Text books commonly state that in the natural environment antibiotics are a means by which bacteria (and yeasts) reduce competition for resources, by creating a ‘zone of inhibition’ around themselves—kind of like unleashing a smelly fart to stop people sitting too closely. However, antibiotics can also be seen as part a more complex system of cell to cell communication/signalling in microbial communities, in fact, they can also be food. When used at the concentrations we employ therapeutically, they can either stop bacterial growth, or kill outright. Just because they can have this effect, doesn’t mean that this is what they evolved to do—’antibiotic’ is simply the name we give to the few (of many) small organic molecules produced by bacteria that happen to have an effect on a particular group of bacteria against which it (along with many other molecules) was screened.
Killing avoidance strategies
A couple of recent research papers remind me that I promised to talk a little about a phenomenon by which bacteria can avoid being killed by antibiotics, without actually being resistant in the classical sense, i.e. they can’t actually grow in elevated concentrations of the antibiotics they survive, and those cells that do survive give rise to populations that are no more, or less, likely to survive next time.
The first paper comes from the lab of Prof. Tony Coates at the Centre for Infection at St. George’s, University of London. Prof. Coates has for a long time been heavily involved in research into the treatment of latent and persistent infections, most notably T.B./tuberculosis. His research team (as indeed are mine) are trying to understand why some antibiotics that kill actively growing bacteria of a particular species have no effect on cells of the same species that aren’t actively growing; almost akin to the bit in Jurassic Park where T. rex kills the lawyer who’s running, but wouldn’t have done had the lawyer stood still §.
One of the reasons for this is that, historically, most drug discovery has been focussed on targeting actively growing cells, but what we are increasingly finding is that persistent infections can be mediated by a recalcitrant population of slow-growing or non-growing cells.
Whilst the idea of targeting non-growing bacteria is not a wholly new idea (you can find a review on the subject by Prof. Coates in my ‘Further reading’), it does seem that together with the report’s first author, Dr Yanmin Hu, their spin-out company (Helperby Therapeutics) has developed a platform and proof of principle drug that is now in trials, demonstrating the utility of such an approach. They have identified an antibiotic compound that has potent anti-Staphylococcal activity, but importantly, acts specifically against non-multiplying cells.
In a second paper, brought to my attention by Ed Yong, the Collins lab in Boston has identified that a sub-population of super-resistant bacteria act in a charitable manner to other members of the colony that are less resistant. Whilst the super-resistant cells could satisfy their own selfishness by merely allowing all their less-resistant siblings to die out, the bacteria in this case have a mutation that maintains their production of indole (a signalling molecule) when normally its production would be shut down on exposure to the antibiotic. When released by the cell, indole stimulates non-resistant cells to enter a state of phenotypic resistance or ‘antibiotic survival’, even though continued-production of indole incurs a fitness cost.
Why might they do this?
Well, for one of the reasons that is very much the subject of my blog, bacterial fitness. As I have mentioned before, antibiotic resistance can have a fitness cost, which means that cells committing themselves to this ‘path of resistance’ may find themselves at a disadvantage come the time when the antibiotic is no longer around. The subject of my research is to document the various ways in which antibiotic resistant Staph. aureus mitigate these fitness costs so that they get to remain resistant and just as competitive as they ever were in the absence of antibiotic. It seems that in the case of the Collins’ lab’s charitable bacteria, they may mitigate the fitness cost of antibiotic resistance at a population level by maintaining the presence of non-resistant cells that can come to the fore once the antibiotic is removed.
“These few drug-resistant mutants, by enhancing the survival capacity of the overall population in stressful environments, may also help to preserve the potential for the population to return to its genetic origins should the stress prove transient. Efforts to monitor and combat antibiotic resistance are complicated by these bet-hedging survival strategies and other forms of bacterial cooperation.”
So what I want to do is briefly introduce the types of ‘antibiotic survival’ strategies seen in bacteria. It goes without saying that future drug discovery that targets the molecular/physiological underpinning for these strategies (once we’ve identified what these are!) will be important for the clinical management of infection.
Resistance or ‘killing avoidance’?
