Antimicrobial resistance level and conjugation permissiveness shape plasmid distribution in clinical enterobacteria.

Conjugative plasmids play a key role in the dissemination of antimicrobial resistance (AMR) genes across bacterial pathogens. AMR plasmids are widespread in clinical settings, but their distribution is not random, and certain associations between plasmids and bacterial clones are particularly successful. For example, the globally spread carbapenem resistance plasmid pOXA-48 can use a wide range of enterobacterial species as hosts, but it is usually associated with a small number of specific Klebsiella pneumoniae clones. These successful associations represent an important threat for hospitalized patients. However, knowledge remains limited about the factors determining AMR plasmid distribution in clinically relevant bacteria. Here, we combined in vitro and in vivo experimental approaches to analyze pOXA-48-associated AMR levels and conjugation dynamics in a collection of wild-type enterobacterial strains isolated from hospitalized patients. Our results revealed significant variability in these traits across different bacterial hosts, with Klebsiella spp. strains showing higher pOXA-48-mediated AMR and conjugation frequencies than Escherichia coli strains. Using experimentally determined parameters, we developed a simple mathematical model to interrogate the contribution of AMR levels and conjugation permissiveness to plasmid distribution in bacterial communities. The simulations revealed that a small subset of clones, combining high AMR levels and conjugation permissiveness, play a critical role in stabilizing the plasmid in different polyclonal microbial communities. These results help to explain the preferential association of plasmid pOXA-48 with K. pneumoniae clones in clinical settings. More generally, our study reveals that species- and strain-specific variability in plasmid-associated phenotypes shape AMR evolution in clinically relevant bacterial communities.

The distribution of fitness effects of plasmid pOXA-48 in clinical enterobacteria.

Antimicrobial resistance (AMR) in bacteria is a major public health problem. The main route for AMR acquisition in clinically important bacteria is the horizontal transfer of plasmids carrying resistance genes. AMR plasmids allow bacteria to survive antibiotics, but they also entail physiological alterations in the host cell. Multiple studies over the last few years have indicated that these alterations can translate into a fitness cost when antibiotics are absent. However, due to technical limitations, most of these studies are based on analysing new associations between plasmids and bacteria generated in vitro, and we know very little about the effects of plasmids in their native bacterial hosts. In this study, we used a CRISPR-Cas9-tool to selectively cure plasmids from clinical enterobacteria to overcome this limitation. Using this approach, we were able to study the fitness effects of the carbapenem resistance plasmid pOXA-48 in 35 pOXA-48-carrying isolates recovered from hospitalized patients. Our results revealed that pOXA-48 produces variable effects across the collection of wild-type enterobacterial strains naturally carrying the plasmid, ranging from fitness costs to fitness benefits. Importantly, the plasmid was only associated with a significant fitness reduction in four out of 35 clones, and produced no significant changes in fitness in the great majority of isolates. Our results suggest that plasmids produce neutral fitness effects in most native bacterial hosts, helping to explain the great prevalence of plasmids in natural microbial communities.

Within-patient evolution of plasmid-mediated antimicrobial resistance.

Antimicrobial resistance (AMR) in bacteria is a major threat to public health; one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been studied extensively in vitro but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalized patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug-resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed compensatory evolution of plasmid-associated fitness cost and the evolution of enhanced plasmid-mediated AMR in bacteria evolving in the gut of hospitalized patients. Crucially, we observed that the evolution of pOXA-48-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.

Variability of plasmid fitness effects contributes to plasmid persistence in bacterial communities.

Plasmid persistence in bacterial populations is strongly influenced by the fitness effects associated with plasmid carriage. However, plasmid fitness effects in wild-type bacterial hosts remain largely unexplored. In this study, we determined the fitness effects of the major antibiotic resistance plasmid pOXA-48_K8 in wild-type, ecologically compatible enterobacterial isolates from the human gut microbiota. Our results show that although pOXA-48_K8 produced an overall reduction in bacterial fitness, it produced small effects in most bacterial hosts, and even beneficial effects in several isolates. Moreover, genomic results showed a link between pOXA-48_K8 fitness effects and bacterial phylogeny, helping to explain plasmid epidemiology. Incorporating our fitness results into a simple population dynamics model revealed a new set of conditions for plasmid stability in bacterial communities, with plasmid persistence increasing with bacterial diversity and becoming less dependent on conjugation. These results help to explain the high prevalence of plasmids in the greatly diverse natural microbial communities.

Collateral sensitivity associated with antibiotic resistance plasmids.

Collateral sensitivity (CS) is a promising alternative approach to counteract the rising problem of antibiotic resistance (ABR). CS occurs when the acquisition of resistance to one antibiotic produces increased susceptibility to a second antibiotic. Recent studies have focused on CS strategies designed against ABR mediated by chromosomal mutations. However, one of the main drivers of ABR in clinically relevant bacteria is the horizontal transfer of ABR genes mediated by plasmids. Here, we report the first analysis of CS associated with the acquisition of complete ABR plasmids, including the clinically important carbapenem-resistance conjugative plasmid pOXA-48. In addition, we describe the conservation of CS in clinical E. coli isolates and its application to selectively kill plasmid-carrying bacteria. Our results provide new insights that establish the basis for developing CS-informed treatment strategies to combat plasmid-mediated ABR.

Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid.

The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.