AbstractPhage therapy can be used in combination with antibiotics to combat infections with bacterial pathogens1–3. However, bacteria can rapidly evolve phage resistance via receptor mutation, or using their CRISPR-Cas adaptive immune systems4, which insert short phage-derived sequences into CRISPR loci in the bacterial genome5 to guide sequence-specific cleavage of cognate sequences6. Unlike CRISPR-Cas immunity, mutation of the phage receptor leads to attenuated virulence when the opportunistic pathogen Pseudomonas aeruginosa is infected with its phage DMS3vir7, which underscores the need to predict how phage resistance evolves under clinically relevant conditions. Here, using eight antibiotics with various modes of action, we show that bacteriostatic antibiotics (which inhibit cell growth without killing) specifically promote evolution of CRISPR-Cas immunity in P. aeruginosa by slowing down phage development and providing more time for cells to acquire phage-derived sequences and mount an immune response. Our data show that some antimicrobial treatments can contribute to the evolution of phage-resistant pathogens with high virulence.

AbstractThe acquisition of antibiotic resistance genes via horizontal gene transfer is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of antibiotic resistance. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of antibiotic resistance. We analysed ~40,000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens including Enterococcus, Staphylococcus, Acinetobacter and Pseudomonas. We modelled the association between CRISPR-Cas and indicators of horizontal gene transfer, and found that pathogens with a CRISPR-Cas system were less likely to carry antibiotic resistance genes than those lacking this defence system. Analysis of the mobile genetic elements targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of antibiotic resistance. These results suggest a potential “immunocompromised” state for multidrug-resistant strains that may be exploited in tailored interventions that rely on mobile genetic elements, such as phage or phagemids, to treat infections caused by bacterial pathogens.

AbstractAntimicrobial resistance (AMR) in bacteria is commonly encoded on conjugative plasmids, mobile elements which can spread horizontally between hosts. Conjugative transfer disseminates AMR in communities but it remains unclear when and how high transfer rates evolve, and with which consequences. Here we studied experimentally the evolution of two antibiotic resistance encoding plasmids when confronted to different immigration rates of susceptible, plasmid-free hosts. While plasmid RP4 did not evolve detectably, plasmid R1 rapidly evolved up to 1000-fold increased transfer rates in the presence of susceptible hosts, at a cost to its host. Unexpectedly, most evolved plasmids also conferred to their hosts the ability to grow at high concentrations of antibiotics. The most common mutations in evolved plasmids were contained within the copA gene which controls plasmid replication and copy number. Evolved copA variants had elevated copy number, leading to both higher transfer rates and AMR. Due to these pleiotropic effects, host availability and antibiotics were each sufficient to select for highly transmissible plasmids conferring high levels of antibiotic resistance.

Conjugative plasmids are mobile elements which spread horizontally between bacterial hosts, and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favour plasmids with higher transfer rates, whereas selection for plasmid carriage favour 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 1000-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.

AbstractIn bacteria, cooperative genes encoding public good molecules are preferentially located on mobile genetic elements (MGEs), and horizontal transfer of MGEs favours the maintenance of public good cooperation. The rate of horizontal transfer itself can evolve in response to selective pressures acting on both MGEs and bacterial hosts: benefits and costs of infectious spread, but also indirect effects of MGE genes to the host. We show here that carriage of public good genes on MGEs can generate another indirect selection for MGE transfer. Transfer increases public good production and, when relatedness is sufficiently high, public goods benefit preferentially genotypes with high transfer ability. Both our simulations and experiments indicate that transfer is not required to occur among kin, provided that public goods still benefit kin. Public good gene mobility thus aligns the interests of chromosomes and MGEs concerning transfer, promoting gene exchange among bacteria.

