Juan Carlos is a PhD researcher in the Cellular and Molecular Medicine Department at the University of Bristol. Over the last 10 years he has been focused on studying multidrug-resistant bacteria and so far has been involved in specific topics such as: resistant bacteria from food for human consumption, in skin from asymptomatic people, diabetic food, etc.
Currently, he is studying the combination of two main mechanism of resistance: intrinsic and acquired, with the ultimate aim of tackling multidrug-resistant bacteria (MDR). Here he takes a few moments to tell us a little about it…

Multidrug-resistant bacteria are an increasing worldwide public health problem, now recognised by the World Health Organisation as a life-threatening issue. The ability of bacteria to overcome the effect of antibiotics – a phenomenon known as resistance – has forced us to find new and innovative ways to fight back against MDR.

One way for bacteria to avoid the effect of antibiotics is by changing their envelope permeability. This process is mainly driven by proteins known as efflux pumps. These systems are very complex and dynamic. Once the antibiotic enters the cell, efflux pumps will detect it and the antibiotic will be extruded from within the cell which in turn will reduce the necessary concentration of antibiotic needed to kill bacteria. On the other hand, bacteria can also develop resistance by using enzymes (another type of proteins) that can degrade or destroy the antibiotic. Both mechanisms have been thoroughly studied; however, we are still trying to figure out how can we inhibit or stop the activity of such molecules.

Further understanding about the biology and the activity of these mechanisms is necessary. The classical approach to do this is by making knock outs (KOs) – deletions within DNA that will produce changes in proteins – which then can be combined with other molecular biology tools to fully understand the bacteria behaviour. In molecular biology, KOs are a genome-editing tools that have been used for decades now and the number of techniques available is countless. It is a reliable method to modify DNA; however, the cost and time that it takes to generate a single KO with classical techniques is very high. Recently, a new gene-editing tool known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was introduced. CRISPR allows us to generate multiple changes within the genome of bacteria, but what it is remarkable is that we can do it simultaneously and at very low cost and effort.

A main problem that we face in the lab is that proteins, such as efflux pumps or drug/antibiotic degrading enzymes, are very big and complex structures. In order to fully understand the biology of the bacteria, first we need to generate the KOs at different locations. Once a KO is generated, we then use other techniques such as proteomics to measure the activity of that particular protein. We can see now that one major pitfall is to construct all the possible KOs (known as a library) that can have an impact in the protein in an efficient and low-cost way.

By introducing CRISPR we can not only systematically generate multiple KOs at different regions at a much faster pace than the classical methods but also do it with low cost and with high efficiency. Generating an extensive library will allow us to understand, more quickly, which are the main regions involved in MDR, facilitating the design of antibiotics and/or inhibitors less susceptible to efflux or the action of enzymes which then can be used to treat MDR bacterial infections in combination with existing antibiotics.