I’ve discussed in a previous post what I might describe as mechanisms of antibiotic resistance, i.e. producing a enzyme that modifies or chews up the antibiotic; or changing the component of the cell so that the antibiotic targeted to that component no longer has any effect, or pumping the antibiotic out of the cell before it does any damage.
It was recognised early on, in the heyday of antibiotics, that penicillin could kill most bacteria in a culture, but could not sterilise a culture. This has been observed with numerous antibiotic compounds, thus at a practical level you cannot achieve a 100% kill with antibiotics. Now this isn’t generally a problem for a healthy individual, as it is at this point the immune system takes over and clears away the remaining cells. However, many people receiving antibiotics aren’t well, they may be immuno-compromised, or suffering from a deep-seated infection. The persistence of a bacterial infection becomes a perfect breeding ground for classical antibiotic resistance, with each resurgence of the infection from surviving cells increasing the probability that resistance may evolve; and thus is thought to play a significant role in the failure of antibacterial treatment.
1. INDIFFERENCE. Bacteria can avoid being killed by being in a stationary phase (non-growing or metabolically inactive). This is actually the default repose of bacteria in the environment, only submitting to bursts of growth in the presence of nutrients. Most current (and old) antibiotics are specific to the particular cell components and processes of actively growing cells, there is no reason to expect that such antibiotics would have any killing effect on cells not engaging in these processes.
2. TOLERANCE. Those antibiotics that do kill bacteria don’t necessarily do so directly; they initiate a series of events, a cascade of physiological responses, which ultimately result in cell death. Unlike indifference, tolerance is not linked to the growth/metabolic state of the bacteria, but instead result from genetic changes that uncouple the killing activity of the drug from its inhibitory activity. In the clinic, tolerance seems to be specific to certain bacteria, and even then only in response to particualr antibiotics targeting the bacteria cell-wall.
3. PERSISTENCE. In a bacterial population there exists a sub-population of ‘persister’, cells that regardless of the growth state of the population as a whole, continue to exist in a stationary or growth-retarded state. It may be that persisters avoid antibiotic killing in the same way that indifferent bacteria do, but whilst there are some antibiotics that can kill indifferent cells, they don’t kill persisters; this suggests that something different is going on in these cells, and there is increasing evidence to suggest that there are defined genetic differences implicated in persistence, including changes within the stress-response pathways, but what these are (and what they do exactly) remains to be seen.
4. BIOFILMS. Finally, and most stubbornly, there is the issue of biofilms. Biofilms are like a condominium (or halls of residence) of bacteria, a structured environment where the bugs are surrounded by a gelatinous matrix of sugar chains and many other macromolecules. They are involved in some 80% of human infections and represent a major cause of antibiotic treatment failure. Within the matrix the bacteria avoid antibiotic killing through indifference and persistence, thought to be brought on by the low oxygen and low nutrient environment; the matrix also provides some protection from certain classes of antibiotics, as well as the immune system. Even if a large number of matrix surface cells are killed off, the matrix structure survives and can be re-populated by the surviving cells. For some bacteria the biofilm environment stimulates them to massively increase their rate of mutation, which can increase the rate at which antibiotic resistance can evolve.
So what do we do?
Well again it comes down to idealism versus pragmatism. The current system of drug discovery is fraught and inefficient enough without an additional burden of esoteric and poorly understood mechanisms of bacterial antibiotic survival. I do think there is some merit in drug discovery targeted at non-growing indifferent bacteria, this is particularly important in the treatment of T.B. The problem is going to be that many of these killing avoidance strategies differ between pathogens and between the particular environment in which they’re found, and also that in the absence of any ongoing preventative treatment, such as potential vaccines, by the time an infection manifests itself the antibiotic survival systems are likely to already be in place.
Other than indifference, biofilms are a system worth addressing in the immediate term. We have amassed a huge amount of data on biofilms, and demonstrated that they are of great clinical importance, thus efforts should be made to increase the number of biofilm busting compounds we have available.
Many people are familiar with antibiotic resistance, but I’m interested to hear (especially from other biologists) how much people knew about such antibiotic survival strategies. Also, as ever, please feel free to ask questions at any level. This (rather long) post barely touches the surface of this subject, there’s plenty more to be said!
