Interaction of primary mammary bovine epithelial cells with biofilm-forming staphylococci associated with subclinical bovine mastitis

Document Type : Full paper (Original article)

Authors

1 Ph.D. Student in Applied Microbiology, Department of Microbiology, Faculty of Natural and Life Sciences, University of Bejaia, Bejaia, Algeria

2 Institute of Veterinary and Agricultural Sciences, University of Batna, Batna, Algeria

3 Department of Microbiology, Faculty of Natural and Life Sciences, University of Bejaia, Bejaia, Algeria

Abstract

Background: Staphylococci are recognized worldwide as one of the most important etiological agents of bovine mastitis due to their virulence factors such as their ability to penetrate inside mammary epithelial cells and their ability to form biofilm. Aims: The objectives of this study were to establish a model of primary mammary epithelial cells originating from the secretory tissue of the bovine udder in order to evaluate the invasion ability of 42 staphylococci isolated from subclinical bovine mastitis cases. Methods: Two techniques were used to establish a model of primary mammary epithelial cells, the explant technique and the enzymatic method. Biofilm formation was detected using a quantitative spectrophotometric assay. When compared with the enzymatic digestion method, the epithelial cells obtained by the explant technique grew faster and reached quickly to confluence. Results: The results showed that 60% of Staphylococcus aureus isolates (n=12) were able to invade the epithelial cells and 72.7% of coagulase negative staphylococci (CNS) isolates were invasive (n=16). Staphylococcus xylosus isolates showed higher invasion values compared to S. aureus isolates and non-biofilm forming staphylococci were able to invade primary epithelial cells, but no significant difference was found between the internalization capabilities of biofilm positive and negative isolates. Conclusion: The results show that the explant technique is a valuable method for developing primary epithelial cells without damaging the cells, and provides new insights regarding the ability of staphylococci to penetrate inside primary mammary epithelial cells.

