The previous post mentioned the onset of antibiotic resistance as one of the scarier, if less sensationalist, outbreaks of our time. We generally discuss antibiotic resistance in medically relevant microbes (on this blog and in the news), where resistance means the ability of disease-causing microbes to grow in the presence of a drug that previously prevented, or at least slowed microbial reproduction.
Microbes (and in the case we're discussing, bacteria) have different strategies to resist drug action. Some mechanisms are inherently “selfish,” in that they only help the cell with that particular gene to escape drug action: think of an efflux pump or a mutation in the drug target. Other mechanisms are more “social,” in that they can help additional, nearby cells that might not themselves contain the necessary gene: think of a secreted enzyme that can degrade the drug. A new research paper published in Antimicrobial Agents and Chemotherapy shows selection for selfish resistance can occur at a lower drug concentration than selection for social resistance.
Many mBiosphere readers with cloning experience may be familiar with social resistance. If you’ve used ampicillin resistance to select for bacterial plasmids, you’ve likely seen small, slow-growing colonies (satellite colonies) surrounding the larger, faster-growing ones – these satellite colonies don’t have the necessary b-lactamase-encoding plasmid, but can grow because the large colonies are secreting this enzyme into the surrounding medium (see example of satellite colonies almost grown to a lawn, right). This is why some researchers prefer a more stringent selection method, such as tetracycline (or b-lactamase-resistant carbenicillin), in which it’s harder to get selection artifacts.
The research team, lead by Dr. Michael Brockhurst of the University of York, hypothesized that sociality of drug resistance would alter the concentration of drug needed to select for a particular mechanism. To test this, they looked at selection conditions for the RK2 plasmid, which encodes both a social resistance mechanism (b-lactamase for ampicillin) and a selfish resistance mechanism (efflux pump for tetracycline), expressed in E. coli.
In the absence of antibiotics, the plasmid-containing bacteria decreased E. coli fitness, slowing the plasmid-containing bacterial growth relative to plasmid-free. Replicating, transcribing, and translating plasmid DNA costs the cells energy that would otherwise go toward cell division, so it makes sense that the plasmid-containing cells grow slower. The researchers determined the minimum inhibitor concentration (MIC) for each drug, and then asked at what drug concentration was the growth equal between plasmid-containing and plasmid-free cells.
Ampicillin resistance was positively selected at drug concentrations higher than the minimum inhibitory concentration (MIC) of sensitive E. coli. This could be due to the invasion of “cheater” cells – those that have lost the plasmid, but are able to grow because the drug concentration is lowered due to enzyme produced by neighboring cells (see figure, right).
Contrasting those results were the findings that tetracycline resistance was positively selected at drug concentrations nearly 1/100th the MIC of sensitive E. coli. This means at a very low drug concentration, plasmid-containing cells replicate as quickly or quicker than plasmid-free cells, even though the latter are still able to grow at this drug concentration.
What about microbes that have acquired multiple means of resistance toward the same drug? The researchers tested ampicillin and tetracycline in combination, and found there was no significant change in the selective concentration. So in bacteria containing multiple drug-resistant genes, a problem seen in multidrug resistant bacterial infections, the selection will be based on the gene requiring lower drug concentration.
Resistance selection is often associated with the use of high drug concentrations (sometimes unnecessarily), which confers benefit to the bacteria containing the resistance gene. The researchers here showed that even an extremely low concentration, much lower than the inhibitory concentration, can select for the presence social antimicrobial resistance genes. This may explain the common presence of resistance mechanisms, which, after all, originated well before the use of antibiotics in medicine.
A drug like penicillin is naturally produced by the saprophytic fungus Penicillium. Penicillin production benefits the fungus by clearing out a niche in its environmental milieu. Nearby bacteria have a better chance at reproduction if they have an antimicrobial resistance gene, but because the soil has both a low microbial concentration (106- 107 per gram of soil) and a very heterogeneous mix of microbes, the bacteria are unlikely to experience exposure to drug concentrations nearly as high as those used in modern medicine. The penicillin concentration in soil is very low, similar to the concentration shown by the authors here to select for tetracycline (selfish) resistance.
Finally, how can we use this information to better guide the decision to use antibiotics in the clinic? The findings suggest that even very low concentrations of antibiotic drug – concentrations that are often found in water – will select for selfish resistance genes, and social genes can ride along with the selfish genes too. The best we can do is to continue to use antibiotics only when we know they’re needed (e.g., not for the common cold). Stay tuned to mBiosphere to learn more research on differentiating proper antibiotic use!
-- Julie Wolf