AbstractPlasmids may maintain antibiotic resistance genes in bacterial populations through conjugation, in the absence of direct selection pressure. However, the costs and benefits of conjugation for plasmid and bacterial fitness are not well understood. Using invasion and competition experiments with plasmid mutants we explicitly tested how conjugation contributes to the maintenance of a plasmid bearing a single extended-spectrum ß-lactamase (ESBL) gene (blaCTX-M-14). Surprisingly, conjugation had little impact on overall frequencies, although it imposed a substantial fitness cost. Instead, stability resulted from the plasmid conferring fitness benefits when rare. Frequency dependent fitness did not require a functional blaCTX-M-14 gene, and was independent of culture media. Fitness benefits when rare are associated with the core plasmid backbone but are able to drive up frequencies of antibiotic resistance because fitness burden of the blaCTX-M-14 gene is very low. Negative frequency dependent fitness can contribute to maintaining a stable frequency of resistance genes in the absence of selection pressure from antimicrobials. In addition, persistent, low cost resistance has broad implications for antimicrobial stewardship.

CRISPR-Cas systems provide sequence-specific immunity against selfish genetic elements in prokaryotes. Now, two studies show that transposon-encoded variants can guide sequence-specific transposition. These findings have important practical implications but also raise questions of why and how this strategy would benefit transposons.

Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance.

The breakdown of antibiotics by β-lactamases may be cooperative, since resistant cells can detoxify their environment and facilitate the growth of susceptible neighbours. However, previous studies of this phenomenon have used artificial bacterial vectors or engineered bacteria to increase the secretion of β-lactamases from cells. Here, we investigated whether a broad-spectrum β-lactamase gene carried by a naturally occurring plasmid (pCT) is cooperative under a range of conditions. In ordinary batch culture on solid media, there was little or no evidence that resistant bacteria could protect susceptible cells from ampicillin, although resistant colonies could locally detoxify this growth medium. However, when susceptible cells were inoculated at high densities, late-appearing phenotypically susceptible bacteria grew in the vicinity of resistant colonies. We infer that persisters, cells that have survived antibiotics by undergoing a period of dormancy, founded these satellite colonies. The number of persister colonies was positively correlated with the density of resistant colonies and increased as antibiotic concentrations decreased. We argue that detoxification can be cooperative under a limited range of conditions: if the toxins are bacteriostatic rather than bacteridical; or if susceptible cells invade communities after resistant bacteria; or if dormancy allows susceptible cells to avoid bactericides. Resistance and tolerance were previously thought to be independent solutions for surviving antibiotics. Here, we show that these are interacting strategies: the presence of bacteria adopting one solution can have substantial effects on the fitness of their neighbours.

Mobile genetic elements in bacteria are enriched in genes participating in social behaviors, suggesting an evolutionary link between gene mobility and social evolution. Cooperative behaviors, like the production of secreted public good molecules, are susceptible to the invasion of non-cooperative individuals, and their evolutionary maintenance requires mechanisms ensuring that benefits are directed preferentially to cooperators. In order to investigate the reasons for the mobility of public good genes, we designed a synthetic bacterial system where we control and quantify the transfer of public good production genes. In our recent study, we have experimentally shown that horizontal transfer helps maintain public good production in the face of both non-producer organisms and non-producer plasmids. Transfer spreads genes to neighboring cells, thus increasing relatedness and directing a higher proportion of public good benefits to producers. The effect is the strongest when public good genes undergo epidemics dynamics, making horizontal transfer especially relevant for pathogenic bacteria that repeatedly infect new hosts and base their virulence on costly public goods. The promotion of cooperation may be a general consequence of horizontal gene transfer in prokaryotes. Our work has an intriguing parallel, cultural transmission, where horizontal transfer, such as teaching, may preferentially promote cooperative behaviors.

Many bacterial species are social, producing costly secreted "public good" molecules that enhance the growth of neighboring cells. The genes coding for these cooperative traits are often propagated via mobile genetic elements and can be virulence factors from a biomedical perspective. Here, we present an experimental framework that links genetic information exchange and the selection of cooperative traits. Using simulations and experiments based on a synthetic bacterial system to control public good secretion and plasmid conjugation, we demonstrate that horizontal gene transfer can favor cooperation. In a well-mixed environment, horizontal transfer brings a direct infectious advantage to any gene, regardless of its cooperation properties. However, in a structured population transfer selects specifically for cooperation by increasing the assortment among cooperative alleles. Conjugation allows cooperative alleles to overcome rarity thresholds and invade bacterial populations structured purely by stochastic dilution effects. Our results provide an explanation for the prevalence of cooperative genes on mobile elements, and suggest a previously unidentified benefit of horizontal gene transfer for bacteria.