How to combat antibiotic-resistant bacteria

[Hermit]

...if the environment is already occupied, by harmless strains...

Again, is there any evidence that one kind of bacterium can crowd out another?

[mistermack]

...if the environment is already occupied, by harmless strains...

Again, is there any evidence that one kind of bacterium can crowd out another?

It's not another kind, it's the same kind. The same bacteria as the resistant ones, but bred for minimum resistance and virulence.

With any environment, there is a size limit to the population it can support. Otherwise, the population would just keep growing for ever. That applies to bacteria, or beavers.

In any case, you have to take account of the effect of gene-exchange.
If resistant bacteria encounter thousands of harmless bacteria, to each ONE of resistant bacteria, then they eventually get some ''harmless'' genes. True, the harmless ones will acquire some of the resistant genes, but as they are constantly cleaned away, and killed and replaced with more harmless ones, that won't matter. You will be constantly diluting the pool of resistant genes, until the resident population is identical to the harmless ones that you are constantly releasing.

Like the example I gave earlier. If you have an island, with all black sheep on it, and constantly release an equal number of white sheep every year, within a few decades, you will end up with a population of all white sheep.

[JimC]

I think the problem is your concept of "harmless strains". They are not going to have genes for "being harmless", they are going to be infectious pathogens missing certain genes; but they will soon be able to pick them up...

It seems to me you have taken the idea from work that has been done on certain insect pets; breeding large numbers, sterilising them by radiation, and releasing them, so they can trigger a massive decrease in reproductive rates in the wild population. I don't think the concept extends well to pathogenic bacteria.

The "swamping" idea has some validity in terms of gut bacteria. There is some evidence, I gather, that a diverse biota of harmless bacteria can reduce the chances of pathogenic forms becoming established.

[mistermack]

I think the problem is your concept of "harmless strains". They are not going to have genes for "being harmless", they are going to be infectious pathogens missing certain genes; but they will soon be able to pick them up...

That could happen. But as they constantly get cleaned away and killed, and replaced with fresh harmless ones, that won't happen.

The mosquito thing is very different. If you bred a mosquito that was resistant to the malarial parasite, and released billions of them into the environment, to compete with the native population, that might be an equivalent practice. Hey, that might work !

edit. Probably not though. It might for a while, till the malarial parasite evolved a way to infest the new mosquitoes. It's not the same situation.

[Calilasseia]

Slight fly in the ointment here. Staphylococcus aureus became resistant, by mopping up plasmids containing antibiotic resistance genes from other bacteria, and integrating them into its genome, as bacteria are able to do. What's to stop your introduced bacteria doing exactly the same?

[Babel]

Slight fly in the ointment here. Staphylococcus aureus became resistant, by mopping up plasmids containing antibiotic resistance genes from other bacteria, and integrating them into its genome, as bacteria are able to do. What's to stop your introduced bacteria doing exactly the same?

I more or less asked the same question (less specific though) on page one and got this answer which reads, to a layman like me, as wishful thinking at best.

Until those massively distributed bacteria evolve into a more resistent strain? Then you have actively infected every hospital with bacteria that have the potential to become at least equally dangerous to the current 'superbacteria', because you have not explained how you would prevent these domesticated bacteria from developing resistance against the 'weak antibiotics'.

Humans have had a few fuck ups in the past when it comes to introducing organisms in a new environment to curtail an existing issue with the flora/fauna of a specific location. You'd hope someone would pay attention and think twice before suggesting it again as some miracle cure.

That wouldn't happen. Because evolution happens when certain genes PROLIFERATE in the overall population. You are artificially ensuring that the least harmful genes proliferate. So long as you constantly introduce fresh low-resistance bacteria, they can't evolve resistance.

It's like saying that there is no point in breeding dogs, they will revert to wolves. Yes they would, if you let them.

[JimC]

Slight fly in the ointment here. Staphylococcus aureus became resistant, by mopping up plasmids containing antibiotic resistance genes from other bacteria, and integrating them into its genome, as bacteria are able to do. What's to stop your introduced bacteria doing exactly the same?

Having done so, and therefore expressing a protein they didn't before, wouldn't they be at a slight selective disadvantage compared to bacteria without that gene, as long as they were in an environment (or body) without antibiotics present?

Not that I think such a small difference would be a major factor...

