Working with antibiotic resistance and microplastics at NORCE at my internship

Written by Eirill Spadoni, bachelor student at department of arctic and marine biology.

Microplastic pollution has become a serious issue in the last decades, impacting the health of entire ecosystems as well as our own. The roughened surface of down-graded plastic particles released into the waters makes them potential growth sites for bacteria to reproduce and easily transfer genetic material to each other, facilitating the emergence and spread of antibiotic resistant strains.

Antibiotics are limited and old ones are gradually meeting more and more resistant strains due to the careless use and disposal of antibiotics. As a result, as we go forward, we may find ourselves unable to cure even the most common bacterial infections.

The project I have been assigned consists of studying the effect of different types of microplastic on the antibiotic resistance of a common bacterium.

Me at the NORCE lab in Tromsø. Photo: private.

My task is to transform and grow bacteria with particles of nylon, a plastic polymer unfortunately very commonly found in our environment, and observe how its presence affects the antibiotic resistance of the bacterium Acinetobacter baylyi. These past weeks I was taught techniques to carry out the experiments. To get some practice before starting the actual experiments, we ran some control experiments without using microplastics. We started from basic things, like the preparation of LB (Lysogeny Broth) medium, a nutrient solution in which bacteria can multiply, and agar plates, gelatinous disks essential for the in-lab growth of bacterial colonies. Although these are very simple (and essential) procedures, they were new to me: during laboratory sessions at the university, all material had been previously prepared for us. As some of the plates contained antibiotics to allow only the bacteria with the resistance gene to grow, we were taught how to properly handle and dispose of them, along with other biohazardous waste, and about the importance of risk assessments.

We learned how to maintain a sterile environment by using laminar hoods, gas burner and the autoclave. Photo: private.

At the same time, we learned of more complex procedures, such as DNA extraction, where bacteria are treated with different chemicals to dissolve their membrane and remove everything other than the DNA containing the antibiotic resistance gene to be transferred to the other strain of bacteria.

The next step of the experiment was to grow overnight cultures of bacteria. I learned that bacteria can survive temperatures of -80C degrees (!), as they are stored for further use. In this case, we took frozen bacteria left from a previous run of the same experiments and suspended them in LB medium to allow them to grow. These were successively mixed with the extracted DNA to induce them to take up the antibiotic resistance genes in a process called transformation. I was familiar with the technique, as I had done it before in a molecular biology lab, but it was interesting to use the protocol provided along the material kit.

Once the bacteria had taken the resistance genes, we made different diluted solutions. This was also something I was familiar with from some microbiology labs I had had in year 2 of the bachelor program. But repeating it and doing it independently made me gain more confidence in the procedure.

After some days we counted the colonies. The colony counting was done using a different technique that the one I was used to. Where they were too many, only a portion was counted. The colonies were big enough to be counted without using a microscope and I used instead a clicker I had never seen before.

Lately I’ve also had the opportunity to observe and help with another related project that aims to observe the difference in antibiotic resistance related to the presence of microplastics in compost and manure from Italy and Norway. The two countries have different regulations related to the use of antibiotics in farming and plastic disposal, as well as (probably) different bacteria strains and different climate. This allows us to study whether the temperature also plays a role in the antibiotic resistance spread. The samples for the experiments in Tromsø are taken from Holt and a local farm. Both compost and manure are combined with microplastics and antibiotics to be analyzed and the presence of different bacteria measured and compared to the data coming from Italy.

My tasks in this project were pretty basic: transferring samples to testing tubes and rinsing them to make them ready to be analyzed, but I enjoyed learning about this different project. I found particularly interesting how institutions from different parts of the world can cooperate on one project for a common important goal!

This internship at NORCE is part of the UiT course BIO-2014 Praksis i næringslivet for biologistudenter.