The Evolution of Drug Resistance

Researchers use whole-genome sequencing to keep tabs on the development of antibiotic resistance in bacteria.

By | December 18, 2011

image: The Evolution of Drug Resistance A digitally colorized micrograph image of E. coliWikimedia Commons, Mattosaurus

A digitally colorized micrograph image of E. coliWIKIMEDIA COMMONS, MATTOSAURUS

Using whole-genome sequencing to track the evolution of bacteria as they are exposed to ever-increasing levels of antibiotics, researchers have identified some consistent—and potentially practicable—genetic mutations, pointing to new possibilities for conquering resistant bugs, according to a study published in today’s (December 18) Nature Genetics. And a second study in the journal, also using whole-genome sequencing, examines how drug-resistant bacteria continue to evolve after antibiotic treatment stops, which may have implications for overall resistance management.

“Both of these studies are fine examples of the utility of whole genome sequencing to test, as well as generate, hypotheses,” Bruce Levin of Emory University in Atlanta and his PhD student Pierre Ankomah said in an email. “They both address important questions about antibiotic resistance.”

Antibiotic resistance is a growing problem in medicine, and many studies have tried to understand the molecular basis of such resistance in an attempt to render the bacteria susceptible once again. But typical laboratory studies of drug-resistance use a fixed concentration of the antibiotic, said Roy Kishony of Harvard University. “What happens in these conditions is that the bacteria acquire typically a single mutation that allows them to cope… and then the evolution process becomes stagnated.”

So Kishony and his colleagues constructed a device, called a morbidostat, that maintains the growth rate of bacteria as antibiotic levels rise. “You have a culture of bacteria and a computer that is monitoring how happy they are, namely how fast they grow,” explained Kishony. “If they are growing too fast it adds more drug.” In essence, it keeps the bugs unhappy and unhealthy, thus pressuring them continuously to adapt.

“It’s a dream of biologists to watch evolution in real time and to observe the actual genetic changes,” said Michael Elowitz of the California Institute of Technology in Pasadena. “This device, together with the amazing power of whole genome sequencing, allows you to actually do that.”

Kishony’s team used the morbidostat to culture genetically identical, drug-susceptible Escherichia coli strains in the presence of one of three antibiotics—chloramphenicol, doxycyclin, or trimethoprim—five cultures for each. After a few weeks of forced evolution with the morbidostat, the researchers performed whole-genome sequencing on all fifteen cultures. While the ten cultures grown in chloramphenicol and doxycyclin contained a range of mutated genes, all five trimethoprim cultures had mutations in the gene encoding dyhydrofolate reductase, the target of the drug.

The consistent outcome seen with trimethoprim prompted the team to ask whether all five cultures followed similar evolutionary paths. Genome sequencing of bacterial samples taken each day from the trimethoprim cultures revealed that indeed they did. “What they see in this study is that in some circumstances you can get an incredibly deterministic sequence of mutational events,” said Elowitz.

Such predictability could in theory offer doctors the ability to monitor patient infections and stay one step ahead of the bacteria. “If we know what are the mutations that are going to appear, we might be able to get smart and think about how to prepare for them in advance,” said Kishony.

A similar monitoring strategy could be used for determining if and when drug-resistant bacteria switch to more vigorously growing forms—the subject of the second paper by Sebastian Gagneux of the University of Basel in Switzerland and colleagues. Drug-resistant bacteria tend to have a considerable growth disadvantage compared to their drug-susceptible counterparts when grown without antibiotics in a lab—a fact that led some researchers to predict lower transmission rates for drug-resistant strains.

Occasionally however, the growth disadvantage seen in the lab can be overcome, and bacteria can maintain their resistance even in the absence of antibiotics. Furthermore, some drug-resistant bacteria isolated from patients also display no sign of growth disadvantage.

Sequencing the genomes of both lab-evolved and clinically isolated rifampicin-resistant Mycobacterium tuberculosis, Gagneux’s team revealed that the more vigorously growing bugs had developed a set of mutations that seem to confer a growth advantage, even in the face of the genetic changes associated with resistance.

“Rather than revert to the higher-fitness, sensitive state, the bacteria remain resistant and the costs of resistance are compensated for by mutations,” explained Bruce Levin and Pierre Ankomah of Emory University in Atlanta, Georgia, in an email. This means, said the pair, that “expectations that resistance will wane because it imposes a burden on bacteria may be overly optimistic.”

