Les staphylocoques d’origine animale : génétique de la résistance à la méticilline et évaluation thérapeutique des bactériophages dans le cadre des mammites bovines | Staphylococci from animal origin: genetics of methicillin resistance and therapeutic assessment of bacteriophages in the context of bovine mastitis

Milk production, one of the main foodstuffs in Europe and particularly in Belgium, is often faced with problems which each have significant impacts on economies. Indeed, decreases in milk production, penalties imposed for the high number of somatic cells in milk (> 4x105 cells / ml in tanks), the slaughter of cows with incurable mastitis, and antibiotic treatment each contribute to these economic losses (Halasa et al., 2007; Stevens et al., 2016; Ashraf and Imran, 2018). Antibiotic treatment will lead to the emergence of antimicrobial resistances such as methicillin resistance which is one of the most encountered in bovine mastitis and concerns staphylococci species (S. aureus and non-aureus) called methicillin resistant staphylococci (MRS) (Cohn and Middleton , 2010). The MRS represents a threat for public health due to their zoonotic potential and the transferability of the resistance between different staphylococcal species (Becker et al., 2018; Lakhundi and Zhang, 2018). Following these antibiotic resistance problems, farmers are increasingly exploring non-antibiotic alternatives for the treatment of bovine mastitis (Ciorba et al., 2015; Leimbach, 2017). These alternatives nowadays mainly involve the preventive or curative use of new products among which bacteriophages are some of the most promising because of their high advantages relative to antibiotics (Angelopoulou et al., 2019). In addition, recent studies have suggested the efficacy of this approach when using phages and phage endolysins against bovine mastitis both in in vitro assays and in mouse models (Zhou et al., 2017; Iwano et al., 2018; Angelopoulou et al., 2019). Building on these observations, the main objective of this thesis was to carry out an evaluation of the therapeutic use of bacteriophages as an alternative to antibiotics against bovine mastitis mediated by S. aureus. To achieve this objective, several studies have been conducted consisting of building up a collection of staphylococci from various origins (study 1), identifying and characterizing the MRS particularly responsible for bovine mastitis (study 2), and finally evaluating the therapeutic efficacy of some bacteriophages on S. aureus strains from our collection responsible for bovine mastitis (study 3). The study 1 revealed that 83% of the staphylococci strains in our collection were isolated from milk sample of cows with mastitis. Most of these samples were collected in Belgium and showed a certain presence of MRS reflecting the overuse of antibiotics in animal production. The majority of the strains responsible for bovine mastitis in this study were identified as S. aureus, although coagulase negative staphylococci (CNS) also constitute a large bacterial population, in particular among strains resistant to methicillin in Belgium. This study also reveals that the comparison of two bovine mastitis diagnostic tests results, classical bacteriology (MALDI-TOF) and qPCR (Pathoproof®), showed a concordance rate of around 67%. The two methods, each with advantages and disadvantages, were considered to be complementary for a better diagnosis. In study 2, the characterization of MRS in our collection enabled the identification of the SCCmec (staphylococcal cassette chromosome) type IV as predominantly present in cattle in Belgium. The limits of typing tests (phenotypic and genetic), the identification of potential new SCCmec as well as the need to think about establishing a new classification of cassettes within the CNS were also highlighted in this study. The study 3, has made it possible to observe the in vitro activity of phages against the strains of S. aureus tested, mediated by a bacterial lysis both in agar and broth media. Efficacy was also observed in vivo, as shown by a decrease of the bacterial titers both in Galleria (G.) mellonella larvae and murine mastitis model and a partial infection of the mice mammary glands in the groups infected and treated with phages. In conclusion, this study provides some relevant finding as well as for the diagnostic methods of bovine mastitis and the MRS involved. Our work complements the studies carried out with regard to the assessment of phage therapy in bovine mastitis by in vitro and in vivo tests. It also makes it possible to show the interest of the experimental intermediate model between in vitro and in vivo using G. mellonella larvae when assessing the phage efficacy against S. aureus strains responsible for bovine mastitis. Several perspectives are possible following this study in order to improve our results and deepen our knowledge on the subject. Among others, a modification of some parameters such as the phage / bacteria ratio and their inoculation route by additional in vivo studies could make it possible to improve the results of the phages efficacy. Others factors such as immune system as well as the presence in milk of some proteins or lipids which can influence the phages effectiveness could be also studied in order to control them during the in vivo evaluation of the phages efficacy. Finally, the successful establishing a new classification of cassettes within the CNS in the future will appear as one of the achievements of this work.