Keywords


Alge, KS; Hauck, SM; Priglinger, SG; Kampik, A and Ueffing, M (2005). Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival. J. Proteome. Res., 5: 862-878.
Almeida, RA; Matthews, KR; Cifrian, E; Guidry, AJ and Oliver, SP (1996). Staphylococcus aureus invasion of bovine mammary epithelial cells. J. Dairy Sci., 79: 1021-1026.
Almeida, RA; Matthews, KR and Oliver, SP (1997). Eukaryotic and prokaryotic cell functions required for invasion of Staphylococcus aureus into bovine mammary epithelial cells. J. Vet. Med. B., 44: 139-145.
Almeida, RA and Oliver, SP (1995). Invasion of bovine mammary epithelial cells by Streptococcus dysgalactiae. J. Dairy Sci., 78: 1310-1317.
Almeida, RA and Oliver, SP (2001). Interaction of coagulase-negative Staphylococcus species with bovine mammary epithelial cells. Microb. Pathog., 31: 205-212.
Anaya López, JL; Contreras Guzmán, OE; Cárabez Trejo, A; Baizabal Aguirre, VM; López Meza, JE; Valdez Alarcón, JJ and Ochoa Zarzosa, A (2006). Invasive potential of bacterial isolates associated with subclinical bovine mastitis. Res. Vet. Sci., 8: 358-361.
Bardiau, M; Detilleux, J; Farnir, F; Mainil, JG and Ote, I (2014). Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis. Vet. Microbiol., 169: 74-79.
Barkema, HW; Schukken, YH and Zadoks, RN (2006). Invited review: the role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J. Dairy Sci., 89: 1877-1895.
Berlutti, F; Morea, C; Battistoni, A; Sarli, S; Cipriani, P; Superti, F; Ammendolia, MG and Valenti, P (2005). Iron availability influences aggregation, biofilm, adhesion and invasion of Pseudomonas aeruginosa and Burkholderia cenocepacia. Int. J. Immunopathol. Pharmacol., 18: 661-670.
Brouillette, E; Grondin, G; Shkreta, L; Lacasse, P and Talbot, BG (2003). In vivo and in vitro demonstration that Staphylococcus aureus is an intracellular pathogen in the presence or absence of fibronectin-binding proteins. Microb. Pathog., 35: 159-168.
Buehrain, G; Eby, E and Michael, J (2014). Cell line cross-contamination: how aware are mammalian cell culturists of the problem and how to monitor it? In Vitro Cell. Dev.Biol. Anim., 40: 211-215.
Buzzola, FR; Quelle, L; Gomez, MI; Catalano, M; Steele Moore, L; Berg, D; Gentilini, E; Denamiel, G and Sordelli, DO (2001). Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina. Epidemiol. Infect., 126: 445-452.
Carrel, A (1912). On the permanent life of tissues outside of the organism. J. Exp. Med., 15: 516-528.
Cifrian, E; Guidry, AJ; O’Brien, CN; Keys, JE and Marquardt, WW (1994). Bovine mammary teat and ductal epithelial cell cultures. Am. J. Vet. Res., 55: 239-246.
Freshney, RA (2010). Culture of animal cells. 6th Edn., Glasgow, UK. PP: 248-249.
Gibson, CA; Vega, JR; Baumrucker, CR; Oakley, CS and Welsch, CW (1991). Establishment and characterization of bovine mammary epithelial cell lines. In Vitro Cell. Dev. Biol., 27(A): 585-594.
Haran, KP; Godden, SM; Boxrud, D; Jawahir, S; Bender, JB and Sreevatsan, S (2012). Prevalence and characterization of Staphylococcus aureus, including Methicillin-resistant Staphylococcus aureus, isolated from bulk tank milk from Minnesota dairy farms. J. Clin. Microbiol., 50: 688-695.
Harrison, RG; Greenman, MJ; Mall, FP and Jackson, CM (1907). Observations of the living developing nerve fiber. Anat. Rec., 1: 116-128.
Hensen, SM; Pavicić, MJ; Lohuis, JA and Poutrel, B (2000). Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains. J. Dairy Sci., 83: 418-429.
Huynh, HT; Robitaille, G and Turner, JD (1991). Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp. Cell. Res., 197: 191-199.
Ishige, I; Nagamura-Inoue, T; Honda, MJ; Harnprasopwat, R; Kido, M; Sugimoto, M; Nakauchi, H and Tojo, A (2009). Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton’s jelly explants of human umbilical cord. Int. J. Hematol., 90: 261-269.
Kaur, G and Dufour, JM (2012). Cell lines valuable tools or useless artifacts. Spermatogenesis. 2: 1-5.
Kedjarune, U; Pongprerachok, S; Arpornmaeklong, P and Ungkusonmongkhon, K (2001). Culturing primary human gingival epithelial cells: comparison of two isolation techniques. J. Craniomaxillofac. Surg., 29: 224-231.
Latasa, C; Roux, A; Toledo Arana, A; Ghigo, JM; Gamazo, C; Penadés, JR and Lasa, I (2005). BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enteric serovar Enteritidis. Mol.Microbio., 58: 1322-1339.
Quinn, PJ; Markey, BK; Carter, ME; Donnelly, WJ and Leonard, FC (2002). Veterinary microbiology and microbial disease. 8th Edn., London, UK, Blackwell Science. PP: 465-475.
Oliveira, M; Bexiga, R; Nunes, SF and Vilela, CL (2011). Invasive potential of biofilm-forming staphylococci bovine subclinical mastitis isolates. J. Vet. Sci., 12: 95-97.
Pal, K and Grover, PL (1983). A simple method for the removal of contaminating fibroblasts from cultures of rat mammary epithelial cells. Cell. Biol. Int. Rep., 7: 779-783.
Pan, C; Kumar, C; Bohl, S; Klingmueller, U and Mann, M (2009). Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol. Cell. Proteom., 8: 443-450.
Pereyra, EL; Picech, F; Renna, MS; Baravalle, C; Andreotti, CS; Russi, R; Calvinho, LF; Diez, C and Dallard, BE (2016). Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells. Vet. Microbiol., 183: 69-77.
Rose, MT and McConochie, H (2006). The long road to a representative in vitro model of bovine lactation. JIFS., 3: 67-72.
Souza, FN; Piepers, S; Della Libera, AMMP; Heinemann, MB; Cerqueira, MMOP and DeVliegher, S (2016). Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior. J. Dairy Sci., 99: 2867-2874.
Spath, L; Rotilio, V; Alessandrini, M; Gambara, G; De Angelis, L; Mancini, M; Mitsiadi, TA; Vivarelli, E; Naro, F; Filippini, Aand Papaccio, G (2010). Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials. J. Cell. Mol. Med., 14: 1635-1644.
Stepanovic, S; Vukovic, D; Hola, V; Di Bonaventura, G; Djukic, S; Cirkovic, I and Ruzicka, F (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS., 115: 891-899.
Yang, X; Qu, L; Wang, X; Zhao, M; Li, W; Hua, J; Shi, M; Moldovan, N; Wang, Hand Dou, Z (2007). Plasticity of epidermal adult stem cells derived from adult goat ear skin. Mol. Reprod. Dev., 74: 386-396.
Zhang, X; Sun, H; Tang, X; Ji, J; Li, X; Sun, J; Ma, Z; Yuan, J and Han, ZC (2005). Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells. Exp. Eye. Res., 80: 227-233.