Imagine a world without antibiotics, where any bacterial infection is potentially life threatening. The World Health Organization (WHO) declared in a report in 2014 (1) that a post-antibiotic era, “in which common infections and minor injuries can kill,” could start this century. A sobering thought.
In the golden age of antibiotic discovery, from the introduction of penicillin in 1940 until about 1960, a fertile source of new antibiotics was soil bacteria. However, only a tiny fraction of the wild bacteria can be easily cultured in the laboratory. These were soon examined for antibiotic compounds. The number of new antibiotic structures that were being discovered dropped off. In the 1980’s a new approach was tried: large scale screen of libraries of chemical compounds. This approach had worked well for general drug discover but didn’t work as well for the discovery of antibiotics. Now scientists are renewing attempts to isolate and study the bacteria that do not grow well in laboratory cultures (in vitro). A study published in January 2015 in the journal Nature (2, see also 3) describes the isolation and characterization of a new antibiotic, teixobactin using a newly developed technology that allows the cultivation of soil bacteria in their natural environments.
Why is this important?
As antibiotics are used in humans, in animals, and in food, the targeted bacteria begin to develop resistance. The scientific and medical communities have been unable to devise strategies to halt the development and spread of resistant strains of bacteria. A well known example of an antibiotic-resistant bacterium is methicillin-resistant Staphylococcus aureus (MRSA), which is any strain of S. aureus that has developed resistance to the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins. Together the penicillins and the cephalosporins are known as the β-lactam antibiotics because they share a common β-lactam chemical structure.
Another well know example is drug-resistant tuberculosis. Multidrug-resistant tuberculosis (MDR TB) is defined as any strain that is resistant to the two most effective TB drugs, rifampin and isoniazid. Extremely drug-resistant tuberculosis (XDR TB) consists of strains that are resistant to the two drugs just mentioned plus fluoroquinolone and one of three injectable drugs (amikacin, kanamycin, or capreomycin).
The rise of drug resistant bacteria and the dearth of new antibiotics are behind the World Health Organization’s warning of the coming of a post-antibiotic era. The recent report by Ling et al. (2) approaches the problem by expanding the search for new antibiotics to bacteria that heretofore had been difficult or impossible to culture.
What allowed this work to be successful?
The authors describe the use of a device, the isolation chip, or ichip, that allows bacteria to be isolated and cultured in their native environments. This device was described in an earlier publication (4), which is available free in PubMed Central (PMC), link below. The ichip is made of Delrin. It has a central plate containing 384 holes of 1 mm diameter. This plate is dipped into a bacterial suspension that is diluted so that approximately one bacterial cell is expected per hole. The holes are sealed with four 0.03-µm-pore-size, 47-mm polycarbonate membranes. Top and bottom plates with matching holes are attached with screws and hold the membranes in place. There are drawings of the ichip in (3,4) and there is a photograph which allows a good appreciation for the size of the device in (5). The assembled ichip is placed back into the environment from which the bacteria were originally isolated. The idea is that being in the natural environment allows the bacteria access to growth factors and nutrients that would be missing in an in vitro culture plate.
It was shown earlier (4) that the ichip increases the recovery of bacteria by at least five-fold over the use of petri dishes and results in greater phylogenic diversity in the bacteria so isolated. In the ichip 40% of the bacteria from seawater formed colonies and 50% from soil. Part of the success of this paper stems from this novel approach.
An interesting aspect of the ichip is that bacteria cultivated for one or more rounds in the ichip are much more likely to be able to grow in culture dishes (4). Twenty-six percent of the ichip-reared colonies domesticated after the first round and 40% after the second. The nature of this bacterial domestication is not understood (4).
What was shown in the study?
Ten thousand bacterial colonies were isolated from the ichip. Extracts from these colonies were tested for antibacterial activity against Staphylococcus aureus, which is a common gram-positive bacterium of the skin. The drug resistant strain, methicillin-resistant Staphylococcus aureus (MRSA), is the bacteria causing trouble in hospitals and clinics.
