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.
After growing cells in the presence of low concentrations of antibiotic for five days (either norfloxacin, ampicillin or kanamycin – each representing an antibiotic with a different primary cellular target), they periodically tested samples for their resistance to a range of the same, and different classes, of antibiotics. The aim was to determine whether treatment with one antibiotic could confer cross-resistance resistance to other antibiotics.
Whilst antibiotic resistance mechanisms against one drug can often confer resistance to different molecule of the same class, development of resistance to a drug molecule in a different class is less likely, and would be an important finding.
The figure (left) produced for the accompanying preview article, by Ben Kaufmann and Deborah Hung 5, summarises the findings. The researchers found that true enough, growth in one of the antibiotics – most notably ampicillin – resulted in cross-resistance to a number of the other antibiotics. In contrast, when grown in the absence of any antibiotics, only spontaneous mutants could be expected when then exposed to high levels of antibiotic. Similar findings were observed with an S. aureus strain and a clinical E. coli strain, so were not just an artefact of laboratory E. coli strains, which can be poor models for in situ clinical infection.
Interestingly, in some cases the ampicillin-treated isolates that were resistant to another drug actually remained sensitive to ampicillin, indicating that sub-lethal antibiotic exposure results in a random mutation process, rather than selective.
Those cells newly resistant to ampicillin did not revert if grown for several days in the absence of the antibiotic, so represent a stable mutation rather than transient adaptation to growth in the presence of ampicillin. The positions of the mutations conferring cross-resistance were found in the expected genes of drug targets, comparable to those documented in other clinical bacteria, rather than by mutation of a general efflux pump capable of pumping out multiple drugs. However, they did identify one isolate with a mutation in the acrAB gene, a multidrug efflux pump that is linked with ROS-responses, and they propose that the mutation may contribute to multidrug resistance; they haven’t discussed this further in this paper.
Of course, the proposition is that the development of such mutants is mediated by the hydroxyl radical causing damage to the cell DNA. If the same experiments were performed in the absence of oxygen, it would be expected that no resistance to antibiotic would be observed, which was indeed the case. Whether or not it is the hydroxyl radical that directly mediates the mutation to the genes encoding the targets of the antibiotics is yet to be unequivocally demonstrated. It may be that DNA damaged by hydroxyl radicals initiates other cellular processes, such as the bacterial SOS response, which is known to repair DNA at the cost of introducing mutations and recombination.
In this case, the proposed mechanism may act as perhaps an alternative means of generating the necessary genetic variation needed to dig themselves out of a tight spot. Indeed, in bacterial populations there may also be naturally occurring mutator cells, which can occupy anywhere between 0.5 – 30% of the cell population. These cells are usually defective in DNA repair, and may undergo a 1000-fold increased rate of mutation compared with the background population 7.
The broader implications of the study are pertinent as current recommendations for antimicrobial chemotherapy is, ideally, to use cocktail antibiotics to preclude evolution of resistance that can result from monotherapies; though whether or not this is widely practised is another matter. Furthermore, the incubation of these strains in sub-lethal concentrations of antibiotic is relevant in clinical practice where bacteria can commonly experience such sub-lethal drug concentrations. The dosing regime for antibiotics means that the concentration of the drug will periodically drop to a low level for short periods, or longer if there is poor patient compliance with the regime instructions. Furthermore, some areas of the body are poorly supplied with drug, receiving less than the plasma concentrations, e.g. skin, joints and prostate. Such conditions may permit the evolution of cross-resistance as described in this work.
There remains a great deal of work to do in this area however. First and foremost, it will be important to determine the clinical relevance of the hydroxyl radical mechanism in situ. One of the drugs the researchers tested, kanamycin, is an aminoglycoside; another study has previously shown that the sub-lethal levels of another aminoglycoside, tobramycin, induces biofilm formation 6. For both Gram-negative and Gram-positive bacteria, subinhibitory antibiotic treatment can stimulate production of exopolysaccharides necessary for biofilm formation – and the significance of biofilms is such that it protects cells from lethal levels of antibiotics, but the low oxygen environment may make hydroxyl radicals less significant.
Furthermore, the phase of growth will be important. Does the mechanism suggested work as well on stationary cells as it does on actively growing cells. It is well known that bacteria can become ‘indifferent’ to bactericidal drugs, simply because current drugs are designed to target processes that are active only in growing cells, rather than non-growing cells. This, in fact, will be the subject of my next post.
Research on antimicrobials since the 1960’s has focussed on identifying the mechanisms by which bacteria can physically modify a drug’s structure, disrupt the interaction of drug and target, or alter the activity of transport machinery that keeps the drug away from its target. An increased research focus to characterise the downstream physiological responses of bacteria to sub-lethal, as well as lethal, levels of antibiotics may help provide new impetus to inform future drug dsicovery 8.
1. Kohanski et al. (2007) A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell 130: 797-810. DOI: 10.1016/j.cell.2007.06.049.
2. Hassett & Imlay (2007) Bactericidal Antibiotics and Oxidative Stress: A Radical Proposal. ACS Chem. Biol. 2: 708–710. DOI: 10.1021/cb700232k.
3. Kohanski et al. (2008) Mistranslation of Membrane Proteins and Two-Component System Activation Trigger Antibiotic-Mediated Cell Death. Cell 135: 679-690. DOI: 10.1016/j.cell.2008.09.038.
*4. Kohanski, M., DePristo, M., & Collins, J. (2010). Sublethal Antibiotic Treatment Leads to Multidrug Resistance via Radical-Induced Mutagenesis Molecular Cell, 37 (3), 311-320 DOI: 10.1016/j.molcel.2010.01.003
5. Kaufmann & Hung (2010) The Fast Track to Multidrug Resistance. Molecular Cell 37: 297-298. DOI: 10.1016/j.molcel.2010.01.027.
6. Hoffman et al. (2005) Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436: 1171-1175. DOI: 10.1038/nature03912.
7. Chopra et al. (2003). The role of mutators in the emergence of antibiotic-resistant bacteria. Drug Resistance Updates 6: 137-145. DOI: 10.1016/S1368-7646(03)00041-4.
8.Dwyer et al. (2009) Role of reactive oxygen species in antibiotic action and resistance. Current Opinion in Microbiology 12: 482-489. DOI: 10.1016/j.mib.2009.06.018.