Antimicrobial agents, particularly antibiotics, are estimated to increase life expectancy across the globe by around 20 years. They are a mainstay of modern medicine, used to treat a range of infections from urinary tract infections to life-threatening diseases like tuberculosis. These drugs are also vital for preventing infection, for example before and after operations or during cancer treatments.
The first antibiotics were discovered less than a century ago, yet resistance to these drugs amongst bacteria is already alarmingly prevalent and continues to rapidly spread. In fact, antimicrobial resistance is a major public health threat and failure to address the issue will have a drastic cost on lives and economies. By 2030, drug-resistant diseases could force up to 24 million people into poverty and, by 2050, are predicted to cause an additional 10 million deaths a year.
In order to understand this pandemic of antimicrobial resistance, we must first understand what antimicrobial agents are. In this article, we will explore the history of antimicrobials and the general mechanisms by which antibiotics kill bacteria.
Antimicrobials vs. antibiotics
First of all, what are antimicrobials and antibiotics, and what is the difference between the two?
The word ‘antimicrobials’ is derived from the Greek word anti (meaning “against”), mikros (“little”) and bios (“life”). As the name suggests, antimicrobials agents are simply natural or synthetic substances that act against microbes.
On the other hand, antibiotics are a group of antimicrobials which are technically defined as substances produced by one microorganism that selectively inhibits growth or kills another organism.
So basically, all antibiotics are antimicrobials, but not all antimicrobials are antibiotics. But don’t worry too much about these definitions because ‘antimicrobials’ and ‘antibiotics’ are generally used interchangeably.
A brief history of antimicrobials
The pre-antibiotic era
The idea of using certain compounds to treat diseases has been around for centuries.
Historical evidence indicates that natural treatments like herbs, honey and even animal faeces were used to treat infections. Remains of tetracycline, an antibiotic used today, have been found in 1,500-year-old human skeletons in Egypt; the most likely explanation for this is that people had somehow consumed tetracycline in their diet. Besides that, another traditional remedy used in Ancient Serbia, Greece, Egypt and China was pressing mouldy bread onto wounds. We now know that the mould would have contained antimicrobials, which were responsible for treating these infections.
These historical remedies had variable efficacy and safety and people using them had no knowledge of the microorganisms causing the diseases. This all changed in the second half of the 19th century with the development of the Germ Theory of disease.
The works of Antoine van Leeuwenhoek contributed significantly to the theory and earned him the title ‘Father of Microbiology’. Using some of the earliest microscopes, which he made himself, van Leeuwenhoek was one of the first to observe bacteria. Meanwhile, in 1861, Louis Pasteur published the Germ Theory, stating that germs, such as those seen by van Leeuwenhoek, were responsible for infectious disease. Robert Koch furthered this theory by identifying the aetiological agents responsible for causing significant human diseases such as tuberculosis, cholera and anthrax. Once the pathogen responsible for disease was determined, specific chemotherapies could be developed to target it.
In 1883, Rudolf Emmerich and Oscar Löw discovered pyocyanase, which became the first antibiotic drug to be used clinically. They found that green bacteria, which is now known to be Pseudomonas aeruginosa, could be isolated from bandages and used to inhibit the growth of other pathogens such as Bacillus anthracis (the causative agent of anthrax). However, various issues with the treatment - such as its associated toxicity - meant the use of pyocyanase was soon halted.
Not long after, Paul Ehrlich discovered Salvarsan, the first antimicrobial used to treat a specific disease - syphilis. At the time, this sexually transmitted disease, caused by Treponema pallidum, was widespread, incurable and had a relatively high mortality rate, so Salvarsan soon became the most widely prescribed drug across the globe.
The golden age of antibiotics
The antibacterial properties of mould had been harnessed by ancient civilisations for centuries, but it was never known what the exact compounds produced by the mould were that actually targeted the bacteria.
Sir Alexander Fleming’s ground-breaking discovery in 1928 solved this mystery. When growing Staphylococcus aureus on agar plates, his culture was contaminated with a blue mould (a fungus of the genus Penicillium). To Fleming’s surprise, he saw a ‘zone of inhibition’ surrounding the contaminating fungus. For some context, a zone of inhibition is an area on the media where bacterial growth is inhibited, so Fleming’s observation indicated that the mould was producing an antibacterial substance that was preventing the growth of S. aureus. He named the antibiotic ‘penicillin’ and his discovery eventually won him the Nobel Prize in 1945.
Check out Figure 1 for an illustration of what Fleming’s agar plate would’ve looked like.
Figure 1: An illustration representing Alexander Fleming’s original culture plate. A zone of inhibition of bacterial growth surrounding the contaminating Penicillium (fungus) colony can be seen due to the antibiotic compound it produced. Outside of this zone, normal staphylococcal colonies (Staphylococcus aureus) can grow. This illustration is based on a photograph taken by Fleming of his original culture plate, which can be found in his paper ‘On The Antibacterial Action Of Cultures Of A Penicillium, With Special Reference To Their Use In The Isolation Of B. influenzae’ (1929). The figure was made using BioRender.
