Surviving antibiotics…

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.

Further reading

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 ]

_Lee, H. et al. (2010) Bacterial charity work leads to population-wide resistance. Nature 467, 82-85._
[Sorry, article behind a paywall] – You can read Ed Yong’s post on it though.

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.

Bio suit…

HOW human do you think you are?

100% ?

90% ?

Well, let’s play a numbers game: in terms of cell numbers, you have in the order of a trillion cells in your body, though this value varies greatly between people and is constantly changing within each of us. However, you have some ten times this number of bacterial cells within (and on) your body 1. So at one level at least, you are only 10% human.

Of course, bacterial cells are quite a bit smaller than your cells, so there’s room for the both of you, in you.

bladder lactobacillus mouth1_scale_final
Images of human epithelial (tissue surface) cells coated with bacteria.

In terms of genes, the instructions that make you you, humans have about 30,000. Again, there are in the order of a hundred times this number of bacterial genes operating within and on your body 2. So at another level you are only 1% human.

Don’t worry though, of course you’re 100% human. Instead, we need to consider the extent of what being human actually is. Being human comes part and parcel with being a super-organism. We live in a symbiotic relationship with hundreds of different species of bacteria, without which we could not survive. Think of them as an invisible extension of your body’s innate defences, occupying every external surface, your skin, your gut, your eyes, ear, nose, and various other orifices.

There is mounting evidence to suggest that they influence our development; our physiology; our nutrition and metabolism; and immunity, where they play an important role from birth in educating our immune systems. They are your interactive suit of armour, both part of the environment and part of you. These communities of bacteria are referred to as the microbiome, and they are being investigated as part of the Human Microbiome Project, an effort by many research labs coordinated by the National Institute of Health.

Continue reading “Bio suit…”