biotechnology, PO Box 1524, El M’Naouer, Oran 31000 (DZ)
Halima ZADI-KARAM University of Oran1, Laboratory of Micro-organisms Biology and Biotechnology, PO Box 1524, El M’Naouer, Oran 31000 (DZ)
Nour-Eddine KARAM University of Oran1, Laboratory of Micro-organisms Biology and Biotechnology, PO Box 1524, El M’Naouer, Oran 31000 (DZ)
Manuel MARTÍNEZ-BUENO Universidad de Granada, Departamento de Microbiología, Avda. Fuentenueva, s/n, Granada 18071 (ES)

DOI:

https://doi.org/10.55779/nsb16111792

Keywords:

enterocins, Enterococcus, lactic acid bacteria, probiotics, virulence factors

Abstract

In this work, we sought to identify and characterize three strains of Enterococcus isolated from camel milk and fermented wheat, based on the capacity to produce bacteriocins and the probiotic potential. Polymerase chain reaction analyses were used to identify bacterial isolates and structural genes of bacteriocins, and also to detect potential enterococcal virulence genes (cylA, esp, gelE, efaAfs, hyl, ace, asa1, vanA, and vanB). The antimicrobial activity of the strains was investigated in solid media by the agar spot method against several pathogenic bacteria. The probiotic potential of the strains was also analysed using low pH (pH 3.0), bile salt resistance, DNAase, and antibiotic susceptibility assays. The strains were identified using rRNA16S sequencing gene, showing their belonging to E. faecium species. Based on PCR results, E. faecium CM9 and CM18 strains included in the genome the structural gene of enterocin A, enterocin B, and enterocin P, while E. faecium H3 possessed enterocin MR10A/B structural gene. The sequence analysis revealed that the H3 strain included in the genome the structural gene of enterocin L50A/B. All the pathogenic bacteria (Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, E. coli, and Pseudomonas aeruginosa) were inhibited by the three strains E. faecium CM9, CM18, and H3. In the well diffusion test, the supernatants of the three strains exhibited inhibitory activity against Listeria monocytogenes CECT 4032. The strains showed high tolerance to low pH and bile salts, did not possess DNase, were susceptible to the majority of antibiotics assayed, and did not possess any of the virulence genes analyzed in this study. A promising candidate strains were identified as potential probiotics with anti-bacterial action against pathogenic bacteria.

Metrics

Metrics Loading ...

References

Abriouel H, Ben Omar H, Lucas R, Martinez-Cañamero M, Gálvez A (2006). Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources. Letters in Applied Microbiology 42(4):331-337. https://doi.org/10.1111/j.1472-765X.2006.01865.x

Achemchem F, Cebrián R, Abrini J, Martínez-Bueno M, Valdivia E, Maqueda M (2012). Antimicrobial characterization and safety aspects of the bacteriocinogenic Enterococcus hirae F420 isolated from Moroccan raw goat milk. Canadian Journal of Microbiology 58(5):596-604. https://doi.org/10.1139/w2012-027

Anagnostopoulos DA, Bozoudi D, Tsaltas D (2018). Enterococci isolated from cypriot green table olives as a new source of technological and probiotic properties. Fermentation 4(2):48. https://doi.org10.3390/fermentation4020048

Aymerich T, Holo H, Havarstein LS, Hugas M, Garriga M, Nes IF (1996). Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Applied and Environmental Microbiology 62(5):1676-1682. https://doi.org/10.1128/aem.62.5.1676-1682.1996

Ben Braïek OB, Cremonesi P, Morandi S, Smaoui S, Hani K, Ghrairi T (2018). Safety characterisation and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain. Microbial Pathogenesis 118:32-38. https://doi.org/10.1016/j.micpath.2018.03.005

