An Investigation into the Impact of Culture Method on Biofilm Recalcitrance from the Perspective of Chronic Ischemic Wounds

Antimicrobial resistance is a global crisis that requires urgent attention. Although there is significant investment into drug development few of these drugs actually make it to market. There are shortcomings with some of the currently available testing methods, as these do not often take into account the in vivo environment in which the infections form. This requires a better understanding of the parameters that affect resistance and susceptibility, such as hypoxia or the recalcitrance of biofilms. Biofilms (communities of surface associated microorganisms) present a significant challenge for researchers and clinicians alike, as they are reported to be up to 1000 times more resistant to antimicrobials (Van Acker et al, 2014). These complex communities have a wealth of factors that induce increased recalcitrance to antimicrobials, and are reported to be present in up to 80% of chronic infections (Uruén et al., 2021). Yet, antimicrobial testing of biofilms is not yet widely adopted, and the use of biofilm testing in drug development is often not reported. This work aims to investigate these parameters to gain insight on how antimicrobial discovery and testing can be refined. The first aim of this project was to determine the influence of culture method and hypoxia on S aureus susceptibility in several in vitro antimicrobial susceptibility testing methods. This compared broth microdilution methods (BMM), the Calgary Biofilm device (CBD), and the biofilm microtiter assay (MTA). Hypoxia was revealed to impact S aureus susceptibility in the BMM and CBD methods, and there was a large difference in the density of biofilms formed in the CBD and MTA. The second aim of this project was to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus, which is simple, cost-effective, high throughput, and reproducible. The establishment of wound infection was confirmed by an increase in viable bacterial counts compared to the inoculum. This enabled the final aim to assess if the ex vivo model formed biofilms and if hypoxia would have an impact on the susceptibility of the biofilms within the ex vivo model. The presence of biofilms was confirmed through scanning electron microscopy, histology, and antimicrobial challenge of the biofilms. Hypoxia elicited an impact on the susceptibility of the ex vivo associated biofilms in an antimicrobial specific manner. This work provides insight into the complex environment that is an ischemic infected wound, and has given insight into the parameters that can affect antimicrobial drug development in the preclinical stages.