Developing microfluidics and microscopy approaches to investigate bacterial populations with single-cell resolution

Failures in antibiotic treatments for bacterial infections are typically attributed to antibiotic resistance. However, it has long been realised that bacteria can also employ other mechanisms to aid survival in the presence of antibiotics. Indeed, some bacterial cells react to the presence of antimicrobial drugs by blocking or retarding growth. These cells are named persister cells and can survive bactericidal antibiotics that require active growth for killing. This property is known as “persistence”. Increasing evidence suggests a link between specific environmental conditions and the development of persistence. In this study, the temporal windows during the growth cycle when the fraction of persisters to β-lactams, quinolones or aminoglycoside increases have been investigated. Our results confirmed that the environment plays a significant role in persister cell formation. Indeed, cells treated with antibiotics but without fresh media, regrow at a higher density than the cells in the nutrient-rich environment. Nowadays classic microbiological approaches are not sufficient for the study of persister cells, since the persister frequency is often low and dynamic changes in phenotype cause cells to switch between the persister and normal phenotype. Over the past decade, microfluidic techniques have gained increasing interest in the research community. Integrated micro- and nanofluidic lab-on-a-chip systems able to handle samples in a picoliter range are now available, and are certain to become an everyday item in biotechnology and biomedicine within a few years. Here we present a novel microfluidic-microscopy assay for the study of persister cell formation under controlled delivery of antibiotics. Our device is fabricated from polydimethylsiloxane, PDMS, and is able to trap single cells. The microfluidic setup has been integrated with a microscope in order to obtain high-resolution imaging and quantitatively measure growth and shape changes of large numbers of individual cells. Our device allowed us to challenge a culture of E. coli with three different antibiotics, and record the individual response of thousands of bacteria. When fresh media was given we distinguished not only dividing cells, as per classic microbiological techniques, but also two further phenotypes: elongating and non-growing. Furthermore, a preliminary study has been conducted regarding the possibility of combining DNA-PAINT with microfluidics for the future investigation of persister cells, with sub-cellular resolution. Together, the results from this thesis enable future investigations, with the goal of advancing our understanding of persistence in response to antibiotics