Comment by hga | Hacker News Reader

Comment by hga | Hacker News Reader

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[ Deleted β-Lactam stuff. ]

Nature asked me for $$$ to read the article, so ... from your description, that's leaving out the hard, fast test of just culturing several billion of those, adding a lethal concentration and seeing if there are any survivors. Which is how I gather one found spontaneous transport mutations, at least circa 1977. The state of the art has likely improved, and these guys used novel microbiological methods to find the bacteria that produce teixobactin in the first place.

ADDED: thanks to betatim's link to the full text, I've skimmed it and read the discussion, and teixobactin sound quite promising. They haven't found any protein to which it binds, and they think it binds to an "Achilles's heel" in the outer cell wall. The method described to generate resistance was their most extreme attempt, so I assume they tried the fast way, and I can see why it didn't work.

Ecologically, they believe there's little gene (e.g. plasmid) transfer between these soil bacteria and human pathogens, and the "30 year" bit came from experience with vancomycin, to which it has a lot of similarity. And they've done lots of tests for human toxicity and effectiveness in mammals. It's still early in the process, but a degree of fuss is warranted, especially due to their discovery method.

Here are the pertinent bits regarding resistance, in summary it implies that spontaneous resistance will be difficult, But... "life has a way":

>We were unable to obtain mutants of S. aureus or M. tuberculosis resistant to teixobactin even when plating on media with a low dose (4 X MIC [minimal inhibitory concentration]) of the compound. Serial passage of S.aureus in the presence of sub-MIC levels of teixobactin over a period of 27 days failed to produce resistant mutants as well (Fig. 2d, Supplementary Discussion)

From the Supplementary information:

>Cells were added to teixobactin present at 0.25xMIC, 0.5xMIC,1xMIC,2xMIC and 224xMIC. At 24 hour intervals, the cultures were checked for growth. Cultures from the second highest concentrations that allowed growth (OD600≥2) were diluted 1:100 into fresh media containing 0.25xMIC, 0.5xMIC,1xMIC,2xMIC and 4xMIC of teixobactin. This serial passaging was repeated daily for 30 days. Any cultures that grew at higher than the MIC levels were passaged on drug free MHA plates and the MIC was then determined by broth microdilution. No resistant mutants were obtained. This experiment was repeated, and produced the same negative result. In order to maximize the chance of obtaining a resistant mutant, we performed an additional experiment with very small incremental increases in the drug concentration. Cells were added to a series of tubes with small differences in the concentration of teixobactin (0.06xMIC, 0.25xMIC, 0.5xMIC, 0.75xMIC,1xMIC, 1.25xMIC, 1.5xMIC, and 2xMIC). At 24 hour intervals, cultures from the highest concentration that allowed growth to a minimum OD600 of 0.2 were diluted 1:100 into fresh medium containing 0.06xMIC, 0.25xMIC, 0.5xMIC, 0.75xMIC,351xMIC, 1.25xMIC, 1.5xMIC, and 2xMIC. This passaging was repeated for 27 days. Cultures that grew at levels higher than the MIC were passaged on drug free MHA plates, and the MIC was determined. For teixobactin, there were no mutants with an MIC greater than the parent S. aureus ATCC 29213.

What they're describing here is an intensive selection process for resistance. It's debatably much more intensive than nature would ever perform: they're growing bacteria in the presence of various dilutions of the antibiotic, taking a sample of the culture, diluting it 100-fold, and repeating the selection many (~30) times. Then they look for any bugs that develop resistance. None were found. Even if you had a truly pathological patient (i.e. someone who was doing his level best to mis-use an antibiotic), it wouldn't come close to this level of selective pressure for antibiotic resistance.

Moreover, there's a strong biochemical argument against resistance: the region that the antibiotic targets is highly conserved, which means that it's probably functionally necessary. The bug is therefore unlikely to evolve away from the threat. This is important, because it's that most likely avenue for antibiotic resistance, energetically speaking: it doesn't cost anything for an organism to mutate a weakly conserved gene, so they do it once, and pass it on to every subsequent generation with no penalty.