Ben Braïek OB, Ghomrassi H, Cremonesi P, Morandi S, Fleury Y, Chevalier PL, … Ghrairi T (2017). Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei). Antonie van Leeuwenhoek 110:771-786. https://doi.org/10.1007/s10482-017-0847-1

Benmouna Z, Dalache F, Zadi-Karam H, Karam N-E (2018). Optimization and some characteristics of bacteriocin produced by Enterococcus sp. CM9 collected from Mauritanian Camel milk. Emirates Journal of Food and Agriculture 30(4):275-282. https://doi.org/10.9755/ejfa.2018.v30.i4.1662

Benmouna Z, Dalache F, Zadi-Karam H, Karam N-E, Vuotto C (2020). Ability of three lactic acid bacteria to grow in sessile mode and to inhibit biofilm formation of pathogenic bacteria. In: Donelli G (Ed). Advances in microbiology, infectious diseases and public health. Advances In Experimental Medicine and Biology 1282. Springer, Cham pp 105-114. https://doi.org/10.1007/5584_2020_495

Bustos AY, Saavedra L, de Valdez GF, Raya RR, Taranto MP (2012). Relationship between bile salt hydrolase activity, changes in the internal pH and tolerance to bile acids in lactic acid bacteria. Biotechnology Letters 34:1511-1518. https://doi.org/10.1007/s10529-012-0932-5

CA-SFM (Comité de l'Antibiogramme de la Société Française de Microbiologie) (2010). Société française de microbiologie [French microbiology society], Paris.

Cebrián R, Baños A, Valdivia E, Pérez-Pulido R, Martínez-Bueno M, Maqueda M (2012). Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain. Food Microbiology 30(1):59-67. https://doi.org/10.1016/j.fm.2011.12.002

Chajęcka-Wierzchowska W, Zadernowska A, Łaniewska-Trokenheim Ł (2016). Virulence factors, antimicrobial resistance and biofilm formation in Enterococcus spp. isolated from retail shrimps. LWT- Food Science and Technology 69:117-122. https://doi.org/10.1016/j.lwt.2016.01.034

Cintas LM, Casaus P, Herranz PE, Håvarstein LS, Holo H, Hernández PE, Nes IF (2000). Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. Journal of Bacteriology 182(23):6806-6814. https://doi.org/10.1128/jb.182.23.6806-6814.2000

Cintas LM, Casaus P, Holo H, Hernández PE, Nes IF, Håvarstein LS (1998). Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to Staphylococcal hemolysins. Journal of Bacteriology 180(8):1988-1994. https://doi.org/10.1128/jb.180.8.1988-1994.1998

De Vuyst L, Moreno MRF, Revets H (2003). Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. International Journal of Food Microbiology 84(3):299-218. https://doi.org/10.1016/S0168-1605(02)00425-7

Du Toit M, Franz CMAP, Dicks LMT, Holzapfel WH (2000). Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. Journal of Applied Microbiology 88(3):482-494. https://doi.org/10.1046/j.1365-2672.2000.00986.x

Facklam RR, Collins MD (1989). Identification of Enterococcus species isolated from human infections by a conventional test scheme. Journal of Clinical Microbiology 27(4):731-734. https://doi.org/10.1128/jcm.27.4.731-734.1989

FAO/WHO (Food and Agriculture Organization/World Health Organization) (2002). Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. FAO/WHO working group, Rome.

Favaro L, Basaglia M, Casella S, Hue I, Dousset X, de Melo Franco BDG, Todorov SD (2014). Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from homemade white brine cheese. Food Microbiology 38:228-239. https://doi.org/10.1016/j.fm.2013.09.008

Fernández M, Hudson JA, Korpela R, de los Reyes-Gavilán CG (2015). Impact on human health of microorganisms present in fermented dairy products: an overview. BioMed Research International 2015:1-13. https://doi.org/10.1155/2015/412714

Fleming HP, Etchells JL, Costilow RN (1975). Microbiological inhibition of isolate of Pediococcus from cucumber brine. Applied and Environmental Microbiology 30(6):1040-1042. https://doi.org/10.1128/am.30.6.1040-1042.1975