[Calilasseia]

Slight fly in the ointment here. Staphylococcus aureus became resistant, by mopping up plasmids containing antibiotic resistance genes from other bacteria, and integrating them into its genome, as bacteria are able to do. What's to stop your introduced bacteria doing exactly the same?

I more or less asked the same question (less specific though) on page one and got this answer which reads, to a layman like me, as wishful thinking at best.

Until those massively distributed bacteria evolve into a more resistent strain? Then you have actively infected every hospital with bacteria that have the potential to become at least equally dangerous to the current 'superbacteria', because you have not explained how you would prevent these domesticated bacteria from developing resistance against the 'weak antibiotics'.

Humans have had a few fuck ups in the past when it comes to introducing organisms in a new environment to curtail an existing issue with the flora/fauna of a specific location. You'd hope someone would pay attention and think twice before suggesting it again as some miracle cure.

That wouldn't happen. Because evolution happens when certain genes PROLIFERATE in the overall population. You are artificially ensuring that the least harmful genes proliferate. So long as you constantly introduce fresh low-resistance bacteria, they can't evolve resistance.

It's like saying that there is no point in breeding dogs, they will revert to wolves. Yes they would, if you let them.

Ah, didn't see that.

Except, of course, that evolution happens whenever any change in allele frequency occurs, for whatever reason. The acquisition of new genes horizontally would be an example thereof. Introducing a large number of new organisms, all capable of integrating the same plasmids into their genomes, seems to me a fairly silly way of trying to eliminate those plasmids from the population.

[Calilasseia]

Slight fly in the ointment here. Staphylococcus aureus became resistant, by mopping up plasmids containing antibiotic resistance genes from other bacteria, and integrating them into its genome, as bacteria are able to do. What's to stop your introduced bacteria doing exactly the same?

Having done so, and therefore expressing a protein they didn't before, wouldn't they be at a slight selective disadvantage compared to bacteria without that gene, as long as they were in an environment (or body) without antibiotics present?

Not that I think such a small difference would be a major factor...

Not necessarily. Experiments conducted with Salmonella typhimurium nailed this one. Because those bacteria retained the same level of fitness as the antibiotic susceptible ones. I covered the relevant paper when dealing with "reduced fitness" canards erected by creationists over at RDF about 3 years ago. Here you go ... from that post (which I saved after carpet bombing Robert Byers with it) ...

Novel Ribosomes Affecting Translational Accuracy, Antibiotic Resistance and Virulence of Salmonella typhimurium by Hohanna Björkman, Patrik Samuelsson, Dan. I. Andersson and Diarmid Hughes, Molecular Microbiology, 31: 53-58 (1999).

The abstract reads:

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.

The paper continues:

Results and Discussion

Selection of the rpsD mutations

Streptomycin-dependent mutants were isolated in S. typhimurium using the underlay technique (Bjare and Gorini, 1971). Three different mutant forms of rpsL, each with a single amino acid substitution in ribosomal protein S12, were identified after DNA sequencing, P90L, P90R and P91D. Co-transduction with a linked Tn10 and sequencing of the rpsL gene in several transductants in each case supports the conclusion that the SmD phenotype is caused by these mutations in rpsL. These mutant forms of rpsL are also associated with streptomycin dependence in Escherichia coli (Timms et al., 1992). From each of the SmD strains, we isolated several spontaneous streptomycin-independent mutants and chose 10 for further study. Based on previous studies, we expected streptomycin independence to be associated with ram mutations in rpsD or rpsE (Birge and Kurland, 1970; Deuser et al., 1970; Kreider and Brownstein, 1972; Hasenbank et al., 1973). For each of the 10 mutants, we sequenced their rpsL, rpsD and rpsE genes. Nine different mutations associated with the streptomycin-independent phenotype were identified (Table 1). Six different mutations were isolated in rpsD, two in rpsE and one in rpsL. The mutation in rpsL is AAA to AGA at codon 42, which alone has previously been shown to cause a non-restrictive streptomycin-resistant phenotype (Tubulekas and Hughes, 1993). Co-transduction with a linked Tn10 showed no linked expression of the SmD phenotype, suggesting that no other mutation outside of rpsL is required for streptomycin independence. To our knowledge, this is the first case in which an SmD phenotype has been shown to be reversible by a second-site mutation within rpsL. We also isolated an rpsD and an rpsE mutation by selecting for improved growth rate in LB of a strain carrying a restrictive rpsL mutation (Table 1). The isolation of so many unique mutations in these selections suggests that the number of possible compensating mutations is much larger. We used the seven different rpsD mutations to construct a set of 24 isogenic strains carrying different combinations of rpsL and rpsD alleles (see Experimental procedures). The rpsL, rpsD and rpsE genes were resequenced in each strain after construction to confirm the presence of the expected allele in each case. These strains were tested for growth rate, translational elongation rate, translational accuracy, minimal inhibitory concentration (MIC) for streptomycin and virulence in mice. The results are presented in Table 2.

