Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance.

Conjugative plasmids are mobile elements that spread horizontally between bacterial hosts and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favor plasmids with higher transfer rates, whereas selection for plasmid carriage favors less-mobile plasmids. However, little is known about the mechanisms leading to variation in transmission rates in natural plasmids or the resultant effects on their bacterial host. We investigated the evolution of AMR plasmids confronted with different immigration rates of susceptible hosts. Plasmid RP4 did not evolve in response to the manipulations, but plasmid R1 rapidly evolved up to 1,000-fold increased transfer rates in the presence of susceptible hosts. Most evolved plasmids also conferred on their hosts the ability to grow at high concentrations of antibiotics. This was because plasmids evolved greater copy numbers as a function of mutations in the copA gene controlling plasmid replication, causing both higher transfer rates and AMR. Reciprocally, plasmids with increased conjugation rates also evolved when selecting for high levels of AMR, despite the absence of susceptible hosts. Such correlated selection between plasmid transfer and AMR could increase the spread of AMR within populations and communities.

Strong Environment-Genotype Interactions Determine the Fitness Costs of Antibiotic Resistance In Vitro and in an Insect Model of Infection.

The acquisition of antibiotic resistance commonly imposes fitness costs, a reduction in the fitness of bacteria in the absence of drugs. These costs have been quantified primarily using in vitro experiments and a small number of in vivo studies in mice, and it is commonly assumed that these diverse methods are consistent. Here, we used an insect model of infection to compare the fitness costs of antibiotic resistance in vivo to those in vitro Experiments explored diverse mechanisms of resistance in a Gram-positive pathogen, Bacillus thuringiensis, and a Gram-negative intestinal symbiont, Enterobacter cloacae Rifampin resistance in B. thuringiensis showed fitness costs that were typically elevated in vivo, although these were modulated by genotype-environment interactions. In contrast, resistance to cefotaxime via derepression of AmpC ß-lactamase in E. cloacae resulted in no detectable costs in vivo or in vitro, while spontaneous resistance to nalidixic acid, and carriage of the IncP plasmid RP4, imposed costs that increased in vivo Overall, fitness costs in vitro were a poor predictor of fitness costs in vivo because of strong genotype-environment interactions throughout this study. Insect infections provide a cheap and accessible means of assessing the fitness consequences of resistance mutations, data that are important for understanding the evolution and spread of resistance. This study emphasizes that the fitness costs imposed by particular mutations or different modes of resistance are extremely variable and that only a subset of these mutations is likely to be prevalent outside the laboratory.

Shifts along the parasite-mutualist continuum are opposed by fundamental trade-offs.

Theory suggests that symbionts can readily evolve more parasitic or mutualistic strategies with respect to hosts. However, many symbionts have stable interactions with hosts that improve nutrient assimilation or confer protection from pathogens. We explored the potential for evolution of increased parasitism or decreased parasitism and mutualism in a natural gut symbiosis between larvae of Plutella xylostella and the microbe Enterobacter cloacae. We focused on interactions with the pathogen, Bacillus thuringiensis: selecting for parasitism in terms of facilitating pathogen infection, or increased mutualism in terms of host protection. Selection for parasitism led to symbionts increasing pathogen-induced mortality but reduced their competitive ability with pathogens and their in vitro growth rates. Symbionts did not evolve to confer protection from pathogens. However, several lineages evolved reduced parasitism, primarily in terms of moderating impacts on host growth, potentially because prudence pays dividends through increased host size. Overall, the evolution of increased parasitism was achievable but was opposed by trade-offs likely to reduce fitness. The evolution of protection may not have occurred because suppressing growth of B. thuringiensis in the gut might provide only weak protection or because evolution towards protective interactions was opposed by the loss of competitive fitness in symbionts.

Biofilms facilitate cheating and social exploitation of ß-lactam resistance in Escherichia coli.

Gram-negative bacteria such as Escherichia coli commonly resist ß-lactam antibiotics using plasmid-encoded ß-lactamase enzymes. Bacterial strains that express ß-lactamases have been found to detoxify liquid cultures and thus to protect genetically susceptible strains, constituting a clear laboratory example of social protection. These results are not necessarily general; on solid media, for instance, the rapid bactericidal action of ß-lactams largely prevents social protection. Here, we tested the hypothesis that the greater tolerance of biofilm bacteria for ß-lactams would facilitate social interactions. We used a recently isolated E. coli strain, capable of strong biofilm formation, to compare how cooperation and exploitation in colony biofilms and broth culture drives the dynamics of a non-conjugative plasmid encoding a clinically important ß-lactamase. Susceptible cells in biofilms were tolerant of ampicillin-high doses and several days of exposure were required to kill them. In support of our hypothesis, we found robust social protection of susceptible E. coli in biofilms, despite fine-scale physical separation of resistant and susceptible cells and lower rates of production of extracellular ß-lactamase. In contrast, social interactions in broth were restricted to a relatively narrow range of ampicillin doses. Our results show that ß-lactam selection pressure on Gram-negative biofilms leads to cooperative resistance characterized by a low equilibrium frequency of resistance plasmids, sufficient to protect all cells.