The next few decades saw the discovery of numerous other antibiotic classes and was hailed as the golden age of antimicrobial chemotherapies. One of these classes was sulphonamides, of which the first member was discovered in the mid-1930s by Gerhard Domagk. The drug, known as Protonsil, significantly reduced the mortality of fatal diseases such as pneumonia and meningitis and saved the lives of many prominent figures including Winston Churchill.
Another new drug was streptomycin, which was isolated from a soil bacterial species called Streptomyces griseus in 1944. This discovery triggered a widespread search for other naturally occurring antibiotics produced by microorganisms that had therapeutic potential. For example, in 1952 vancomycin (a glycopeptide antibiotic) was obtained from Streptomyces orientalis in a soil sample from Borneo.
The era of antimicrobial resistance
In 1945, Fleming predicted that if penicillin wasn’t used appropriately, bacteria resistant to the antibiotic would emerge and spread.
Despite his caution, the antibiotic became widely available with very little concern for preventing resistance by controlling dosing and ensuring complete courses were taken. Just as Fleming had said, resistance quickly emerged following this intensive usage. Furthermore, the issue of antimicrobial resistance is not restricted to penicillin.
One way to treat infections by bacteria that are resistant to penicillin, or any other antibiotic, is to administer another type of antibiotics that the pathogen is susceptible too. However, since the golden era of antimicrobials, very few novel antibiotics are now being developed and pathogens are becoming resistant to many of the existing drugs.
Instead, the classes of antibiotics we already know about are growing, but the discovery of new classes is rare. In other words, most new antimicrobials are variations of pre-existing ones. Chemical modifications to antibiotics can be incredibly useful, expanding their antibacterial spectrum and overcoming particular mechanisms of resistance.
Let’s use penicillins as an example of this. The natural penicillin, penicillin G, discovered by Fleming, was only effective against gram-positive bacteria like S. aureus. Bacteria are classified as gram-positive or gram-negative based on the structure of their cell wall (have a read of our Shigella article for a more detailed explanation of this). S. aureus developed resistance by producing an enzyme called penicillinase, which in turn, degrades penicillin G and renders it ineffective.
In response to this, a second generation of penicillins was produced, known as anti-staphylococcal penicillins. These were not susceptible to penicillinase-mediated degradation. In addition, further generations of penicillins which target Gram-negative pathogens such as Pseudomonas aeruginosa and Salmonella have also been developed.
Check out Figure 2 for a summary of these generations of penicillin.
Figure 2: Generations of penicillin. Penicillins are all beta-lactam antibiotics as they all have a beta-lactam ring (highlighted in red). In addition, chemical modifications made at each generation are highlighted in different colours. This figure was adapted from ‘Penicillin’s Discovery and Antibiotic Resistance: Lessons for the Future?’,Lobanovska and Pilla (2017)’.
Classifying antibiotics
Antibiotics are commonly categorised based on their spectrum of activity, effect on bacteria and mechanism of action. Now you’re familiar with the background of antibiotics, we can look at how they work in a bit more detail by discussing these 3 groupings.
1. Spectrum of activity
An antibiotic can be described as broad or narrow spectrum depending on the range of bacterial species it is active against.
Broad spectrum antibiotics can inhibit a variety of bacteria and tend to be effective against gram-positive and gram-negative species. In contrast, narrow spectrum antibiotics are specific to a few particular species. For example, glycopeptides (such as vancomycin) only target gram-positive species. Alternatively, sulphonamides are specific to aerobic bacteria (these bacteria need oxygen to survive).
Typically, using a narrow-spectrum antibiotic is preferable as it will target the pathogen with minimal effect on non-pathogenic bacteria (for example, bacteria in the gut microbiota). However, rapidly and correctly diagnosing the bacteria causing the infection can be very challenging, so doctors often have no choice but to use broad-spectrum antibiotics instead.
2. Effect on bacteria
Depending on its mechanism of action, which we will discuss later, antibiotics can affect bacteria differently.
Firstly, antibiotics can be bactericidal, meaning they will kill the target microorganisms. Alternatively, bacteriostatic antibiotics inhibit the growth and replication of bacteria. These drugs keep the size of the pathogen population low enough, giving the immune system the chance to clear the rest itself.
In certain cases, some antibiotics can actually be bacteriostatic and bactericidal depending on the context of use (e.g. the dosage and length of treatment). For example, aminoglycosides and fluoroquinolones are concentration-dependent bacterial agents, meaning their rate of killing increases with their concentration. At lower concentrations these antibiotics are bacteriostatic but they can become bactericidal at higher concentrations.
3. Mechanism of action
Different classes of antibiotics can have different mechanisms of action, meaning they act on bacteria in various ways. This variation is generally due to differences in chemical structures and affinities to bacterial cell structures, such as the cell wall or nucleic acids.