rpsD and streptomycin resistance

A surprising outcome from our experiments was the discovery that three of the seven rpsD alleles (Q53L, [chr]916[/chr]V200 and 201 UAG termination) are associated with resistance to streptomycin in the presence of the wild-type rpsL allele (Table 2). This linkage between a streptomycin resistance phenotype and an rpsD mutation was supported by sequencing rpsL and rpsD from several transductants in each strain construction experiment. This is the first demonstration of resistance to streptomycin associated with alleles of rpsD. The Q53L mutation has been identified previously in E. coli but was not investigated with respect to streptomycin resistance and was assumed to have a ram phenotype (Van Acken, 1975). Resistance to streptomycin has until now only been connected with alterations in the amino acid sequence of the S12 protein and the sequence of 16S rRNA (reviewed in Kurland et al., 1996). The level of streptomycin resistance associated with the rpsD alleles is lower than that conferred by classic rpsL mutations such as K42T and K42N (Table 2), but is nevertheless significantly above the wild-type level. The primary binding site for streptomycin on the ribosome is in a region of 16S rRNA (Abad and Amils, 1994; Spickler et al., 1997), close to the decoding centre where ribosomal proteins S4 and S12 proteins are close neighbours (Brimacombe, 1991). Thus, the finding that S4 mutants can mediate streptomycin resistance, while novel, seems perfectly reasonable. The remaining four rpsD alleles were sensitive to streptomycin at close to the wild-type level (Table 2).

Streptomycin-resistant rpsD alleles are restrictive

The three rpsD alleles causing resistance to streptomycin also restrict translational errors, as measured by nonsense readthrough, and have slow translation elongation rates (Table 2). These are classic phenotypes of streptomycin resistant rpsL alleles (Bohman et al., 1984; Ruusala et al., 1984; Tubulekas and Hughes, 1993) and have not previously been associated with mutations in the rpsD gene. Indeed, the original designation of the gene for ribosomal protein S4 was ram(for ribosomal ambiguity), because of the finding that mutations in this gene increase translational errors (Rosset and Gorini, 1969). Our results show that the connection between rpsD mutants and ribosomal ambiguity is not absolute. Indeed, our results would suggest that a significant proportion of rpsD mutants selected on the basis of streptomycin independence for SmD strains (the classic way to select ram mutants) are not error-prone mutants, but error-restrictive mutants. The remaining four rpsD mutants isolated have the classic phenotypes associated with ram mutants of rpsD: increased error levels and normal elongation rates (Piepersberg et al., 1979; Andersson et al., 1982).

Restrictive rpsD alleles compensate restrictive and dependent alleles of rpsL

We expected that combining restrictive alleles of rpsL with ram alleles of rpsD would result in strains showing phenotypical compensation, having intermediate pseudowild-type phenotypes with regard to growth rate, translation elongation rate and readthrough of nonsense codons (Bjare and Gorini, 1971; Andersson et al., 1982; Andersson et al., 1986). These expectations were borne out (Table 2). What was not expected was that combining the restrictive alleles of rpsL, K42T and K42N, with the restrictive alleles of rpsD, Q53L, [chr]916[/chr]200 and UAG201, would also result in double mutant strains that showed phenotypical compensations and pseudo-wild-type phenotypes with regard to growth rate, translation elongation rate and nonsense codon readthrough (Table 2). Indeed, some ram rpsD alleles (K205T and K205N) relieve the restriction of translational errors to a lesser extent than restrictive rpsD alleles do (Table 2). The readthrough level for these double mutants is only 2.5 times higher than for the rpsL alleles alone, which is to be compared with 10–15 times higher for the restrictive rpsD mutant combinations. Also, the growth rates of the combinations between K205T, K205N and the two restrictive rpsL alleles are the highest among all the double mutants tested (in spite of the continuing error restriction), suggesting that other parameters not measured here are influencing the growth rates.

