Francisco Dionisio

Positive Epistasis Drives the Acquisition of Multidrug Resistance

The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact—epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics. Surprisingly we find that a significant fraction of resistant mutations can be beneficial in certain resistant genetic backgrounds, that some double resistances entail no measurable cost, and that some allelic combinations are hotspots for rapid compensation. These results provide additional insight as to why multi-resistant bacteria are so prevalent and reveal an extra layer of complexity on epistatic patterns previously unrecognized, since it is hidden in genome-wide studies of genetic interactions using gene knockouts.

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations.

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)—including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.

Immune Subversion and Quorum-Sensing Shape the Variation in Infectious Dose among Bacterial Pathogens

Many studies have been devoted to understand the mechanisms used by pathogenic bacteria to exploit human hosts. These mechanisms are very diverse in the detail, but share commonalities whose quantification should enlighten the evolution of virulence from both a molecular and an ecological perspective. We mined the literature for experimental data on infectious dose of bacterial pathogens in humans (ID50) and also for traits with which ID50 might be associated. These compilations were checked and complemented with genome analyses. We observed that ID50 varies in a continuous way by over 10 orders of magnitude. Low ID50 values are very strongly associated with the capacity of the bacteria to kill professional phagocytes or to survive in the intracellular milieu of these cells. Inversely, high ID50 values are associated with motile and fast-growing bacteria that use quorum-sensing based regulation of virulence factors expression. Infectious dose is not associated with genome size and shows insignificant phylogenetic inertia, in line with frequent virulence shifts associated with the horizontal gene transfer of a small number of virulence factors. Contrary to previous proposals, infectious dose shows little dependence on contact-dependent secretion systems and on the natural route of exposure. When all variables are combined, immune subversion and quorum-sensing are sufficient to explain two thirds of the variance in infectious dose. Our results show the key role of immune subversion in effective human infection by small bacterial populations. They also suggest that cooperative processes might be important for successful infection by bacteria with high ID50. Our results suggest that trade-offs between selection for population growth-related traits and selection for the ability to subvert the immune system shape bacterial infectiousness. Understanding these trade-offs provides guidelines to study the evolution of virulence and in particular the micro-evolutionary paths of emerging pathogens.

Temperate Bacterial Viruses as Double-Edged Swords in Bacterial Warfare

It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a “replicating toxin”. However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.

Small-world networks decrease the speed of Muller's ratchet

Mullers ratchet is an evolutionary process that has been implicated in the extinction of asexual species, the evolution of non-recombining genomes, such as the mitochondria, the degeneration of the Y chromosome, and the evolution of sex and recombination. Here we study the speed of Mullers ratchet in a spatially structured population which is subdivided into many small populations (demes) connected by migration, and distributed on a graph. We studied different types of networks: regular networks (similar to the stepping-stone model), small-world networks and completely random graphs. We show that at the onset of the small-world network - which is characterized by high local connectivity among the demes but low average path length - the speed of the ratchet starts to decrease dramatically. This result is independent of the number of demes considered, but is more pronounced the larger the network and the stronger the deleterious effect of mutations. Furthermore, although the ratchet slows down with increasing migration between demes, the observed decrease in speed is smaller in the stepping-stone model than in small-world networks. As migration rate increases, the structured populations approach, but never reach, the result in the corresponding panmictic population with the same number of individuals. Since small-world networks have been shown to describe well the real contact networks among people, we discuss our results in the light of the evolution of microbes and disease epidemics.

Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids

Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmids conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such interactions may determine how antibiotic resistance disseminates in bacterial populations.

Multiple plasmid interference – Pledging allegiance to my enemy's enemy

As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmids identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors.

Social behaviour involving drug resistance: the role of initial density, initial frequency and population structure in shaping the effect of antibiotic resistance as a public good

Bacteria sometimes cooperate with co-inhabiting cells. Pathogenic bacteria, for example, often produce and excrete virulence factors, eventually benefitting both producer and non-producer cells. The role of social interactions involving antibiotic resistance, however, has been more elusive. Enzymes that inactivate β-lactam antibiotics such as ampicillin or penicillin (β-lactamases) are good candidates as public goods. Nonetheless, it has been claimed that bacteria harbouring plasmids of natural origin coding for β-lactamase almost do not protect sensitive bacteria. This does not fit with the fact that ampicillin-sensitive bacteria can be isolated from subjects undergoing ampicillin treatment. We hypothesised that there are two non-exclusive explanations for the discrepancy between previous works: (1) the range of values of demographic conditions (such as initial strain frequency, initial total cell density or habitat structure) has not been broad enough to include most scenarios, or (2) there are interactions between some of these factors. We performed experiments with Escherichia coli bacterial cells to measure the degree of protection of sensitive cells when co-cultured with cells harbouring RP4, R16a or the R1 plasmids, all of natural origin and coding for β-lactamases, and in presence of ampicillin. In these co-cultures, performed in structured and non-structured environments, both the initial total cell density and the initial frequency of sensitive cells spanned four orders of magnitude. We found protection of sensitive cells in 63% of tested conditions. All factors (plasmid, structure, frequency and density) significantly affect levels of protection. Moreover, all factors interact, with interactions revealing large or very large effect sizes.

Lower within-community variance of negative density dependence increases forest diversity.

Local abundance of adult trees impedes growth of conspecific seedlings through host-specific enemies, a mechanism first proposed by Janzen and Connell to explain plant diversity in forests. While several studies suggest the importance of this mechanism, there is still little information of how the variance of negative density dependence (NDD) affects diversity of forest communities. With computer simulations, we analyzed the impact of strength and variance of NDD within tree communities on species diversity. We show that stronger NDD leads to higher species diversity. Furthermore, lower range of strengths of NDD within a community increases species richness and decreases variance of species abundances. Our results show that, beyond the average strength of NDD, the variance of NDD is also crucially important to explain species diversity. This can explain the dissimilarity of biodiversity between tropical and temperate forest: highly diverse forests could have lower NDD variance. This report suggests that natural enemies and the variety of the magnitude of their effects can contribute to the maintenance of biodiversity.