On that note, let’s have a look at the five main mechanisms antibiotics use to kill bacteria or inhibit their growth. Also, have a look at Figure 3 for an illustration summarising all these mechanisms.
Inhibition of cell wall synthesis
Cell walls are present in (almost) all bacteria as they have a range of roles that are essential for survival. They provide structural integrity to help maintain the shape of the cell, act as a barrier to entry of certain molecules and prevent osmotic lysis. Osmotic lysis can occur as water moves in and out of cells by osmosis - without the cell wall, too much water in the cell can result in it bursting.
Given the importance of cell walls for bacteria alongside their absence in animal cells, antibiotics which inhibit the production of the cell wall will kill or inhibit the growth of bacteria, without harming our own cells. As a result, cell wall inhibition is the most common mechanism of action amongst antibiotics.
A well-known example of such drugs are beta-lactams; these are compounds which share a beta-lactam ring in their chemical structure, such as penicillins.
Alteration of cell membranes
Another vital cellular structure that is targeted by several antibiotics is the cell membrane. This membrane is an important barrier which controls the passage of molecules between the intracellular and extracellular environment.
Therefore, altering cell membranes damages this barrier, resulting in important molecules escaping from bacterial cells. Unlike bacterial cell walls, which are absent from our own cells, cell membranes are present in animal and bacterial cells. This means that antibiotics that target cell membranes can be toxic to our cells. For this reason, such antibiotics are only administered topically rather than systemically.
Inhibition of protein synthesis
In eukaryotic and prokaryotic cells, proteins have a wide range of functions ranging from enzymes to protein channels to receptors, and are critical for cell survival. Therefore, inhibiting protein synthesis is therefore another useful mechanism of action for antibiotics.
You may be wondering, if protein synthesis is so important for animal cells as well as bacterial ones, then drugs which target protein synthesis should be toxic to humans as well?
Fortunately, this is not the case as these antibiotics can selectively target bacteria. This is because eukaryotic and prokaryotic cells have different ribosomes (the machines which translate mRNA into a polypeptide).
The mass of ribosomes is measured using Svedberg (S) units. Eukaryotes have 80S ribosomes and are made up of 40S and 60S subunits (don’t worry, these values aren’t supposed to add up!). In contrast, prokaryotic ribosomes are 70S ribosomes that consist of a 30S and 50S subunit. Thus, antibiotics can selectively interfere with bacterial protein synthesis by binding to 30S or 50S subunits as these are absent in our own cells.
Inhibition of nucleic acid synthesis
You’re probably starting to see a pattern here - antibiotics tend to target essential processes and structures in bacterial cells. And preferably, the target is absent in eukaryotic cells.
Nucleic acid synthesis is another example of this, whereby antibiotics can target DNA replication or DNA transcription (when a cell’s DNA sequence is copied into RNA).
The enzymes which catalyse the processes are different in prokaryotes and eukaryotes, which prevents non-selective toxicity.
For example, quinolones target DNA gyrase, an enzyme which is absent from eukaryotic cells. Meanwhile, another antibiotic class that targets nucleic acid synthesis is rifamycin. It inhibits RNA polymerase in bacteria to block RNA synthesis. Because the RNA polymerase in eukaryotic and prokaryotic cells have substantial structural differences, rifamycin is specific to bacteria.
Inhibition of metabolic pathways
Other antibiotics can be ‘antimetabolites’, which selectively interfere with important biochemical processes in bacterial cells.
For example, sulphonamides, which we mentioned earlier as one of the first classes to be developed, and trimethoprim both interfere with the bacterial folic acid synthesis pathway by competitively inhibiting different target enzymes in the pathway. Folic acid is needed for nucleic acid synthesis so this mode of action has a bacteriostatic effect. On the other hand, humans aren’t susceptible as we get folic acids from our diet, whilst bacteria have no choice but to make it intracellularly.
Figure 3: Targets of common antibiotics. Different antibiotics have different targets, mechanisms of action and effects on bacteria. The diagram shows the major bacterial processes and components that are targeted by antibiotics. This figure was adapted from ‘Mechanisms of Antibacterial Drugs’, Lumen Learning and made using BioRender.
Why is this important?
By now, it should be clear to you that antibiotics are undoubtedly one of the most effective and important tools in modern medicine, as they have saved millions of lives and increased our life expectancy significantly.
Furthermore, perhaps more surprising is their use outside of human therapeutics. Antibiotic usage is actually greatest in farming where the drugs are used to treat and prevent infections and also as growth enhancers.
Consequently, understanding how antibiotics affect bacteria not only helps us to determine which antibiotics are most appropriate for different infections, but also suggests how bacteria may evolve resistance to the drug, as we will see in the next article.
As we know, antimicrobial resistance is a major public health threat, making research into antibiotics even more important - whether this is to prevent resistance occurring, enhance existing antibiotics or even develop and discover new ones.
Author
Ambar Khan
BSc Biological Sciences
Imperial College London
Disclaimer: All figures created using BioRender are intended solely for educational purposes and not for profit.