What is unique in our data is the evidence that phenotypical reversal of ribosomal restrictiveness does not require an ambiguity mutation, but can apparently also be achieved by another restrictive mutation. It will be interesting to determine the kinetic basis of the restrictive rpsD phenotypes in translation and to relate these to earlier data and interpretations on kinetic compensations between restrictive rpsL and ram rpsD alleles (Andersson et al., 1986). Our current interpretation is that reversal of restrictiveness is not necessarily the sum of two independent events, but may be a consequence of as yet undefined structural alterations to the ribosome. The initial isolation of these restrictive rpsD mutations on the basis that they relieve streptomycin dependence supports this conclusion, in contrast to the earlier assumption that relief of restrictiveness and streptomycin dependency required an increase in ribosomal ambiguity (Bjare and Gorini, 1971; Ruusala et al., 1984). Taken together, our data suggests that the relief of ribosomal restrictiveness or of streptomycin dependency may depend more on the details of structural interactions on the ribosome than on the additive effects of opposing phenotypes.

Streptomycin MICs for rpsL 106 and rpsL 116 depend on the specific allele of rpsD

In the MIC tests, the two rpsLmutants have the same MIC value, >1024, with the upper limit at 16 000 (Tubulekas et al., 1991). With only two exceptions, all of the double mutants have the MIC for streptomycin characteristic of rpsL alone, >1024. The two exceptions are K42T in combination with either rpsDQ53P or rpsD[chr]916[/chr]V200 in which the MIC values are greatly reduced (Table 2). This suggests that the single amino acid difference in S12 distinguishing these two mutants (K42T for rpsL106 and K42N for rpsL116) has implications for the level of resistance to streptomycin in combination with specific rpsD alleles. This is further support for our suggestion that the phenotype of the ribosome depends on the S4 and S12 proteins in a way that cannot be calculated simply as the average of two independent phenotypes.

Combinations of rpsL and rpsD are virulent in mice

Our initial reason for constructing these strains was to test whether ram alleles of rpsD could restore virulence to S. typhimurium strains carrying restrictive alleles of rpsLi in the mouse model. Competition experiments in mice between a wild-type strain and strains carrying each combination of rpsL and rpsD gave the following results (Table 2): (i) each of the two rpsL alleles alone is associated with a severe loss of avirulence, as are four of the rpsD alleles (all three of the restrictive rpsD alleles are associated with loss of virulence); (ii) three of the ram rpsD alleles have no negative effect on virulence; (iii) all combinations of the avirulent rpsL and avirulent rpsD alleles are significantly more virulent than either allele alone; in six out of eight cases, the virulence is restored to within a factor of four, or better, of the wild-type level; (iv) combinations of avirulent rpsL with virulent rpsD alleles are significantly more virulent than rpsL alone; in five out of six cases, the virulence is restored to within a factor of four, or better, of the wild-type virulence level. From these results, we conclude that the acquisition of any one of these seven rpsD mutations by an avirulent rpsL strain would have resulted in a significant restoration of virulence. This leaves open the question of why rpsD alleles were not isolated as virulence-restoring compensatory mutations in selections in the mouse model.

In conclusion, we have shown that rpsD mutations can have either a ribosomal ambiguity (ram) or a restrictive phenotype. Furthermore, both the ram and the restrictive alleles of rpsD can compensate for the phenotypes of restrictive and streptomycin-dependent rpsL alleles. Thus, the relationship between the proteins S4 and S12 on the ribosome is more complex than previously assumed, and a ribosomal phenotype cannot be predicted to be simply an average of the individual phenotypes of mutant S4 and S12 proteins. Our data also show that rpsL, rpsD, double mutant combinations are virulent in mice. The explanation for the bias we observe in the distribution of compensating mutations isolated in mice (all occur within rpsL) must therefore be sought elsewhere. One attractive possibility is that the mutation rates and/or mutational spectrum differ between bacteria grown in mice and that grown in laboratory media.

