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Microbial Evolution and Growth Arena (MEGA-plate), accelerated evolution and co-selection studies with Escherichia coli strains for extended spectrum beta-lactamase (ESBL) production
Török Bence - year 5
University of Veterinary Medicine Budapest, Department of Pharmacology and Toxicology
Supervisor: Dr. Ádám Kerek

Abstract:

One of today's major problems is the spread of antimicrobial resistance (AMR), which could be the leading cause of death worldwide by 2050. Preventing this requires joint thinking and cooperation between animal and human health professionals. Understanding the development of resistance is aided by evolutionary and coselection studies. The first implementation in Hungary of the MEGA-plate (Microbial Evolution and Growth Arena) developed by Harvard University will allow in vitro evolution and coselection studies on Escherichia coli, one of the most important bacteria for the spread of AMR. A 60x30 cm culture dish has been constructed with nine equally divided compartments, providing 1x, 10x, 100x and 1000x concentrations of the test agent, increasing on a decimal basis. An efficient disinfection protocol was developed using 7.5% hydrogen peroxide and UV light and tested with cefotaxime. We determined the minimum inhibitory concentration (MIC) of the test agents. For cefotaxime, we used ¼ of the MIC of 2 µg/ml as a starting point to provide an evolutionary advantage, so that the bacteria grew to the 1000x concentration in 13 days. In the MIC of the new samples, we found that not only the MIC of cefotaxime (0.125, 0.25, 4, 32 µg/ml) increased, but also the 10x concentration of cefotaxime for oxytetracycline (8 µg/ml), potentiated sulphonamide (8 µg/ml), amoxicillin (32 µg/ml) and florfenicol (128 µg/ml); with neomycin (64 µg/ml), ceftriaxone (16 µg/ml), ceftiofur (8 µg/ml) and cefquinom (4 µg/ml), 100x concentrations of cefotaxime caused an increase. The 1000x concentration of cefotaxime caused a further increase, and an effect was already observed with enrofloxacin (0.125 µg/ml), colistin (32 µg/ml). No change in the antimicrobial resistance gene pool was observed by new generation sequencing, but the effect of 100x and 1000x concentrations of cefotaxime resulted in the expression of drug-degrading enzymes due to chromosomal point mutations in the ampC gene, which increased the MIC of beta-lactam drugs. In addition, the activation of several efflux pumps is hypothesized, but further transcriptomic studies are required. However, no resistance gene responsible for ESBL production was identified.

The method allows us to perform evolutionary and coselection studies that can induce AMR and to monitor the impact of drugs on phenotypic susceptibility and the bacterial genome using conventional and molecular biology methods, contributing to a better understanding of one of the most pressing problems of our time, the development of AMR.



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