^§^ The theory that T. rex would only ‘see’ moving objects is probably a little outdated.
As always I will try to find open access material where available, and will update those references that aren’t as and when they do.
_Hu et al. (2010) A New Approach for the Discovery of Antibiotics by Targeting Non-Multiplying Bacteria: A Novel Topical Antibiotic for Staphylococcal Infections. PLoS ONE 5: e11818._
[ Open access ]
_Coates, A. et al. (2002) The future challenge facing the development of new antimicrobial drugs. Nature Reviews Drug Discovery 1: 895-910._
[ Free pdf ]
Levin, B.R. and Rozen, D.E. (2006) Non-inhertied antibiotic resistance. Nat Rev Microbiol 4: 556-562.
[ free pdf ]
– A very useful grounding to the subject of phenotypic resistance, as it was understood back in 2006.
Lewis, K. (2010) Persister cells. Annu. Rev. Microbiol. 64: 357-72.
[Sorry, another paywall paper ]
– Good review of bacterial persistence.
[This post was restored from a WayBackWhen archive. It was originally posted to a blog called ‘The Gene Gym” that began life on the Nature Network in 2010, and then moved to Spekrum’s SciLogs platform.]
[This post was restored from a WayBackWhen archive of an older incarnation of mentalindigestions.net]
“FROM the perspective of a bacterium, higher eukaryotes are oversexed, unadventurous and reproduce in an inconvenient way.” So says Pål Johnsen and Bruce Levin in their commentary of today’s article for discussion, and nary a truer word said. Of course, one may state that inconvenient as reproduction may be, bacteria clearly have no sense of fun.
There was once an idea that we could address the problem of antibiotic resistant bacterial strains by removing the ailing antibiotic from clinical use. In the absence of selective pressure it was thought that the evolutionary traits that enable the strain to resist the antibiotic would actually put the strain at a competitive disadvantage compared with a strain that doesn’t have such antibiotic resistance. The proposed cause of this? Fitness costs – these are imposed by a resource-expensive set of mutations, or carriage of alien DNA, that make the resistant strain compete less well once its non-resistant brethren are no longer being killed off by the antibiotic.
However, some years ago now experimental evidence suggested that this is not always the case; it may in fact be often not the case. It is worth mentioning at this point that it has been shown that in some circumstances (alt) the number of infections caused by a particular antibiotic-resistant pathogenic bacterium have become fewer on reduction (or removal) of the antibiotic to which that strain is resistant, but to assume this would be the case with all strains/antibiotics is naïve.
It is true, with few exceptions, that initially both plasmid and chromosomally encoded resistances result in fitness losses. However, when resistance has a cost it is possible for compensatory mutations in a cell to ameliorate these costs, usually without the loss of resistance. The type of compensatory mutations that mitigate the fitness cost of acquiring antibiotic resistance, or any other incoming DNA that encodes potentially useful genes, will depend very much upon the environment in which the bacteria finds itself. These include the availability of resources, i.e. the growth environment of the bacteria, the environment of the genes (mobile or chromosomal), or whether the genes are being selected for by an external factor, such as the presence of antibiotics in the case of resistance genes.
So what sort of ‘nips and tucks’ might a bacterial population undergo in order to maintain a battery of costly genes, but that may provide an ongoing advantage? Well, this is the subject of much ongoing research; one example indicated that, in the absence of selective pressure, costly genes are simply silenced – a molecular mechanism often found in higher organisms that prevents a gene from being ‘switched on’. Thus a reservoir of drug resistance determinants may remain in populations that have compensated for their presence, remaining ‘inactive’ until a selective pressure removes the silencing.
A recent study by Peter Lind (et al.), a grad student working in the lab of Dan Andersson at Uppsala, Sweden, addresses a particularly pertinent question of compensatory mutations: those associated with genes acquired by horizontal gene transfer (HGT). HGT bypasses the slow and haphazard process of evolution (via random mutation, selection and recombination) by offering an opportunity for bacteria to receive fully fledged genes encoding pathogenicity factors (genes that make bacteria better at causing disease) as well as genes that encode resistances to disinfectants and/or antibiotics, amongst others. There is no doubt that such incoming DNA may pose significant fitness costs, so Lind et. al. set out to quantify the nature of compensatory mutations on such incoming DNA.
