Periodic Reporting for period 1 - REPLAY (Analysing the role of history, chance and selection on the evolution of plasmid-mediated antibiotic resistance)
Período documentado: 2020-09-01 hasta 2022-08-31
What is the problem addressed by REPLAY?
Antibiotic resistance (AMR) in bacteria is one of the major health problems facing modern medicine. AMR can emerge by de novo mutations or by horizontal gene transfer (HGT) events. The latter is the predominant mode of acquisition of resistance in clinical settings. The specialized genetic vectors driving HGT in bacteria are known as mobile genetic elements (MGE). The most relevant MGE for the dissemination of AMR are plasmids, which are self-replicating DNA molecules capable of conjugating among different cells, and of transferring several resistance determinants in a single event
In REPLAY we have studied how plasmid-mediated antibiotic-resistance evolves in bacteria isolated from human patients. Particularly, we have focused in the plasmid pOXA48 and in the Enterobacteriaceae family of bacteria. pOXA 48 bears a carbapenemase, making that the bacteria that bears this plasmid very difficult to be treated. Carbapenem resistant Enterobacteriaceae are one of the most concerning threats in clinical settings around the world. Resistance to carbapenems, which are considered by the WHO as Critically-Important Antimicrobials, is mainly driven by conjugative plasmids encoding carbapenemases, which are enzymes able to hydrolyse penicillins, cephalosporins, monobactams and carbapenems.
Why is REPLAY important for society?
As AMR is one of the biggest problems from a sanitary point of view, increasing our understanding of how bacteria become resistant can help us to better treat resistant-infections. Therefore, the ultimate goal of REPLAY is to pave the way to have better and more rationalised treatments to combat the emergence and spread of antibiotic resistances.
What are the overall objectives of REPLAY?
As plasmid-mediated AMR is arguably the main mechanism of acquisition of AMR in vivo, understanding the evolutionary forces that determine the emergence of plasmid-bacterium associations is urgently needed. The overall goal of REPLAY is to gain an understanding of how plasmid-mediated AMR evolves in clinically relevant bacteria. Mainly, we have focused on understanding the effect of different bacteria from the Enterobacteriaceae family on plasmid-bacterium interactions and compensatory evolution using a range of plasmids and bacterial clones of clinical relevance in a full factorial design.
Antibiotic resistance (AMR) in bacteria is one of the major health problems facing modern medicine. AMR can emerge by de novo mutations or by horizontal gene transfer (HGT) events. The latter is the predominant mode of acquisition of resistance in clinical settings. The specialized genetic vectors driving HGT in bacteria are known as mobile genetic elements (MGE). The most relevant MGE for the dissemination of AMR are plasmids, which are self-replicating DNA molecules capable of conjugating among different cells, and of transferring several resistance determinants in a single event
In REPLAY we have studied how plasmid-mediated antibiotic-resistance evolves in bacteria isolated from human patients. Particularly, we have focused in the plasmid pOXA48 and in the Enterobacteriaceae family of bacteria. pOXA 48 bears a carbapenemase, making that the bacteria that bears this plasmid very difficult to be treated. Carbapenem resistant Enterobacteriaceae are one of the most concerning threats in clinical settings around the world. Resistance to carbapenems, which are considered by the WHO as Critically-Important Antimicrobials, is mainly driven by conjugative plasmids encoding carbapenemases, which are enzymes able to hydrolyse penicillins, cephalosporins, monobactams and carbapenems.
Why is REPLAY important for society?
As AMR is one of the biggest problems from a sanitary point of view, increasing our understanding of how bacteria become resistant can help us to better treat resistant-infections. Therefore, the ultimate goal of REPLAY is to pave the way to have better and more rationalised treatments to combat the emergence and spread of antibiotic resistances.
What are the overall objectives of REPLAY?
