8: Antibiotic Discovery
- Page ID
- 124421
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Discovery of Antibiotics
Prior to the discovery of antibiotics, huge numbers of people died from bacterial infections. For example, during the American Civil War one third of the deaths were due to battle while two thirds died of infection. Treatment had improved by World War One, however, approximately one third of the fatalities were still due to infection. Scientists were actively looking for better antiseptics, cleaning solutions to prevent bacterial growth. However, most of these antiseptics were very corrosive and damaged the patient tissue while preventing the bacterial growth.
In 1928, Alexander Flemming returned from vacation to his messy lab and discovered a mold contaminant that had killed the neighboring staphylococci bacteria on one of his plates. There was a zone of inhibition or area of non-growth surrounding the mold contamination area. Fleming was intrigued by this observation and eventually showed that it was caused by a compound that he named penicillin after the fungus that produces it: Penicillium notatum.
To test the effectiveness of penicillin, Fleming tested it against several common pathogens. Penicillin inhibited the growth of Gram-positive bacteria associated with scarlet fever, pneumonia, gonorrhea, meningitis, and strep throat, while leaving eukaryotic cells unharmed (Brown, 2005). It exhibited the qualities of the miracle drug Fleming had been pursuing for years!
Work on penicillin lay dormant for ten years until scientists with extensive chemistry experience read Flemming's paper and used their background to purify the active compound and test it on infectious bacteria in patients (Amyes, 2001). This allowed penicillin to be mass-produced and administered orally and systematically to patients. Penicillin was commercialized in time to reach wounded soldiers in the battlefields of World War II, and significantly contributed to the Allied victory. The collaborative development of the first natural product antibiotic won Fleming, Florey, and Chain the Nobel Prize in Physiology or Medicine in 1945.
- Selectively toxic toward microbes
- Generally antibacterial
- Small molecules (secondary metabolites)
- May be a synthetic or a natural product
Antibiotics
Antibiotics describe any small molecule produced by a microbe that kills or inhibits the growth of other microbes (Clardy, Fischbach, & Currie, 2009). “Small molecules” refers to the fact that antibiotics are smaller than the macromolecules that constitute a cell. Antibiotics greatly vary in their chemical structure and those with similar structures are grouped into classes of antibiotics. New classes of antibiotics are even more challenging to discover than new antibiotics themselves. One common class of antibiotic is the β-lactams, all of which have a four-member ring structure. This core structure allows β-lactams like penicillin and penicillin-derived antibiotics to inhibit bacterial cell wall synthesis (Figure 1).
Figure 1. The β-lactam class of antibiotics, such as penicillin (1) and cephylosporin (2), have the important core of a four-member β-lactam ring (in red). This structure allows β -lactam antibiotics to inhibit bacterial cell wall synthesis. From: Fvasconcellos 19:02, 23 October 2007 (UTC), Public domain, via Wikimedia Commons
Strictly speaking, true antibiotics are naturally-occurring antimicrobial compounds including penicillin, streptomycin, and chloramphenicol, which were originally isolated from living microorganisms. Natural product antibiotics make up 60–80% of all antimicrobials and most of the rest have been developed synthetically based on the natural product versions. Interestingly, the genus of bacteria, Streptomyces, which have complex lifecycles and look like fungi on solid media are the source of ~60% of the clinical antibiotics used today.
Antibiotic Targets
Antibiotics that interrupt the same target in the bacterial cell are also typically classified by their mechanism of action. Antibiotics need to target features that are specific to bacterial cells and are either not present in eukaryotic cells or are different enough that the drug doesn't disrupt on the eukaryotic version. One common target absent in eukaryotic cells is the bacterial cell wall. This cell wall is made of peptidoglycan (a macromolecule containing amino acids and a complex sugar). This material surrounds the cell, provides structure to the cell, defines the cell shape and is tough enough to prevent the cell from changes in osmotic potential or mechanical force. Gram-positive bacteria have a thick peptidoglycan layer as the outer surface of the cell, while Gram-negative bacteria have only a thin layer of peptidoglycan positioned in between two phospholipid membranes.
