Bacteriophages for biofilm biocontrol and detection of VBNC bacteria in invasive biomedical device-associated infections

There are currently more than 500000 types of invasive biomedical devices (IBD) in use worldwide, that have importantly improved the quality of medical treatments and the overall life quality of patients. Device-associated infections represent a serious complication and account for 25.6% of all health care–associated infections, while surgical-site infections account for an additional 21.8%. IBD infections are frequently associated with biofilm formation on device biomaterial, which cause persistent infections that are difficult to treat and to diagnose. Bacteria within biofilms are harder to eradicate due to incomplete or slow penetration of antimicrobials through biofilm layers, reduced antibiotic action due to the micro-environment in biofilm, and the presence of resistant phenotypes - persisters and viable but non-culturable (VBNC) bacteria. The inaccurate use of antibiotics due to misdiagnosis or administration of inadequate doses additionally contribute to the appearance of persisters, VBNC bacteria and emerging multidrug resistant strains as bacteria transition into those states as a mechanism of survival. Currently, conventional sample culturing still remains the gold standard for microbiological diagnosis of IBD. However, VBNC bacteria lack the ability to grow on routine culture medium and contribute importantly to culture-negative results. VBNC bacteria retain viability and virulence, thus remaining ready to resuscitate from this state when the environment permits and cause recurring infections. IBD infections associated with biofilm and VBNC bacteria are difficult to treat and to diagnose, which represent important clinical issues. Bacteriophages are bacterial viruses that specifically recognize and infect bacteria as part of their replication cycle. From the medical point of view, lytic phages are interesting as they are virulent and quickly lyse the infecting bacterium. The knowledge about phages is increasing the belief that these viruses are a strong weapon against bacteria, especially nowadays, when antibiotic resistance among bacteria rapidly increase. For that, more and more studies are focused on possibilities of phages use, not only for treatment, but also in diagnosis of bacterial infections. In the proposed project, we would like to demonstrate that phages are useful agents in biofilm and VBNC bacteria eradication from device-associated infections and to develop an alternative to conventional microbiological diagnostic procedure, that will allow specific detection of all viable bacteria in SF, with the use of phages and qPCR. To prove this, we are going to target for phage's use against some of the most relevant bacterial pathogens involved in implant related infections: S. aureus, S. epidermidis, P. aeruginosa, E. faecalis, E. faecium, E. coli and C. acnes, using bacteria isolated from samples of patients undergoing revision surgery at Valdoltra Orthopaedic Hospital due to PJI. Bacterial biofilms will be grown on solid surface (on membrane) as a static biofilm system and the VBNC state in bacteria will be induced. We will evaluate the influence of the VBNC physiological state of bacteria on phage growth parameters and determine if phage-infected VBNC bacteria can resuscitate from the VBNC state and if phages infecting VBNC bacteria, are able to continue their reproductive cycle after bacteria resuscitate from the VBNC state. We will research the synergistic action of phages and antibiotic on biofilm and VBNC bacteria. During the project, 100 culture-negative sonicate fluid samples from patients with a diagnosed PJI will be analyzed with the developed and optimized method with phages and qPCR. Data collected in this very ambitious project will help in designing future clinical trials needed to further develop phage therapy for the management of device associated biofilm infections and, more broadly, difficult-to-treat infections, as well as the development of new fast and effective diagnostic tools.