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Dr. Tim Stinear, a Senior Research Fellow in the Department of Microbiology and Immunology at the University of Melbourne, discusses a recent paper he co-authored, "Genomics of Drug Resistant S. Aureus". Dr. Stinear did whole-genome sequencing using the Ion PGM sequencer.
Howden BP, McEvoy CRE, Allen DL, Chua K, Gao W, et al. (2011) Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR. PLoS Pathog 7(11): e1002359. doi:10.1371/journal.ppat.10023¬59
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Video transcript:
One of the greatest public health threats that we have in our hospitals is MRSA, Methicillin-resistant staph aureus. We have some drugs that are effective against this super bug. One of them is Vancomycin. You can imagine that it's a serious problem when this bacteria in MRSA develops resistance to Vancomycin. Fortunately, high level resistance to the antibody Vancomycin is rare. But what is more common is this low level resistance where the bacteria evolve in some way to become a little bit resistant, sufficient that they fail therapy when clinicians try and treat the patient with this drug. So this is the topic that we've undertaken to try and understand what happens to MRSA in a patient when we treat them with this last line drug, Vancomycin and the bugs develop resistance. What is going on inside the microbe?
This resistance to Vancomycin is called intermediate level resistance, and staph aureus strains that have this resistance have the acronym VISA. VISA is becoming more common, particularly in Australian hospitals and the problem with it is that we have very few other drugs left. When an MRSA strain develops the VISA phenotype, intermediate we only have one or two other drugs that we can use and there are issues with toxicity of these drugs and cost. So we need to find out what the bacteria are doing and is there something that we can do with the way we use Vancomycin, perhaps, that would prevent the evolution of intermediate Vancomycin resistance.
The basic design of our study is to take carefully collected bacteria that have been obtained from the patient at the time that they started Vancomycin therapy and then 20 days later when they failed therapy. So we have sequential pairs of staph aureus strains that have been grown from the blood of patients when they first developed a blood strain staph infection, and 20 days later when they still had their blood strain staph infection but they're now being treated with Vancomycin. The basic idea is that you take the genomic DNA from these bacteria at day zero when you have your first isolate and then day 20 when you have your resisting strain and you sequence the genomes of first strains and you compare them and you look at the differences in the DNA, what has happened in the strain over the course of that Vancomycin therapy.
The VISA phenotype is pleiotropic, different mutations can occur to give you the same phenotype. This is why it's important to sequence the whole genome of these strains because it's hard to predict from the phenotype where a particular mutation might be occurring. In 4 out of 5 of these clinical pairs that we sequenced, there was a consistent mutation in a two component regulator. That means there was a mutation in the regulatory gene that controls many other genes in staph aureus and this was the breakthrough that we could see for the first time. Here's a novel mechanism by which staph aureus is developing resistance to Vancomycin.
These mutations also appear to down-regulate virulence and this is an unexpected finding. When we submitted our paper for review, the reviewer said perhaps when we did our gene swapping experiment, we introduced another mutation into another regulator that controls virulence gene expression. They said we need to do a complementation experiment- add the gene we're studying back in to show we haven't introduced a secondary mutation. This is complicated and sometimes gives you very ambiguous results. Better to use whole genome sequencing to sequence the strain where you've swapped the gene in and then show that is the only mutation present in the swapped strain. We used the Ion Torrent sequencing PGM to good effect because within the time frame of when the paper was under review, we were able to, within one week, sequence our mutants and show that the only mutation we've introduced was the one we claimed that was responsible for the phenotype changes in both drug resistance and also down-regulation of virulence.
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Mark Pallen Discusses His Work on Ion PGM Sequencerby iontorrent129 views
this video is so helpful in giving knowledge about staph aureus resistance to antibiotics and how a mutation causes the resistance
lydiaglam92 2 months ago
Howden BP, McEvoy CRE, Allen DL, Chua K, Gao W, et al. (2011) Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR. PLoS Pathog 7(11): e1002359. doi:10.1371/journal.ppat.1002359
tstinear 3 months ago