Some common antibiotics, including penicillin and vancomycin, inhibit cell wall synthesis resulting in eventual cell lysis. Another common antibiotic target is the bacterial ribosome which completes the process of translation. Although both bacteria and eukaryotic cells both do translation using a very similar process, the physical machinery of the ribosome is different enough that it can be targeted specifically. The antibiotics erythromycin and streptomycin specifically bind to the bacterial ribosome and prevent it from accurately building proteins. In a similar way, the antibiotic ciprofloxacin binds to the bacterial DNA gyrase enzyme involved in DNA replication preventing it's function while leaving the eukaryotic DNA gyrase unharmed. Similarly, rifampin inhibits RNA polymerase, which is necessary for the process of transcription.
Figure 2. Schematic of antibiotic cellular targets (modes of action) and major classes of antibiotics (and examples of drugs). From OpenStax Microbiology
However, many bacteria have evolved mechanisms to render these antibiotics useless. There are two common mechanisms by which bacteria can acquire resistance to an antibiotic:
1. Bacteria may acquire a new resistance gene as the result of a random mutation, or
2. Bacteria pick up a preexisting resistance gene passed to them from other bacteria in the environment.
This rapid increase in antibiotic resistance is a major threat to human health. Unfortunately, due to evolution, the development of resistance is inevitable, but we can change our behavior to slow the rate while simultaneously developing new antibiotics.
- Cellular target (e.g., the bacterial cell wall) is altered in the resistant population so that antibiotics that would normally target them are no longer able to bind properly (e.g., vancomycin).
- Antibiotics may be actively secreted out of the resistant bacterium to prevent them from reaching their target (e.g., tetracyclines).
- The resistant bacterium may block the antibiotic entry into the cytoplasm (e.g., antibiotics).
- The antibiotic may be modified or destroyed by the resistant bacterium (e.g., β-lactams).
Protocols
Finding New Antibiotics
You have isolated a collection of soil bacteria with the hopes that some of them will produce antibiotics and now it is time to test them to see if they do indeed produce antibiotics. You will be conducting an assay (aka test) to identify which of your bacteria are antibiotic producers.
Before doing this, it is critical that you make a copy of your master plate so that you have an uncontaminated sample of each of your bacteria.
Protocol 1: Replicate Your Master Plate
To keep your bacterial isolates happy and healthy, you need to feed them regularly. To do that, the first thing that you will do today is to replicate your master plate by picking up a very small amount of bacteria from each patch and transferring them to the same spot on a new sterile
master plate. Make sure to note the phenotype for each colony in your lab notebook (use the same colony descriptors as last week). Also be sure to note any contamination or if you have two different appearances within one patch. Use sterile technique to be sure to keep everything sterile.
- Media plates of choice
- Master plates from last week
- Square grid
- Sterile toothpicks
Protocol
- Obtain your master plate(s) and as many new sterile plates as you already have for your master(s).
- Label your new plates with the same information as last week:
- Group Name
- Media Type
- Temperature
- Plate Number
- and mark the orientation on your plate
- Line up the square grid with your new plate.
- Using one sterile toothpick, pick up a VERY small amount of the patch. Just touch the patch, you don’t need to see the bacteria on the toothpick and you’ll still have thousands!
- Touch it to the new plate (in the same spot as the first patch) and color in the square being sure not to contaminate between two neighbors.
- Choose the temperature you wish to grow at, record your decision in your notebook, and place your plates upside down at that temperature (food side up).
Protocol 2: Spread/Patch Screen for Isolate Antibiotic Production
It is possible to make modifications to this suggested protocol (your imagination is the limit), be sure to accurately report your techniques in your lab notebook.
Theory
The spread-patch protocol assays for bioactivity in bacteria that are in close physical contact with the tester strain. In this protocol, the tester strain is spread on a plate, dried, and then the isolates are patched on top the spread. If the isolate is active against the tester strain, it should theoretically have no trouble growing on the spread. This protocol assumes that microbe-microbe interactions may induce antibiotic production and will increase our chances of identifying a producer. One challenge you may run into is that an antibiotic producer may need time to establish itself on the medium or may not be successful at invading a growing culture of susceptible bacteria.
Figure 3. Spread-patch schematic. Isolates are patched (in the same grid arrangement as master plate) onto an agar plate that has been spread with the ESKAPE pathogen.
