Evaluating the Safety of Indigenous Enterococcus species isolated from dairy products and Breast milk by Biochemical and Molecular Methods

Document Type : Research Paper

Authors

1 Azad university of Karaj-Iran

2 Animal Science Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

3 Razi Vaccine and Serum Research Institute.Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

Abstract

Enterococcus plays an important role in various industries. Some Enterococci isolated from dairy and breast milk has been reported as probiotics. However, these species selected as starter cultures or as probiotics should be evaluated for their safety and the absence of different virulence traits including virulence genes and antibiotic resistance. The aim of this study was to determine the safety of locally isolated Enterococcus species biochemically and targeting essential virulence genes by PCR method. A number of suspected Enterococcus species isolated from ewe milk, traditional cheese and mothers' milk were identified to genus level by phenotypic characteristics and 16SrRNA sequencing. The selected isolates were screened for their probiotic properties by determining their acid and bile resistance, antibacterial activity against a number of pathogens and antibiotic resistance. Phenotypic virulence parameters including lipase, DNAse and hemolysis of red blood cells, was determined. The presence or absence of a number of virulence genes including asa1, hyl, esp, agg, gelE, cylA, cylB, cylM, efaAfm, efaAfs and vancomycin resistance genes including vanA, vanH, vanR and vanY was evaluated. The results of this study determined the probiotic properties of some of these species. Four isolates showed acid and bile salt tolerance and showed significant antimicrobial effects. All isolates from Breast milk, lacked virulence genes. In virulance phenotypic experiments, all strains were alpha hemolytic. All strains were susceptible to Tetracycline, Cephalothin Amoxicillin, Cefazolin and Doxycycline and all of them except the TA00154 strain, were resistant to Nalidixic acid and Trimethoprim sulfamethoxazole.