THERE you are, stood in a green grocers poring over your favourite variety of apple. Suddenly you catch the scent of something heavenly; a smell not unlike the apple you have in your hand, only better. You abandon your apple and follow the scent to the next aisle where you find more apples of the same variety. They smell superior to the others. You pick one up and are compelled to take a bite; on doing so you realise something – it’s pretty bloody awful. You put down the unpalatable apple and move on to alternative apples.
I could be describing a situation reminiscent of the selectively bred, brightly coloured, sweet smelling fruits that line our supermarket shelves; those that in fact taste like tasteless facsimiles of the original spots-and-all varieties. In this situation we are being manipulated by the supermarkets, but in nature it may be viruses doing the manipulating.
Viruses are parasites, making use of infected host cells to replicate more virus. Of course, it isn’t enough just to replicate, viruses also need to spread to new cells, and new hosts. Plant viruses are often carried from plant to plant by insects; the insects become known in this context as ‘vectors’. The study of the biology of insect vectors is, as you may imagine, fundamentally important to understanding the transmission of a whole range of parasites (viral, bacterial and protozoan) between plants, or between humans and animals. Of particular interest is how parasites, such as viruses, manipulate their insect vectors by altering the physical properties of the host they infect.
A Penn State based group, headed by Mark Mescher, have been using Cucumber Mosaic Virus (CMV), a known generalist plant pathogen, to study the effect it has on the interaction between cultivated squash plants and aphids (sap sucking bugs). The results of this study are reported by Kerry Mauck et al. in a recent paper.
They show that CMV-infected plants have elevated volatile (readily dispersing in air) emissions that attract aphid vectors. This in itself is not a revelation; the authors cite two well documented examples of this phenomenon, from Potato leaf roll virus (PLRV) and Barley yellow dwarf virus (BYDV), where infected plants release volatiles that attract aphids. However, these other viruses employ a different method of transmission to CMV, and the main thrust of this paper is to identify how the mode of transmission modifies the host-insect interaction.
A FEW years ago, a Boston University team headed by Jim Collins published findings that suggested the means by which bactericidal antibiotics result in cell death. Rather than the cause being the cellular target of the drug, the team showed it was the secondary effects of stimulating the production of hydroxyl radicals, a reactive oxygen species 1. The hydroxyl radical is known to cause significant damage to cellular DNA, proteins and cell wall, leading to cell death.
Their 2007 study 1 was initially met with a few raised eyebrows in some quarters, coming in for some criticism for having a few gaps; namely whether the role of the hydroxyl radical was even pertinent in a real world infections settings, which are often in the low-oxygen environment of biofilms 2. There was also some question of whether it was adequately demonstrated that the oxidative stress was a source or the result of cell damage. However, subsequent studies reported by Kohanski, as well as other labs, have described a more defined link between a bactericidal drug and resulting hydroxyl radical formation 3.
In the latest edition of Molecular Cell, a new article from Mike Kohanski, Mark DePristo and Jim Collins reports that prolonged exposure to sub-lethal concentrations of antibiotics can induce multiple drug resistance in E. coli and Staphylococcus aureus strains that were initially drug sensitive 4. E. coli strains were tested with sub-lethal levels of three major classes of bactericidal antibiotics (quinolone, B-lactam and aminoglycoside), which were found to significantly increase the mutation rate, confirming their expectations.
IN evolutionary theory there is a phenomenon known as canalisation, a process in which the phenotype (i.e. the outward physical appearance of an organism) remains unchanged, despite genetic or environmental influences. This suggests that a mechanism exists to buffer the physical appearance from such changes, which may explain why some species can remain mostly unchanged for millions of years.
The buffering afforded by this mechanism permits the accumulation of genetic variation, in effect storing it up like an evolutionary capacitor. Also, presumably the accumulated genetic variation may be released by an event that overcomes the evolutionary capacitor, releasing fuel (in the form of variation) that provides a substrate for natural selection and potentially accelerating evolution. But how?