This assay is an elegant way to find antibiotic producers as they seem to appear overnight. This approach takes advantage of the fact that microbes are constantly interacting with each other in the environment. For a long time, scientists have been working to find the signals that bacteria use to comunicate and identify their neighbors. Because these bacteria are so close together, they can directly compete which will hopefully result in one of your isolates producing antibiotics.
- Media plates (same as master plate)
- Liquid culture of safe ESKAPE relative
- Master plates with isolates
- Cotton swabs
- Square grid
- Sterile toothpicks
Protocol
- Obtain your master plate and the cultures containing your safe ESKAPE relative of choice. I recommend choosing at least one Gram positive and one Gram negative ESKAPE relative to test against. This will increase your chances of finding an antibiotic producer.
- Based on the number of patches you have successfully growing on your Master plate, determine how many fresh medium plates you need to test all of your strains.
- Label each plate with:
- Group Name
- Date
- Media Type
- ESKAPE relative used
- Incubation Temperature
- Plate Number
- Obtain a liquid culture of your safe ESKAPE relative.
- Dip your cotton swab into the liquid culture and swipe the swab along the edge of the tube to prevent dripping and remove extra liquid.
- Completely but gently (don't rip the plate) color over the surface of the plate to spread the ESKAPE pathogen over the whole surface. Turn the plate 90 degrees and recolor with the same swab completely. Finally, turn the plate an additional 45 degrees and color for a third and final time. This will ensure that you've gotten the ESKAPE pathogen onto every surface on the plate evenly. The liquid should be absorbed into medium within minutes.
- Wait for the plate to dry 10–15 minutes.
- Place the dry plate spread with your ESKAPE relative face-up on top of the grid and align line of orientation with grid.
- Pick isolates from the master plate and patch onto safe ESKAPE relative spread arranged in the same orientation as on the as master plate.
- Place in incubator at the temperature of your choice and allow them to grow for one week.
When you are finished patching your samples, please put your new plates food side up at the appropriate temperature. You should have 1-2 newly made duplicate master plates, and at least 2 antibiotic production test plates.
Stack your initial master plates from last week and put them in a plate bag with your dilution plates that have been stored from last week. Label the correctly colored tape with your group name and the date and use this to seal your bags. These plates will be stored in the fridge in case you need them next week. The used sticks should go in the appropriate container to be sterilized for re-use. Any tubes, tips, and cotton swabs that you used can be thrown in the red biohazard bin. Please put the ESKAPE pathogen tubes in the labeled area and return all reagents and tools to the place that you got them.
Using the 70% ethanol sprayers on your bench, spray down your bench space to disinfect it and wipe it clean with a paper towel
References
Amyes, S. G. B. (2001). Magic Bullets, Lost Horizons: The Rise and Fall of Antibiotics. New York: Taylor & Francis.
Brown, K. (2005). Penicillin Man: Alexander Fleming and the Antibiotic Revolution. Stroud, Gloucestershire: Sutton.
Clardy, J., Fischbach, M. A., & Currie, C. R. (2009). The natural history of antibiotics. Curr Biol, 19(11):R437–R441. doi: 10.1016/j.cub.2009.04.001.
D'Costa, V. M., Griffiths, E., & Wright, G. D. (2007). Expanding the soil antibiotic resistome: exploring environmental diversity. Curr Opin Microbiol, 10(5):481–489. doi: 10.1016/j.mib.2007.08.009
Hernandez, S., Tsang, T., Bascom-Slack, C., Broderick, N., & Handelsman, J. (2018). Tiny Earth: A Research Guide for Student Sourcing Antibiotic Discovery. XanEdu Publishing Inc. Print
Linares, J. F., Gustafsson, I., Baquero, F., & Martinez, J. L. (2006). Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A, 103(51):19484–19489. doi: 10.1073/ pnas.0608949103
Parker, N., Schneegurt, M., Tu, A.-H. T., Forster, B. M., & Lister, P. (2018). Microbiology. OpenStax.
Slonczewski, J., & Foster, J. W. (2011). Microbiology: An Evolving Science (2nd ed.). New York: W.W. Norton & Co.
Watve, M. G., Tickoo, R., Jog, M. M., & Bhole, B. D. (2001). How many antibiotics are produced by the genus Streptomyces? Arch Microbiol, 176(5):386–390. doi: 10.1007/s002030100345