Keywords

References

 Al-Saleh  A , Metwalli A. M and Abu-Tarboush H. M.(2006). Bile salts and acid tolerance and cholesterol removal from media by some lactic acid bacteria and bifidobacteria. Journal of the Saudi Society of food and nutrition. 1: 1-16.
Anderson, J. W. and S. E. Gilliland .(1999). Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans. Journal of the American College of Nutrition .18(1): 43-50.
Araújo, T. F. and C. L. d. L. F. Ferreira .(2013). The genus Enterococcus as probiotic: safety concerns. Brazilian archives of biology and technology .56(3): 457-466.
 Başyiğit G, Kuleaşan H, Karahan AG. (2006).Viability of human-derived probiotic lactobacilli in ice cream produced with sucrose and aspartame. Journal of industrial microbiology & biotechnology. 33(9): 796-800.
Bhardwaj A, Kaur G, Gupta H, Vij S, Malik R.( 2011). Interspecies diversity, safety and probiotic potential of bacteriocinogenic Enterococcus faecium isolated from dairy food and human faeces. World Journal of Microbiology and Biotechnology. 27(3): 591-602.
Billström H, Lund B, Sullivan A, Nord CE. (2008). Virulence and antimicrobial resistance in clinical Enterococcus faecium. International journal of antimicrobial agents. 32(5): 374-377.
Britz, T. and R. K. Robinson.(2008). Advanced dairy science and technology, John Wiley & Sons.  the trusted publisher of academic, scientific, and professional books since 1807.
Buntin,N , Chanthachum,S and Hongpattarakere,T (2008). Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. Songklanakarin Journal of Science & Technology. 30 (Suppl.1), 141-148.
Conde-Estévez D, Sorli L, José Antonio Morales-Molina J.A, Knobel H, Terradas R, Mateu-de Antonio J, Horcajada J.P, Grau S. (2010). Características clínicas diferenciales entre las bacteriemias por Enterococcus faecalis y Enterococcus faecium. Enfermedades Infecciosas y Microbiología Clínica. 28(6): 342-348..
Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, Baldassarri L.(2004). Survey for virulence determinants among Enterococcus faecalis isolated from different sources. Journal of Medical Microbiology. 53(1):13-20.
Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. (2003). Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). Journal of Medical Microbiology. 52(6): 491-498.
Dutka-Malen S, Evers S, Courvalin P. (1995). Detection of glycopeptide resistance genotypes and identification of the species level of clinically relevant enterococci by PCR. Journal of Clinical Microbiology. 33:24–27.
Eaton, T. J. and M. J. Gasson ).2001(.Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Applied and environmental microbiology. 67(4): 1628-1635.
Eaton, T. J. and M. J. Gasson .(2002). A variant enterococcal surface protein Espfm in Enterococcus faecium; distribution among food, commensal, medical, and environmental isolates. FEMS microbiology letters .216(2): 269-275.
Elsner HA, Sobottka I, Mack D, Claussen M, Laufs R, Wirth R. (2000). Virulence factors of Enterococcus faecalis and Enterococcus faecium blood culture isolates. European Journal of Clinical Microbiology and Infectious Diseases. 19(1): 39-42.
Flahaut, S., et al. (1996). Relationship between stress response towards bile salts, acid and heat treatment in Enterococcus faecalis. FEMS microbiology letters. 138(1): 49-54.
 Franz C.M,  Muscholl-Silberhorn A.B,  Yousif N.M.K, Vancanneyt M,  Swings J and HolzapfelW.H. ( 2001). Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Journal of Applied and Environmental Microbiology. 67: 4385-4389.
Giraffa, G. (2003). Functionality of enterococci in dairy products. International journal of food microbiology. 88(2): 215-222.
Hafsa S. H, Mendonca A, Brehm-Stecher B, Hassan A. A, Ibrahim S. A. (2015). Probiotic Potential and Antimicrobial Activity of Enterococcus faecium Isolated from Chicken Caecal and Fecal Samples. World Academy of Science, Engineering and Technology, International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering. 9(4): 359-363.
Heikens E, Bonten MJ, Willems RJ. (2007). Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162. Journal of bacteriology. 189(22): 8233-8240.
Holzapfel WH, Haberer P, Geisen R, Björkroth J, Schillinger U.( 2001).Taxonomy and important features of probiotic microorganisms in food and nutrition. The American journal of clinical nutrition. 73(2):365s-73s.
Hong HA, Khaneja R, Tam NM, Cazzato A, Tan S, Urdaci M. (2009). Bacillus subtilis isolated from the human gastrointestinal tract. Research in microbiology. 160(2):134-43.
Iweriebor BC, Gaqavu S, Obi LC, Nwodo UU, Okoh AI. (2015). Antibiotic susceptibilities of Enterococcus species isolated from hospital and domestic wastewater effluents in Alice, eastern cape province of South Africa. International journal of environmental research and public health. 12(4): 4231-4246.
Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW. (2006). Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in northwestern China. Applied and environmental microbiology. 72(6): 3832-3845.
Jiménez E, Ladero V, Chico I, Maldonado-Barragán A, López M, Martín V, Fernández L, Fernández M, Álvarez MA, Torres C, Rodríguez JM. (2013). Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk. BMC microbiology. 13(1): 1.
Kıvanç SA, Kıvanç M, Yiğit T. (2016). Antibiotic susceptibility, antibacterial activity and characterisation of Enterococcus faecium strains isolated from breast milk. Experimental and therapeutic medicine .12(3): 1732-1740
Kos V. N, Desjardins C.A, Griggs A, Cerqueira G, Van Tonder A, Holden  M.T.G, Godfrey  P, Palmer  K.L, Bodi K, Mongodin  E.F, Wortman  J, Feldgarden  M, Lawley  T, Gill S.R, Haas  B.J, Birren  B, Gilmore M.S.(2012). Comparative genomics of vancomycin-resistant Staphylococcus aureus strains and their positions within the clade most commonly associated with Methicillin-resistant S. aureus hospital-acquired infection in the United States. MBio( bimonthly peer-reviewed open access scientific journal). 3(3): e00112-00112.
Lin, M.-Y. and F.-J. Chang .(2000). Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356. Digestive diseases and sciences. 45(8): 1617-1622.
Lobo V, Patil A, Phatak A, Chandra N.( 2010). Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy reviews .4(8): 118-26.
Miele A, Bandera M, Goldstein BP. (1995). Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in VanA isolates. Antimicrobial agents and chemotherapy. 39(8): 1772-1778.
Moraes PM, Perin LM, Todorov SD, Silva A Jr, Franco BD, Nero LA.  (2012). Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese.Journal of Applied Microbiology.113:318-328.
Moreno  M. F, 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.
Ogaki, M, Rocha KR, Terra MR, Furlaneto MC, Maia LF. (2016). Screening of the enterocin-encoding genes and antimicrobial activity in Enterococcus species. Journal of microbiologyand biotechnology. 28;26(6):1026-34.
Ou C.C, Lu T.M, Tsai J.J, Yen J.H, Chen H.W, Lini M.Y.( 2009). Antioxidative Effect of Lactic Acid Bacteria: Intact Cells vsIntracellular Extracts. Journal of Food and Drug Analysis. Vol. 17 Issue 3, p209-216.
Paulsen IT, Banerjei L, Myers GS, Nelson KE, Seshadri R, Read TD, Fouts DE, Eisen JA, Gill SR, Heidelberg JF, Tettelin H,Dodson RJ, Umayam L, Brinkac L, Beanan M, Daugherty S, DeBoy RT, Durkin S, Kolonay J, Madupu R, Nelson W, Vamathevan J, Tran B, Upton J, Hansen T, Shetty J, Khouri H, Utterback T, Radune D, Ketchum KA, Dougherty BA, Fraser CM.(2003). Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science. 299(5615): 2071-2074.
 Pieniz S,  Andreazza R,  Anghinoni T,  Camargo F,  Brandelli A. (2014). Probiotic potential, antimicrobial and antioxidant activities of Enterococcus durans strain LAB18s. Food Control .37: 251-256.
Rabbani M, Shafiee F, Shayegh Z, MirMohammadSadeghi H, Samsam Shariat Z, Etemadifar Z, Moazen F. (2015). Isolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria. Iranian journal of pharmaceutical research: IJPR .14(3): 901-6.
Rahimi F, Talebi M, Saifi M, Pourshafie MR. (2007). Distribution of enterococcal species and detection of vancomycin resistance genes by multiplex PCR in Tehran sewage. Iranian Biomedical Journal. 11(3): 161-167.
Rasmussen M, Johansson D, Söbirk SK, Mörgelin M, Shannon O. (2010). Clinical isolates of Enterococcus faecalis aggregate human platelets. Microbes and Infection. 12(4): 295-301.
Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, Klare I, Nallapareddy SR, Huang W, Murray BE.. (2003). A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. Journal of Infectious Diseases. 187(3): 508-512.
Ryan KJ, Ray CG.(2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 294–5. ISBN: 0-8385-8529-9.
Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS. (1999). Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infection and immunity. 67(1): 193-200.
Todorov, S. and L. Dicks .(2005). Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria. Enzyme and Microbial Technology. 36(2): 318-326.
Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H.( 2004). Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. Journal of Clinical Microbiology .42(10): 4473-4479.
Animal Sciences Journal
Volume 31, Issue 120
December 2018
Pages 89-102

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