Should the identified mutations be proven to offer M. tuberculosis a growth advantage—so far only a strong prediction—it suggests they could be useful, “as markers of drug-resistant strains that might be particularly transmissible,” said Gagneux.

“These studies together show the value of inexpensive whole-genome sequencing for developing insights into microbial strategies for dealing with antibiotic treatments,” says Jim Collins of Boston University. “I think these are going to be the types of studies we are going to see with increasing frequency this coming decade.”

E. Toprak et al., “Evolutionary paths to antibiotic resistance under dynamically sustained drug selection,” Nature Genetics, doi:10.1038/ng.1034, 2011.

I. Comas et al., “Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes, Nature Genetics, doi:10.1038/ng.1038, 2011.

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Comments

Avatar of: cos14mopolitan

cos14mopolitan

Posts: 3

December 19, 2011

Interesting ideas. I see one limitation to the use of continuous culture as a model: Cells collecting in foam or making films at the surface and sides of the reactors (almost impossible to avoid) dominate the population as progenitors for the next generation and will be sequestered from antibiotics. This has perhaps little analogy to natural populations of pathogens. 

Avatar of:

Posts: 0

December 19, 2011

Interesting ideas. I see one limitation to the use of continuous culture as a model: Cells collecting in foam or making films at the surface and sides of the reactors (almost impossible to avoid) dominate the population as progenitors for the next generation and will be sequestered from antibiotics. This has perhaps little analogy to natural populations of pathogens. 

Avatar of:

Posts: 0

December 19, 2011

Interesting ideas. I see one limitation to the use of continuous culture as a model: Cells collecting in foam or making films at the surface and sides of the reactors (almost impossible to avoid) dominate the population as progenitors for the next generation and will be sequestered from antibiotics. This has perhaps little analogy to natural populations of pathogens. 

Avatar of: jvkohl

jvkohl

Posts: 53

December 21, 2011

Does it make sense to anyone else to attempt to use the pheromones responsible for quorum sensing in one strain to suppress growth of closely related ~ compared to more genetically diverse ~ strains? Now that such an effect could potentially be measured, the reason(s) for the typical colonization by one organism (in one tissue) might help to avoid harm done by attempts to entirely eliminate it. This might result in treatments that would suppress infection to levels typically managed by a competent immune system (and reduce problems with mutations).

Avatar of:

Posts: 0

December 21, 2011

Does it make sense to anyone else to attempt to use the pheromones responsible for quorum sensing in one strain to suppress growth of closely related ~ compared to more genetically diverse ~ strains? Now that such an effect could potentially be measured, the reason(s) for the typical colonization by one organism (in one tissue) might help to avoid harm done by attempts to entirely eliminate it. This might result in treatments that would suppress infection to levels typically managed by a competent immune system (and reduce problems with mutations).

Avatar of:

Posts: 0

December 21, 2011

Does it make sense to anyone else to attempt to use the pheromones responsible for quorum sensing in one strain to suppress growth of closely related ~ compared to more genetically diverse ~ strains? Now that such an effect could potentially be measured, the reason(s) for the typical colonization by one organism (in one tissue) might help to avoid harm done by attempts to entirely eliminate it. This might result in treatments that would suppress infection to levels typically managed by a competent immune system (and reduce problems with mutations).

Avatar of:

Posts: 0

February 9, 2012

 This is a valid observation, and not one that has been discussed in the paper, though I'm sure the researchers are aware of it. However, you do seem to suggest that such an occurrence would have little analogy to natural populations, when in fact much of the evidence from the biofilm community is that biofilms play a significant role in infection, and the persistence thereof. In this case, augmenting this system to look directly at the effects on the biofilm population would be very useful.  

Avatar of:

Posts: 0

February 9, 2012

 This is a valid observation, and not one that has been discussed in the paper, though I'm sure the researchers are aware of it. However, you do seem to suggest that such an occurrence would have little analogy to natural populations, when in fact much of the evidence from the biofilm community is that biofilms play a significant role in infection, and the persistence thereof. In this case, augmenting this system to look directly at the effects on the biofilm population would be very useful.  

Avatar of: Jim C

Jim C

Posts: 1457

February 9, 2012

 This is a valid observation, and not one that has been discussed in the paper, though I'm sure the researchers are aware of it. However, you do seem to suggest that such an occurrence would have little analogy to natural populations, when in fact much of the evidence from the biofilm community is that biofilms play a significant role in infection, and the persistence thereof. In this case, augmenting this system to look directly at the effects on the biofilm population would be very useful.  

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