Antimicrobial-induced plasmid-transfer in Escherichia coli

PhD student: Gang Liu, gangliu@sund.ku.dk
Department of Veterinary and Animal Sciences,
Section for Veterinary Clinical Microbiology

Background

Antimicrobial resistance (AMR) is a serious threat to human and animal health, and it is projected that by the year 2050 more people will die due to infections with AMR bacteria than cancer, if we do not find ways to prevent development and spread of AMR. The World Health Organization has published a priority list of bacteria to which it is important to find solutions to the AMR problem, and E. coli that are resistant to broad spectrum antimicrobials of the cephalosporin-classes are considered one of the biggest problems. A particular problem with these bacteria is that they carry their antibiotic resistance genes on mobile genetic elements, so called plasmids, which allows them to spread the resistance to other E. coli by a mechanism known as conjugation.

Purpose

In this thesis work, I have studied how treatment with a cephalosporin drug, cefotaxime, affects the way E. coli spread its resistance by conjugation to other bacteria. First I determined whether treatment with antibiotic activates the plasmids to spread to other bacteria more efficiently, and when I saw that this was indeed the case, I elucidated the underlying mechanisms of cefotaxime-induced plasmid transfer in E. coli.

Results

A new experimental setup was designed in this study to study the efficacy of plasmid borne spread of antibiotic resistance by conjugation. Previous methods have used antibiotics during the transfer experiment, which has made it impossible to decide whether an increase in number of resistant bacteria is due to a more efficient spread of plasmids, or the fact that bacteria with the plasmid are the only ones growing in the presence of the antibiotic. I observed that treatment with high concentrations of cefotaxime up-regulated the genes, as well as the proteins, that controls and guides the spread of the resistance plasmids to other bacteria, and I measured that this indeed resulted in a higher proportion of surrounding bacteria obtaining the resistance plasmid. I performed this first part using a model with one naturally occurring resistance plasmid, called pTF2. Investigation of 25 additional plasmids encoding cephalosporin resistance showed that eight other plasmids among these showed the same increase in plasmid spread due to treatment, and further that the increase could also be induced by a different antibiotic, ampicillin, which does not belong the cephalosporin class, but which has a similar mode of action on the bacteria. This suggests that the phenomena is wide spread among antibiotic resistance plasmids in E. coli.

The up-regulation of the transfer system and the increased spread of resistance depend on the presence of the resistance gene, but how it happens is still not clear. I determined that is not because the bacteria is very stressed and desperately tries to spread it genes before it dies, and I identified 6 genes in the chromosome of the E. coli that are essential for the increased spread, but which were not essential for spread in circumstances without treatment. One gene (rfaH), which is normally involved in enhancing expression of other genes, seemed to be central. Other genes were involved in synthesis of the outer membrane of the bacterium. The exact mechanism by which these genes are involved still remains to be determined.

Perspectives

The results of this PhD study suggest that treatment against infections may enhance spread of cephalosporin-resistance plasmids between bacteria. So far this has only been shown in the laboratory, where bacteria have optimal conditions for mating, and studies in real situations, for example in animal intestines, are needed to assess the importance of the observation. Even though the exact mechanism remains unknown, the study provided some insight into the possible mechanisms that controls the increased spread of AMR. This includes identification of six chromosomal genes that are essential for the increased spread, and with further knowledge, it may be possible to target these genes to prevent increased spread of the resistance plasmids.