A new species of bacteria, named Eleftheria terrae, produced extracts that gave large clearing zones on lawns of growing S. aureus. The antibiotic teixobactin (the structure is in the featured image) was isolated from the extracts and shown to be active in vitro against a variety of pathogenic gram-positive bacteria. The mode of action of teixobactin was shown to be binding to lipid II, a precursor of pepdidoglycan (part of the cell wall) and lipid III, a precursor of the teichoic acid found in the cell wall.
Teixobactin was also shown to have good activity in vivo. In one study mice were infected intraperitoneally (bacteria were injected into the body cavity) as a model of septicemia with a dose of methicillin-resistant Staphylococcus aureus (MSRA) that leads to 90% death. Teixobactin, 1 to 20 mg per kg, was administered i.v. (intravenously) one hour later. All of the mice lived. In another experiment, the dose of teixobactin at which half of the mice live, the PD50 (protective dose 50%), was found to be 0.2 mg/kg. The PD50 of the main antibiotic used to treat MRSA, vancomycin, is 2.75 mg/kg.
The authors looked for the development of resistant mutants by growing S. aureus in the presence of concentrations of teixobactin that would not kill all of the cells. The cells that grew in the second highest concentrations of teixobactin were diluted into fresh media. The cells were passaged every day for thirty days. At the end of the study no resistant mutants had developed. The lack of development of resistance was attributed to the mode of action of teixobactin, which attacks multiple nonprotein targets. Antimicrobials that attack proteins are susceptable to the development of resistance by relatively simple changes in the amino acid sequence of the protein. Such changes usually affect the binding of the drug without significantly affecting the function of the protein. The attack of multiple targets helps prevent resistance since more than one change is required to allow resistance to develop. Vancomycin binds lipid II and it took thirty years for resistance to appear after its clinical introduction.
What implications does this work have for the future?
The ichip technology provides a relatively easy way to pan for rare, hard-to-culture bacteria that might produce novel antimicrobials. Although the present paper provides an example of a gram-negative bacteria producing an antibiotic against a gram-positive bacteria, it would be possible and desirable to run the screen in reverse and look for a antimicrobials against gram-negative bacteria.
The lack of development of resistance is an interesting aspect of the study. It might be expected that evolution would favor the emergence of antimicrobials with killing mechanisms that are difficult for the target organisms to avoid by mutation. However, you might also expect evolution to select for organisms immune to any killing mechanism that develops. After all, it’s every microbe for itself in the wild. Resistance could ultimately arise in pathogenic bacteria by horizontal gene transfer from organisms that had managed to develop resistance in the wild, but, as in the case of vancomycin, this might take years or decades. The work described by Ling et al. certainly brings a glimmer of hope to the war on the bacteria.
References
- WHO (2014), Antimicrobial Resistance Global Report on Surveillance. Full text.
- Ling, L. L., Schneider, T., Peoples, A. J., Spoering, A. L., Engels, I., Conlon, B. P., Mueller, A., Schäberle, T. F., Hughes, D. E., Epstein, S., Jones, M, Lazarides, L, Steadman, V. A., Cohen, D. R., Felix, C. R., Fetterman, K. A., Millett, W. P., Nitti, A. G., Zullo, A. M, Chen, C., Lewis, K. (2015) A new antibiotic kills pathogens without detectable resistance. Nature 517:455-459 doi: 10.1038/nature14098 Link.
- Wright, G. (2015) An irresistible newcomer. Nature 517 : 442-443 doi: 10.1038/nature14193
- Nichols, D., Cahoon, N., Trakhtenberg, E. M., Pham, L., Mehta, A., Belanger, A., Kanigan, T., Lewis, K., and Epstein, S. S. (2010). Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species. Appl. Environ. Microbiol., 76(8), 2445–2450. doi:10.1128/AEM.01754-09 Full text.
- Ledford, H. (2015) Promising antibiotic discovered in microbial ‘dark matter’. Nature News Nature :doi: 10.1038/nature.2015.16675 Link