BIONÆR-Bionæringsprogram

The project's overall goal was to to study the potential of collection and wild-type bacterial phages for the therapy or the prophylaxis of staphylococcal udder infections in cows (bovine mastitis). There is a general increase in antibiotic resistance, and in general, high antibiotic consumption is associated with more resistance in infectious agents. Bovine mastitis is a frequent infection in agriculture, and such infections are usually treated with antibiotics. The most common cause of bovine mastitis is different staphylococci, with Staphylococcus aureus being the most important. An alternative to antibiotic treatments in staphylococcal mastitis, can thus reduce the consumption of antibiotics and thereby reduce the development or spread of resistance. In order to do this, we must know the characteristics of the staphylococci involved, isolate appropriate bacteriophages and investigate whether they have the desired effect on the staphylococci. In the project we have collected 272 staphylococci isolates, in which 100 are from Norway and 172 from Belgium. In all isolates we mapped the phenotypic resistance, and detected the presence of selected virulence genes. All isolates were identified to the species level. Isolation of new bacteriophages proved to be significantly more challenging than expected. No bacteriophages against S. aureus were detected, but two phages against Staphylococcus saprophyticus were isolated. In lack of bacterial phages against S. aureus, the most important staphylococcal pathogen in bovine mastitis, we used four collection phages (received from other institutions) in the trials. After initial testing in , selected S. aureus isolates that showed sensitivity to the bacterial phages were challenged in two live models. Galleria mellonella larvae inoculated with S. aureus were treated with bacterial phages. The result showed that although the treatment with bacterial phages did not influence the mortality rate, a statistically significant decrease of bacteria was observed in the larvae treated with the phages compared to the non-treated controls. One of the phages was further tested against one S. aureus isolate in a mouse mastitis model. Also in this trial, a statistically significant decrease of bacteria in the mammary glands was observed, and a weaker inflammation were observed. The results show that bacterial phages can be active against susceptible bacterial isolates also in tissue. The two isolated phages against S. saprophyticus were isolated from wastewater in Paris, and the genomes of these were sequenced. Also the genomes of 100 of the staphylococcal isolates were sequenced. These isolates were chosen on the basis of their resistance pattern and virulence profile. The sequences were used to study clonality, among other the genetic basis for the methicillin resistance. Methicillin resistance is surveilled in human medicine worldwide because it represents multiresistance and hence an undesired development. The sequencing revealed that resistance genes to methicillin were detected also in isolates that tested negative in other methods. Antimicrobial multiresistance is dreaded because it hampers antibiotic therapy. Multiresistance (defined as resistance towards at least 3 antibiotic families) was detected in 15% of S. aureus isolates. In addition, 94% of the S. aureus isolated had at least three virulence genes. This was not unexpected, as the isolates were isolated from mastitis, and hence had already proven to be able to cause infections (e.g. be virulent), and also probably had been exposed to antibiotic treatments. During the project, all partners have collaborated and exchanged scientific knowledge and technical know-how. Facilities and deliverables were shared and several meetings, coordinator visits and laboratory exchanges were organised as foreseen in the project.

Prosjektet har bidratt til etablering og videreføring av internasjonalt samarbeid. En av partnerne (University of Liege) kjente vi fra tidligere samarbeid, men gjennom prosjektet er samarbeidet utvidet til å omfatte nye forskermiljø ved ytterligere to institusjoner. Prosjektet har gitt erfaring med nye metoder, som vi nå har tilgang til. Samarbeidet har vært raust, inkluderende og fruktbart, og alle partnere er interessert i å videreføre samarbeidet i nye prosjekter. Prosjektet har gitt samfunnseffekt i form av å opprettholde fokus på antibiotikaresistens i veterinærmedisin, og i tillegg har det bidratt til kompetansebygging innen temaet både hos oss og hos partnerne.

Antimicrobial treatment of mastitis has led to the selection of resistant staphylococci, among which the Methicillin Resistant S. aureus (MRSA) are the most frequently studied ones. Still, MR has also been described for non-S. aureus (NSA) species. Bovine veterinary MR(N)SA represent not only a problem in the treatment of mastitis, but also a potential hazard in public health via the inter-Staphylococcus transferability of the mobile genetic «Staphylococcal Cassette Chromosome» (SCC) that carry the mec gene(s) encoding MR and the zoonotic potential of some Staphylococcus species. The overall aim of this project is to study the potential of bacteriophages isolated from farm slurry and milk in different countries for the therapy or the prophylaxis of staphylococcal mastitis in cows, focusing more specifically on their potential activity against MR(N)SA. The potency of these phages as alternative to antimicrobial administration will be unravelled by: a) testing their lytic activity on bovine MR(N)SA isolated from cows with mastitis during in vitro growth (=lysotyping); (b) assessing their therapeutic or prophylactic activity compared to antimicrobials by conducting challenge experiments in mice. The project is divided into 4 workpackages. The main achievements will be: (i) assessment of the potential of phages as alternative therapy or prophylaxis of MR(N)S mammary gland infections and (ii) identification of the genetic criteria of identification of the most effective phages.