As plasmid-mediated AMR is arguably the main mechanism of acquisition of AMR in vivo, understanding the evolutionary forces that determine the emergence of plasmid-bacterium associations is urgently needed. The overall goal of REPLAY is to gain an understanding of how plasmid-mediated AMR evolves in clinically relevant bacteria. Mainly, we have focused on understanding the effect of different bacteria from the Enterobacteriaceae family on plasmid-bacterium interactions and compensatory evolution using a range of plasmids and bacterial clones of clinical relevance in a full factorial design.
First, we have selected a group of pathogens belonging to the Enterobacteriaceae family isolated from human patients from the Hospital Universitario Ramón y Cajal where the plasmid pOXA48 has been found.
Then, we have measured the effects of the plasmid in those bacteria. We have found, that the presence of the plasmid affects both to the resistance to antibiotics and to the physiology of the cell. The bacteria bearing pOXA48 become resistant to b-lactam antibiotics but in the absence of the antibiotic the plasmid impose a cost, reducing the growth of the bacteria bearing them. Additionally, we estimated the plasmid copy number of the pOXA48 in the cells, showing that the plasmid copy number is higher in Klebsiella pneumoniae than in Escherichia coli
Once we have studied the effects of the plasmid in the cells, we have propagated them in controlled environments in the laboratory for 150 generations, allowing the bacteria and the plasmid to coevolve together. In this experiment, called experimental evolution, we have propagated 5 clones of Escherichia coli and 7 clones of Klebsiella pneumoniae both in the presence and in the absence of antibiotics. Then, we sequenced the genome of more than 150 evolved populations to understand the evolutionary dynamics of plasmid/host adaptation. Once the experiment was done, we have focused our attention in 4 main different aspects that are crucial for the biology of the plasmid: i) has the cost of the pOXA48 being compensated after the coevolution with the different clones? ii), what are the mutations driving plasmid/host adaptation?, iii) has the plasmid changed/evolved? and iv) does the plasmid/host adaptation is strain or species-specific or is that adaptation driven by common mechanisms?
i) Our results showed that the initial cost of the plasmid was compensated after 150 generations of coevolution with the host independently of the strains, and of the presence or absence of antibiotic in the environment.
ii) The cost of plasmids is usually associated with the plasmid copy number that the plasmid has in the cell. Therefore, we found that when the cells bearing the plasmid were propagated in the absence of antibiotics, the plasmid copy number was reduced probably reducing the cost of pOXA48. However, in the presence of antibiotics, the plasmid copy number was the same that at the beginning of the experiment.
iii) We found several mutations in the plasmid at the end of the experiment. Some of them were encoded in the machinery that the plasmid uses to spread among different cells. Some other were found affecting the proteins involved in the replication of the plasmid. The most relevant mutations that we found are some that inactivate the carbapenemase, and therefore, making the cell sensitive to antibiotics again.
iv) By sequencing the whole-genome of the evolving populations, we found that the mechanisms driving plasmid/host adaptation are both species-specific and strain-specific. In Escherichia coli, we found that the vast majority of adaptive mutations wer single nucleotide mutations in the chromosome of the bacteria. Further, those mutations varied among the different clones. However, in Klebsiella pneumoniae, the presence of the plasmid induced the expression of some genetic elements known as insertion sequences. The insertion sequences are mobile genetic elements capable of "jump" to different parts of the genome. We have found that the solely presence of pOXA48 induced the movement of those insertion sequences, and that the adaptive mutations are caused by the movements of those insertion sequences and not by single nucleotide polymorphisms. As in Escherichia coli, each different Klebsiella pneumoniae clone adapted to the plasmid in a different way.