Franz CMAP, Muscholl-Silberhorn AB, Yousif NMK, Van-Canneyt M, Swings J, Holzapfel WH (2001). Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Applied and Environmental Microbiology 67(9):4385-4389. https://doi.org/10.1128/AEM.67.9.4385-4389.2001

García-Solache M, Rice LB (2019). The Enterococcus: a model of adaptability to its environment. Clinical Microbiology Reviews 32(2). https://doi.org/10.1128/cmr.00058-18

Ghomrassi H, Ben Braïek O, Choiset Y, Haertlé T, Hani K, Chobert JM, Ghrairi T (2016). Evaluation of marine bacteriocinogenic enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria. Diseases of Aquatic Organisms 118(1):31-43. https://doi.org/10.3354/dao02953

Gutiérrez J, Criado R, Olivera A, Citti R, Martín M, Herranz C, … Hernández P (2002). Cloning, production, secretion and purification of the bacteriocin enterocin P in Escherichia coli. In: Abstracts of the International Symposium on Enterococci in Foods: Functional and Safety Aspects. Berlin pp 16.

Hanchi H, Mottawea W, Sebei K, Hammami R (2018). The Genus Enterococcus: between probiotic potential and safety concerns-an update. Frontiers in Microbiology 9:1791. https://doi.org/10.3389/fmicb.2018.01791

Hu CB, Malaphan W, Zendo T, Nakayama J, Sonomoto K (2010). Enterocin X, a novel two peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides. Applied and Environmental Microbiology 76(13):4542-4545. https://doi.org/10.1128/AEM.02264-09

Javed A, Masud T, ul Ain Q, Imran M, Maqsood S (2011). Enterocins of Enterococcus faecium, emerging natural food preservatives. Annals of Microbiology 61:699-708. https://doi.org/10.1007/s13213-011-0223-8

Karpiński TM, Szkaradkiewicz AK (2013). Characteristic of bacteriocines and their application. Polish Journal of Microbiology 62(3):223-235. https://pubmed.ncbi.nlm.nih.gov/24459827

Kasimin ME, Shamsuddin S, Molujin AM, Sabullah MK, Gansau JA, Jawan R (2022). Enterocin: promising biopreservative produced by Enterococcus sp. Microorganisms 10(4):684. https://doi.org/10.3390/microorganisms10040684

Kavitake D, Devi PB, Delattre C, Reddy GB, Shetty PH (2023). Exopolysaccharides produced by Enterococcus genus-An overview. International Journal of Biological Macromolecules 226:111-120. https://doi.org/10.1016/j.ijbiomac.2022.12.042

Lukášová J, Šustáčková A (2003). Enterococci and antibiotic resistance. Acta Veterinaria Brno 72(2):315-323. https://actavet.vfu.cz/72/2/0315/

Maragkoudakis PA, Zoumpopoulou G, Miaris C, Kalantzopoulos G, Pot B, Tsakalidou E (2006). Probiotic potential of Lactobacillus strains isolated from dairy products. International Dairy Journal 16(3):189-199. https://doi.org/10.1016/j.idairyj.2005.02.009

Marteau P, Minekus M, Havenaar R, Huis In’t Veld JHJ (1997). Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile. International Dairy Journal 80(6):1031-1037. https://doi.org/10.3168/jds.S0022-0302(97)76027-2

Martínez-Bueno M, Valdivia E, Gálvez A, Coyette J, Maqueda M (1998). Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS-48 in Enterococcus faecalis. Molecular Microbiology 27(2):347-358. https://doi.org/10.1046/j.1365-2958.1998.00682.x

Martín-Platero AM, Valdivia E, Maqueda M, Martínez-Bueno M (2009). Characterization and safety evaluation of enterococci isolated from Spanish goats' milk cheeses. International Journal of Food Microbiology 132(1):24-32. https://doi.org/10.1016/j.ijfoodmicro.2009.03.010