The idea of capacitance was first suggested by Rutherford and Lindquist 1 following experiments on a protein called heat shock protein 90 (Hsp90) in fruitflies. Generally speaking, heat shock proteins assist in the maintenance and correct folding of cellular proteins, especially when under temperature stress; Hsp90 plays a particular role in maintaining the unstable signalling proteins that act as key regulators of growth and development.
They suggested that in nature, a stressing event such as high or low temperatures may overcome the protective buffering effect that Hsp90 has on maintaining these key regulators. As Hsp90 becomes diverted from its usual role, due to an increase of stress-damaged proteins in the cell, those cell signalling proteins it normally maintains are free to adopt a range of altered behaviours, interfering with the development of the organism. The result is morphological variants upon which natural selection can act. Rutherford and Lindquist found as much, with chemically and environmentally compromised Hsp90 resulting in flies with abnormal wings, legs or eyes, they observed a broad variety of phenotypes.
Rutherford and Lindquist went on to demonstrate that the capacity for such remarkable variation was pre-existing, i.e. it was encoded genetically prior to the stressing event, but had been silenced. Evolutionary capacitance may therefore provide a mechanism of adaptive evolution in which a population under stress may release previously silent variation, resulting in the appearance of certain individuals with more desirable traits in that changed environment. When such revealed traits are selected for they can become fixed and independently of the buffering action of Hsp90.
This week, in a letter to Nature, Valeria Specchia et al.2 report some fascinating evidence that indicates that beyond merely acting as a gate-keeper to unleash variation, mutations of Hsp90 that compromise its functionality result in new, rather than pre-exisiting, variation. They observed that mutations in Hsp90 affect the production of piRNAs. These are small RNA molecules that are involved in the silencing of genes, particularly those involved in development, i.e. sex cells like eggs and sperm, and all the cell types that give rise to these cells. These piRNAs are also responsible for repressing genetic elements called transposons.
FURTHER to my recent post on why people don’t accept evidence, it turns out that an editorial 1 and an opinion 2 piece in this week’s Nature, the latter unfortunately behind a pay-wall, actually focus on just this issue. The editorial states:
“Empirical evidence shows that people tend to react to reports on issues such as climate change according to their personal values (see page 296). Those who favour individualism over egalitarianism are more likely to reject evidence of climate change and calls to restrict emissions. And the messenger matters perhaps just as much as the message. People have more trust in experts — and scientists — when they sense that the speaker shares their values.”
So people tend to accept the evidence that supports their personal proclivities, and in fact interpret evidence in a manner than does so, thus people tend to persist in cherished beliefs and views even when confronted with contradictory evidence. This of course is something probably appreciated by most of us. Dan Kahan, in his opinion piece, points out:
“People endorse whichever position reinforces their connection to others with whom they share important commitments. As a result, public debate about science is strikingly polarized. The same groups who disagree on ‘cultural issues’ — abortion, same-sex marriage and school prayer — also disagree on whether climate change is real and on whether underground disposal of nuclear waste is safe.”
Another factor that weighs heavily in the public perception, and acceptance, of facts is the messenger. Owing to the fact that most people are ill-equipped to evaluate the raw data from scientific studies, they rely on the position of credible experts; it seems that those experts laypersons see as credible are those perceived to share the same values.
Research into the mental processes involved in such public perception is, Dan tells us, being conducted by Donald Braman at George Washington University Law School in Washington DC, Geoffrey Cohen at Stanford University in Palo Alto, California, John Gastil at the University of Washington in Seattle, Paul Slovic at the University of Oregon in Eugene and Dan Kahan, the Elizabeth K. Dollard professor of law at Yale Law School. These processes are collectively referred to as ‘cultural cognition’.
So what is cultural cognition? Kahan describes it as, ‘the influence of group values (ones relating to equality and authority, individualism and community) on risk perceptions and related beliefs.’ I would imagine that peer-pressure represents one example within a spectrum of influences in cultural cognition.