We have presented the results of REPLAY in the meeting organised by the International Society for Plasmid Biology and Other Mobile Genetic Elements (Tolouse, France 2022). We are drafting the manuscript and it will be submitted during 2023 to a high impact open access journal. Once the paper is submitted, we will announce it in our twitter pages and in our websites. All the data generated by REPLAY and the code used for the analysis of the results has been deposited in https://github.com/jorgEVOplasmids/expev_pOXA-48
Then, we have measured the effects of the plasmid in those bacteria. We have found, that the presence of the plasmid affects both to the resistance to antibiotics and to the physiology of the cell. The bacteria bearing pOXA48 become resistant to b-lactam antibiotics but in the absence of the antibiotic the plasmid impose a cost, reducing the growth of the bacteria bearing them. Additionally, we estimated the plasmid copy number of the pOXA48 in the cells, showing that the plasmid copy number is higher in Klebsiella pneumoniae than in Escherichia coli
Once we have studied the effects of the plasmid in the cells, we have propagated them in controlled environments in the laboratory for 150 generations, allowing the bacteria and the plasmid to coevolve together. In this experiment, called experimental evolution, we have propagated 5 clones of Escherichia coli and 7 clones of Klebsiella pneumoniae both in the presence and in the absence of antibiotics. Then, we sequenced the genome of more than 150 evolved populations to understand the evolutionary dynamics of plasmid/host adaptation. Once the experiment was done, we have focused our attention in 4 main different aspects that are crucial for the biology of the plasmid: i) has the cost of the pOXA48 being compensated after the coevolution with the different clones? ii), what are the mutations driving plasmid/host adaptation?, iii) has the plasmid changed/evolved? and iv) does the plasmid/host adaptation is strain or species-specific or is that adaptation driven by common mechanisms?
i) Our results showed that the initial cost of the plasmid was compensated after 150 generations of coevolution with the host independently of the strains, and of the presence or absence of antibiotic in the environment.
ii) The cost of plasmids is usually associated with the plasmid copy number that the plasmid has in the cell. Therefore, we found that when the cells bearing the plasmid were propagated in the absence of antibiotics, the plasmid copy number was reduced probably reducing the cost of pOXA48. However, in the presence of antibiotics, the plasmid copy number was the same that at the beginning of the experiment.
iii) We found several mutations in the plasmid at the end of the experiment. Some of them were encoded in the machinery that the plasmid uses to spread among different cells. Some other were found affecting the proteins involved in the replication of the plasmid. The most relevant mutations that we found are some that inactivate the carbapenemase, and therefore, making the cell sensitive to antibiotics again.
iv) By sequencing the whole-genome of the evolving populations, we found that the mechanisms driving plasmid/host adaptation are both species-specific and strain-specific. In Escherichia coli, we found that the vast majority of adaptive mutations wer single nucleotide mutations in the chromosome of the bacteria. Further, those mutations varied among the different clones. However, in Klebsiella pneumoniae, the presence of the plasmid induced the expression of some genetic elements known as insertion sequences. The insertion sequences are mobile genetic elements capable of "jump" to different parts of the genome. We have found that the solely presence of pOXA48 induced the movement of those insertion sequences, and that the adaptive mutations are caused by the movements of those insertion sequences and not by single nucleotide polymorphisms. As in Escherichia coli, each different Klebsiella pneumoniae clone adapted to the plasmid in a different way.
We have presented the results of REPLAY in the meeting organised by the International Society for Plasmid Biology and Other Mobile Genetic Elements (Tolouse, France 2022). We are drafting the manuscript and it will be submitted during 2023 to a high impact open access journal. Once the paper is submitted, we will announce it in our twitter pages and in our websites. All the data generated by REPLAY and the code used for the analysis of the results has been deposited in https://github.com/jorgEVOplasmids/expev_pOXA-48
REPLAY is basic science that aims to better understand the problem caused by resistant bacteria worldwide. In REPLAY, we have found for the first time that plasmid-mediated antibiotic-resistance is strains and species-specific using a plasmid and pathogens of high relevance from a clinical point of view. Further, we have designed a pipeline to analyze plasmid/host adaptation, focusing on the movements of insertion sequences, which will be helpful for any evolutionary biologist studying how bacteria evolve in different environments. Our results and our pipeline, hopefully will be very useful to any microbiologist studying how pathogens evolve in different conditions. So, hopefully, our experiments, results, and methods will be useful both for clinical microbiologists and for evolutionary biologists.