The antibiotic is just a peptide, so in theory you could see some sort of specialized peptidase evolve from an existing gene...but the problem is that the bug would have to then carry around that gene and express it constantly (or even less likely: evolve a sensing system that allows for selective expression). It's a highly unlikely thing, and virtually impossible to maintain over multiple generations without constant selective pressure. Bacteria do not like to hold on to genes that they don't need.

Nothing is impossible over evolutionary time, of course, but the researchers don't seem to be overstating their case here.

Removed anthropomorphising:

It's a highly unlikely thing, and virtually impossible to maintain over multiple generations without constant selective pressure. Bacteria are quickly out-competed by genetic variants with fewer expressing genes that they don't need.

BTW: do you have any links/data re the relative 'cost' of expressing unneeded genes?

Relative to what? I don't know if there have been papers on the thermodynamics of it, but it's a complex process that depends on a bunch of factors: the size of the organism, the size of the genome, the size of the new protein, the level of expression, etc.

Empirically, everyone who has done lab work with bacteria knows that they'll quickly (i.e. within a few generations) kick out plasmids unless there's selective pressure to keep them. Genes are more likely to stick around than plasmids (since they're directly integrated in the organism's genome, and there's less of an energetic cost to accumulating DNA sequence), but stable genome transformation is slow/unlikely, and bacteria will still find a way to disable un-needed genes in a relatively short period of evolutionary time.

"Bacteria are quickly out-competed by genetic variants with fewer expressing genes that they don't need." This has not been true for MRSA, where transmission of resistant organisms in the community was sustained despite there not being particularly intense selective pressure in the community. Last I checked (I don't work in community transmission as much) we don't have a firm handle on why that's true, but I'd caution against simply assuming that resistant organisms will be out competed absent selective pressure.

Beyond that, even if they are out competed, that's not sufficient. They need to be out competed to extinction.

MRSA is due to the mecA gene, which is integrated into the S. Aureus genome. It's not plasmid-mediated resistance, which means that it's less likely to disappear from bacterial populations, even in the absence of selective pressure:

http://www.eurekaselect.com/68849/article

True - but the post I was responding to wasn't talking about plasmid-mediated resistance, but rather a broad assertion that a competitive disadvantage will take care of things. That's a strong assumption for which we have a counter example.

More than mecA, which itself doesn't sound like it's grossly maladaptive in the absence of β-Lactam antibiotics (it codes for an "replacement" enzyme that's not β-Lactam sensitive): https://en.wikipedia.org/wiki/Methicillin-resistant_Staphylo...

There's also regulatory genes that may be present, there are 6 known varieties of the whole gene cassette, with 5 further subtypes based on these regulators, so in the absence of β-Lactam antibiotics it sounds like a lot of them will be fit.

Nasty.

If the antibiotic is abused by an entire society, then it'll end up in the water supply just like caffeine, birth control drugs, and Prozac.

Without having read the article too closely, teixobactin appears to be binding the lipids of the bacterial cell wall. It might be hard to evolve simple resistance to this (and the authors support this by experiment) as this would require that the bacterium changes the composition of its membranes, rather than evolve a mutated enzyme, which is how most spontaneous resistance occurs.

However, that does not exclude the possibility of the bacterium acquiring a plasmid or phage carrying an enzyme that inactives teixobactin. This particular mode of acquiring antibiotic resistance is quite common.

Nevertheless, this finding does seem to be quite a big deal. A novel broad-spectum antibiotic where spontaneous resistance is unlikely is a pretty powerful addition to the pharmacopeia.

"acquiring a plasmid or phage carrying an enzyme that inactives teixobactin. This particular mode of acquiring antibiotic resistance is quite common."

Right, which is why I said the bit about bacteria kicking out genes that they don't need. Bacteria will "eject" a plasmid within a generation or two if they no longer need the gene(s) in question. Replicating a big gob of easily-ejectable DNA is not something bacteria do unless there's a good reason to do it.

Basically, for plasmid resistance to propagate, you need to have constant exposure to the antibiotic in question, or the resistance gene needs to be stably integrated into the bacterial genome. The former doesn't happen until an antibiotic is in extremely wide use, and the latter is one more rare step on top of an already unlikely chain of events.

It's not impossible, it's just unlikely.

Tim you can do anything. Omniref or help me understand wtf just happened. :)

lol. this knowledge is just the consequence of a mis-spent youth. ;-)

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