Research

Plasmid-bacteria coevolution

The plasmid-mediated spread of multi-drug resistance (MDR) to human pathogens is threatening our fight against infectious diseases. Hence, we need novel therapies aimed at limiting the spread of new resistance genes. However, while we and others have shown that the long-term persistence of a plasmid can improve in a bacterial host either by mutations in the plasmid, the host or both (De Gelder et al., 2008; Sota et al., 2010; Hughes et al., 2012; Stalder et al., 2017), only in very few cases have the molecular mechanisms been unraveled (Loftie-Eaton et al., 2016; Yano et al., 2016). We have also shown that evolutionary pattenrs in bacterial biofilms differ from those in well-mixed liquid cultures (Ridenhour et al., 2017)

For the molecular mechanisms to become useful targets for alternative therapies to slow down the spread of resistance, they should be common among multiple pathogen. We and others have identified critical mutations in chromosomally encoded accessory helicases (Loftie-Eaton et al., 2017). Plasmid-helicase interactions in bacteria may thus be key to the ability of bacterial pathogens to retain newly acquired MDR plasmids. We also showed for the first time that these mutations pre-adapt the bacteria to other MDR plasmids that they acquire later in time, leading to enhanced retention of mobile resistance genes (designated ‘increased plasmid permissiveness’; Loftie-Eaton et al., 2017). Bacteria with increased permissiveness can thus serve as stable repositories for multiple MDR plasmids, eventually generating strains with an expanded arsenal of resistance genes. Using molecular techniques, experimental evolution and mathematical modeling, we are now testing the following hypotheses: (i) chromosomal mutations can pre-adapt bacteria to other plasmids, leading to greater plasmid permissiveness; (ii) plasmid permissiveness can expand the spectrum of antibiotic resistance traits within a bacterial species; and (iii) accessory helicases are linked to the persistence of newly acquired MDR plasmids across a wide spectrum of bacterial pathogens.

Extant diversity of antibiotic resistance plasmids and their natural reservoirs

Over the past 15 years we have engaged in studies to better understand the vast genetic diversity of natural plasmids that are able to spread antibiotic resistance genes among a wide range of bacteria. This work significantly increased the number of annotated whole plasmid genome sequences that are available in Genbank. We have shown that the core plasmid genomes are surprisingly conserved on a global scale (Schlüter et al., 2007; Sen et al. 2011; Brown et al., 2013). These are accompanied by very different types and numbers of so-called “accessory genes” that can provide the host with multi-drug resistance and other beneficial traits. We developed and optimized a method to predict the hosts and host-range of plasmids based on their genomic signature (Suzuki et al., 2010) . We also built the first robust phylogeny of plasmids (Sen et al., 2013). These are resources that are critical to advance our understanding of the evolutionary history of antibiotic resistance plasmids.

Recently scientists have realized the significance of environmental microbiomes in the emergence and spread of antibiotic resistance, as they try to track down the trajectories of resistance genes from environment to clinic. However, these efforts suffer from one major obstacles: the inability to determine which host(s) carry and spread specific antibiotic resistance genes (ARGs). Because these genes often travel on mobile genetic elements such as plasmids, they can move rapidly between different species. When determining the metagenome sequence of a microbial community with current methods, all associations between specific bacteria and their resistance plasmids are lost. This makes it nearly impossible to determine who hosted which mobile element or ARG. In a joint effort between Phase Genomics and my lab we have shown that we can overcome this limitation. Using a unique cultivation-independent approach based on proximity-ligation (Hi-C), we were able to determine the bacterial hosts of specific ARG and the plasmids and integrons that encode them in a wastewater microbiome (Stalder et al., 2019). Up to now, no peer-reviewed publications have used such an approach to tackle the issue of antibiotic resistance gene or plasmid spread in environmental microbiomes.

The ecology of horizontal gene transfer

We are interested in the effect of spatial structure on plasmid transfer (Fox et al. 2008), and in the frequency and range of plasmid transfer in natural environments (Top et al., 1990).  Most natural bacterial populations are not well mixed but grow in spatially distinct microcolonies or structured biofilms attached to surfaces. We directly addressed the consequences of this common mode of growth on the spread of antibiotic resistance genes using a spatially explicit mathematical model (Krone et al., 2007). Among other findings, we showed that a plasmid can spread from just a few bacterial cells to a dense population of bacteria as long as spatial structure is maintained and there are sufficient nutrients. In contrast to the general belief that biofilms would promote plasmid transfer due to the close proximity of cells, we showed that plasmid transfer was much more frequent at the liquid-air interface than in the biofilm itself (Król et al., 2011; Król et al., 2013; Stalder & Top, 2016) This research is important because we know so little about the critical factors that promote or limit spread of antibiotic resistance genes. A few decades ago assessing plasmid transfer in environments such as soil was done using cultivation-based methods (Top et al., 1990). Today we are trying to get a better understanding of the dissemination of plasmids between bacteria in agricultural soil after manure application using the cultivation-independent method Hi-C.