Martín-Platero AM, Valdivia E, Ruiz-Rodriguez M, Soler JJ, Martin-Vivaldi M, Maqueda M, Martínez-Bueno M (2006). Characterization of antimicrobial substances produced by Enterococcus faecalis MRR 10-3, isolated from the uropygial gland of the Hoopoe (Upupa epops). Applied and Environmental Microbiology 72(6):4245-4249. https://doi.org/10.1128/AEM.02940-05

Moreno MRF, Sarantinopoulos P, Tsakalidou E, De Vuyst L (2006). The role and application of enterococci in food and health. International Journal of Food Microbiology 106(1):1-24. https://doi.org/10.1016/j.ijfoodmicro.2005.06.026

Nami Y, Vaseghi Bakhshayesh R, MohammadZadeh Jalaly H, Lotfi H, Eslami S, Hejazi MA (2019). Probiotic properties of Enterococcus isolated from artisanal dairy products. Frontiers in Microbiology 10:300. https://doi.org/10.3389/fmicb.2019.00300

Nes IF, Diep DB, Holo H (2007). Bacteriocin diversity in Streptococcus and Enterococcus. Journal of Bacteriology 189(4):1189-1198. https://doi.org/10.1128/jb.01254-06

Nes IF, Diep DB, Ike Y (2014). Enterococcal bacteriocins and antimicrobial proteins that contribute to niche control. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (Eds). Enterococci: from commensals to leading causes of drug resistant infection, 1st edn. Massachusetts Eye and Ear Infirmary, Boston, MA. https://www.ncbi.nlm.nih.gov/books/NBK190424

Ogaki MB, Rocha KR, Terra MR, Furlaneto MC, Furlaneto-Maia L (2016). Screening of the enterocin-encoding genes and antimicrobial activity in Enterococcus species. Journal of Microbiology and Biotechnology 26(6):1026-1034. https://doi.org/10.4014/jmb.1509.09020

Özdemir GB, Oryaşın E, Bıyık HH, Özteber M, Bozdoğan B (2011). Phenotypic and genotypic characterization of bacteriocins in enterococcal isolates of different sources. Indian Journal of Microbiology 51(2):182-187. https://doi.org/10.1007/s12088-011-0143-0

Özmen Toğay S, Çelebi Keskin A, Açık L, Temiz A (2010). Virulence genes, antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods. Journal of Applied Microbiology 109(3):1084-1092. https://doi.org/10.1111/j.1365-2672.2010.04763.x

Perez RH, Himeno K, Ishibashi N, Masuda Y, Zendo T, Fujita K, … Sonomoto K (2012). Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system. Journal of Bioscience and Bioengineering 114(5):490-496. https://doi.org/10.1016/j.jbiosc.2012.06.003

Rehaiem A, Belgacem ZB, Edalatian MR, Martínez B, Rodríguez A, Manai M, Guerra NP (2014). Assessment of potential probiotic properties and multiple bacteriocin encoding-genes of the technological performing strain Enterococcus faecium MMRA. Food Control 37:343-350. https://doi.org/10.1016/j.foodcont.2013.09.044

Reviriego C, Eaton T, Martín R, Jiménez E, Fernández L, Gasson MJ, Rodríguez JM (2005). Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. Journal of Human Lactation 21(2):131-137. https://doi.org/10.1177/0890334405275394

Rodríguez-Lucas C, Ladero V (2023). Enterococcal Phages: Food and Health Applications. Antibiotics 12(5):842. https://doi.org/10.3390/antibiotics12050842

Ruiz-Rodríguez M, Valdivia E, Martín-Vivaldi M, Martín-Platero AM, Martínez-Bueno M, Méndez M, … Soler JJ (2012). Antimicrobial activity and genetic profile of enteroccoci isolated from Hoopoes uropygial gland. PLoS One 7(7):e41843. https://doi.org/10.1371/journal.pone.0041843