Oh, look at that. Exeprimental results confirming that specific mutations are associated with streptomycin resistance in Salmonella typhimurium, that these correlate with similar mutations in other bacteria such as Escherischia coli, and that the S. typhimurium variants of streptomycin resistance mutations are in addition associated in several strains with compensatory mutations that restore full wild type fitness with respect to virulence (just to knock another favourite creationist canard on the head all in the one package).

Enjoy along with your gin, Jim.

[JimC]

Thanks, Cali; detailed and comprehensive as ever!

[mistermack]

Ah, didn't see that.

Except, of course, that evolution happens whenever any change in allele frequency occurs, for whatever reason. The acquisition of new genes horizontally would be an example thereof. Introducing a large number of new organisms, all capable of integrating the same plasmids into their genomes, seems to me a fairly silly way of trying to eliminate those plasmids from the population.

But you are favouring the resistant genes there. The same thing will happen in the other direction too, with resistant bacteria acquiring non resistant genes. And because of the much greater numbers of non resistant bacteria, due to their constant introduction, that would be the overall direction of travel.

Also, you have to take into account that in hospital environments, the environment is regularly cleaned.
So transfer of resistant genes to the bacteria you introduce really doesn't matter. They get cleaned away and preferably incinerated. You then start again with a small population of resident bacteria that were missed, and a huge influx of your harmless ones. And the process can begin again.

Like the sheep on the island analogue that I posted earlier.
If you have 100 black sheep on an island, and every year you cull the numbers back to 100, and introduce 100 white sheep, you will end up with all-white sheep.
There will be some isolated stray black genes, but the sheep will be white.

The difference is that this would happen every day, not every year. With the numbers involved astronomically bigger.

[Blind groper]

Just a point about the mutation of resistance to antibiotics. A lot of these already exist.

As I pointed out earlier, antibiotics are made by microorganisms in the on going war with other microorganisms. There are hundreds or thousands of such antibiotics in use all the time in nature. This means that the drive to developing antibiotic resistance is going on all the time in nature. It does not require human intervention.

This helps to explain the speed by which antibiotic resistance sometimes arises. It does not require a novel mutation. The mutation may have occurred millions of years ago, and been maintained in bacterial plasmids all that time, being used to confer resistance to an antibiotic one microbe uses to kill other microbes. When humans start using that antibiotic, the resistance already exists, and gets passed around via plasmid exchange.

[mistermack]

Just a point about the mutation of resistance to antibiotics. A lot of these already exist.

As I pointed out earlier, antibiotics are made by microorganisms in the on going war with other microorganisms. There are hundreds or thousands of such antibiotics in use all the time in nature. This means that the drive to developing antibiotic resistance is going on all the time in nature. It does not require human intervention.

This helps to explain the speed by which antibiotic resistance sometimes arises. It does not require a novel mutation. The mutation may have occurred millions of years ago, and been maintained in bacterial plasmids all that time, being used to confer resistance to an antibiotic one microbe uses to kill other microbes. When humans start using that antibiotic, the resistance already exists, and gets passed around via plasmid exchange.

Copycat ! : http://rationalia.com/forum/viewtopic.p ... 5#p1565242

[mistermack]

One thing about today's technology, there are new ways of developing a harmless bacterium.
If you can't do it by selection, you can do it by GM.

They did it here http://www.bbc.co.uk/news/science-environment-27765974 in an anti-malaria experiment that isn't too far away from what I'm suggesting.

I haven't read right through it, but apparently, they transferred a gene from a slime-mould into mosquitoes to make them produce nearly all male offspring. The idea is to breed lots of them, and release them. Sounds familiar.

[JimC]

One thing about today's technology, there are new ways of developing a harmless bacterium.
If you can't do it by selection, you can do it by GM.

They did it here http://www.bbc.co.uk/news/science-environment-27765974 in an anti-malaria experiment that isn't too far away from what I'm suggesting.

I haven't read right through it, but apparently, they transferred a gene from a slime-mould into mosquitoes to make them produce nearly all male offspring. The idea is to breed lots of them, and release them. Sounds familiar.

A quite different phenomena. In the context of, say, golden staph bacteria, it would mean releasing a genetically-engineered strain that, when it shares plasmids, delivers a gene which stops the recipient reproducing.