Qinqin Wang:
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The existence of cooperative antibiotic resistance genes makes bacteria highly adaptable and greatly impacts bacterial evolution. Cooperative antibiotic evolution is a black box and full of unknowns, it is now time to shed light on the mechanisms behind it. The production of βlactamases by bacteria is the most common mechanism of bacterial resistance to β-lactam antibiotics. As the best representative of cooperative antibiotic resistance, the β-lactamases can be excreted into the extracellular space to degrade antibiotics in the environment, thus providing opportunities for the survival of the nearby sensitive β-lactamase non-producers. Non-producers thus enjoy the benefits of an expensive public good without cost.
There are complex cooperation and competition interactions between the producers and the survived non-producers, which leads to the tragedy of the Commons, that is, selfish individuals violate the common interests of the whole community through excessive consumption of resources. Therefore, the way producers coordinate their dealings with competitors becomes very important. Plasmids have been reported to be the preferred location for many cooperative genes encoding pathogenic secretory proteins. The interactions between cooperative genes and horizontal gene transfer (HGT) are emerging as a hotspot for studying the spread of antibiotic resistance and have only been receiving attention in the past few years, and here they are the overall research aim of this Ph.D. thesis.
The present work evaluates if cooperative antibiotic resistance inflects the selection processes leading to population-wide antibiotic resistance and explores whether the cooperative genes encoded on different replicons resulted in different collaborative behaviors. To do that, we expressed β-lactamases at three different levels: chromosome, conjugative plasmid, and non-conjugative plasmid. Chromosome and plasmids correspond to low-copy and high-copy replicons, because the encoding gene position alters the copy number of the β-lactamase gene.
Our analysis revealed that the plasmid-encoded β-lactamase gene generally had higher β-lactamases activity than chromosome-encoded producers. It confirms that high-copy replicons have higher expression levels. Furthermore, we showed that HGT can enforce collaboration: when the β-lactamase excreted in the environment was scarce and only sustained weak cooperation between producers and susceptible nonproducers (as in the case of the relative ratio of producers is very small), the advantage of enforced cooperation was significant. Specifically, HGT converted non-producers into producers, and the transmission of plasmids carrying resistant genes was critical for stabilizing resistance. We also noted that the cooperative antibiotic resistance can increase the occurrence of HGT by giving more rescued potential recipients. The work shown here can be divided into five parts.
In the first part, we developed a convenient method to facilitate inserting large length foreign genes into natural plasmids, which effectively helped us track the movement of plasmids. We also made plasmid mutants with low, medium, and high transfer efficiency for future research.
In the second part, we reviewed the current researches of public goods related to bacterial resistance, and focused specifically on their involvement in the spread of antibiotic resistance by promoting cooperation among community members.
In the third part, we used a solid agar plate model to systematically study the effect of βlactamase and HGT on bacterial cooperation and showed that cooperative resistance can spread and maintain antibiotic resistance by promoting HGT.
In the fourth part, to deepen our understanding of how β-lactam antibiotic resistance promotes bacterial cooperation and the contribution of HGT to the spread of antibiotic resistance, we shifted our experimental settings from in-vitro agar plate to the in-vivo complex rat intestinal environment. Antibiotic use disrupted the structure of the gut microbiota. However, β-lactamase producers could help to rapidly rebalance the intestinal flora after antibiotic treatment.
In the fifth part, we confirmed the wide existence of β-lactamase genes and the preferred locations (chromosome, non-mobile plasmid, conjugative plasmids) for them by analyzing the E. coli genomes in the GenBank database. Then, the different characteristics of β-lactamases, such as β-lactamase activity, minimum inhibitory concentration, and cooperation ability of seven selected β-lactamases were experimentally compared. We highlighted that HGT and high expression levels due to strong promoters and high gene copy numbers are major factors contributing to efficient cooperation between β-lactamase producers and non-producers. Additionally, we proposed that both intracellular and extracellular high β-lactamase activity is critical for cooperation antibiotic resistance.