Saavedra J, Taranto MP, Sesma F, De Valdez GF (2003). Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains. International Journal of Food Microbiology 88(2-3):241-245. https://doi.org/10.1016/S0168-1605(03)00186-7

Sava IG, Heikens E, Huebner J (2010). Pathogenesis and immunity in enterococcal infections. Clinical Microbiology and Infection 16(6):533-540. https://doi.org/10.1111/j.1469-0691.2010.03213.x

Schillinger U, Lücke FK (1989). Antibacterial activity of Lactobacillus sake isolated from meat. Applied and Environmental Microbiology 55(8):1901-1906. https://doi.org/10.1128/aem.55.8.1901-1906.1989

Shehata MG, El Sohaimy SA, El-Sahn MA, Youssef MM (2016). Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Annals of Agricultural Sciences 61(1):65-75. https://doi.org/10.1016/j.aoas.2016.03.001

Singhal N, Maurya AK, Mohanty S, Kumar M, Virdi JS (2019). Evaluation of bile salt hydrolases, cholesterol-lowering capabilities, and probiotic potential of Enterococcus faecium isolated from rhizosphere. Frontiers in Microbiology 10:1567. https://doi.org/10.3389/fmicb.2019.01567

Sonsa-Ard N, Rodtong S, Chikindas ML, Yongsawatdigul J (2015). Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe. Food Control 54:308-316. https://doi.org/10.1016/j.foodcont.2015.02.010

Strompfová V, Lauková A (2007). In vitro study on bacteriocin production of enterococci associated with chickens. Anaerobe 13(5-6):228-237. https://doi.org/10.1016/j.anaerobe.2007.07.002

Suvorov A (2020). What is wrong with enterococcal probiotics? Probiotics and antimicrobial proteins 12(1):1-4. https://doi.org/10.1007/s12602-020-09633-y

Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S (2001). Quality assurance criteria for probiotic bacteria. The American Journal of Clinical Nutrition 73(2):393-398. https://doi.org/10.1093/ajcn/73.2.393s

Vankerckhoven V, Huys G, Vancanneyt M, Vael C, Klare I, Romond M-B, … Goossens H (2008). Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project. Trends in Food Science & Technology 19(2):102-114. https://doi.org/10.1016/j.tifs.2007.07.013

Wang X, Yang Y, Huycke MM (2020). Risks associated with enterococci as probiotics. Food Research International 129:108788. https://doi.org/10.1016/j.foodres.2019.108788

Wardal E, Markowska K, Żabicka D, Wroblewska M, Giemza M, Mik E, … Sadowy E (2014). Molecular analysis of vanA outbreak of Enterococcus faecium in two Warsaw hospitals: the importance of mobile genetic elements. BioMed Research International 2014:575367. https://doi.org/10.1155/2014/575367

Wu Y, Pang X, Wu Y, Liu X, Zhang X (2022). Enterocins: classification, synthesis, antibacterial mechanisms and food applications. Molecules 27(7):2258. https://doi.org/10.3390/molecules27072258

Zaghloul EH, Abuohashish HM, El Sharkawy AS, Abbas EM, Ahmed MM, Al-Rejaie SS (2023). Probiotic potential of the marine isolate Enterococcus faecium EA9 and In Vivo evaluation of its antisepsis action in rats. Marine Drugs 21(1):45. https://doi.org/10.3390/md21010045

Zommiti M, Cambronel M, Maillot O, Barreau M, Sebei K, Feuilloley M, … Connil N (2018). Evaluation of probiotic properties and safety of Enterococcus faecium isolated from artisanal Tunisian meat “Dried Ossban”. Frontiers in Microbiology 9:1685. https://doi.org/10.3389/fmicb.2018.01685