Since they were first discovered less than a century ago, antibiotics have undoubtedly revolutionised modern medicine, increasing the average human life expectancy by about 20 years.
Unfortunately, the potential of these miracle drugs was soon overshadowed by the realisation that bacteria can quickly become resistant to them and spread their resistant traits to other bacteria. The resulting pandemic of antimicrobial resistance is a serious public health threat, and failure to address the issue does and will continue to, 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 this article, we will explore the basics of antibiotic resistance in bacteria, from the mechanisms underlying resistance to the factors contributing to its increase.
Evolution of a killer
Let’s start with defining resistance: antimicrobial resistance is defined as the ability of a microorganism to survive and replicate in the presence of an antimicrobial agent that would normally kill or inhibit it.
Although antimicrobials and resistance are both relatively new to us, they are by no means new in nature. Many microorganisms produce substances that inhibit or kill competitors, whilst susceptible microbes continue to evolve resistance so they can survive in the presence of these antimicrobials. However, human’s heavy usage of these antibiotics has significantly increased the rate at which resistance evolves.
With this in mind, Sir Alexander Fleming warned that resistance to penicillin, which he had recently discovered, would emerge and quickly spread if it wasn’t used appropriately. His warning was not heeded and as soon as the drug became widely available, resistance quickly followed. This story is a common one - a similar pattern of resistance emerging soon after a drug is introduced has been seen with methicillin, vancomycin, streptomycin and many more.
Where we have gone wrong
With regards to human antibiotic usage, there are several key factors that are currently increasing the rate at which resistance evolves.
Antibiotic overuse
This is a particular issue because of agriculture's reliance on antibiotics. In fact, in the US the majority of antibiotics are given to animals, mainly for growth promotion.
Antibiotic misuse
Another factor is antibiotic misuse, for example, if a patient is prescribed antibiotics for the flu (this is actually caused by viral pathogens, so antibiotics will be ineffective).
Subtherapeutic dosing
This phenomenon occurs when antibiotic dosing is either too low or too far apart in time to exert its effect, which gives time for the pathogenic bacteria to mutate.
Incomplete treatment
Patients not completing their prescribed treatment course is also a common issue. Similar to subtherapeutic dosing, the resting bacterial population can adapt to their environment and become resistant.
Poor regulation
Even with all these social factors contributing to the rise of bacterial strains resistant to antibiotics, many countries selling the drugs over the counter and online.
Furthermore, once a bacterial pathogen becomes resistant to the standard antibiotic used to treat it, the problem doesn’t stop there. Doctors will have no choice but to turn to second-line antibiotics, which are used when the main antibiotic treatment does not work, and will also eventually become useless. This process repeats itself, giving rise to strains that gradually become resistant to more and more of the drugs in our arsenal. In the end, we are left with important human pathogens which cause serious diseases in humans but are essentially untreatable. For example, we are seeing the emergence of Mycobacterium tuberculosis strains (the causative agent of tuberculosis) which are resistant to first and even second-line antibiotics.
Not all resistance is created equal
Antimicrobial resistance is a very broad term that can be broken down into a few groups, based on a variety of factors, some of which we will discuss in the rest of the article.
First, we will look at how resistance can be an innate or adaptive feature of bacteria. Then we will explore the main general mechanisms which enable resistant bacteria to survive in the presence of an antimicrobial.
1. Intrinsic resistance
Intrinsic resistance is an innate feature of a microorganism and is a result of its structural or functional characteristics. Basically, the microorganism has never been susceptible to the antimicrobial but is just naturally resistant.
There are a variety of ways in which a microorganism can be intrinsically resistant to a certain antimicrobial substance. For example, Gram-negative bacteria have an outer membrane in addition to their cytoplasmic (inner) membrane. The outer membrane is impermeable to certain antibiotics, such as vancomycin, so they can’t enter the cell. These bacteria are therefore intrinsically resistant to such antibiotics. In contrast, Gram-positive bacteria like Staphylococcus aureus are susceptible to these antibiotics as they lack the outer membrane barrier.
Alternatively, intrinsic resistance can be due to the absence of an antibiotic’s target, the presence of exporters, membrane proteins that have the ability to pump out the drug or enzymes which degrade it. We will discuss these mechanisms more later on.
2. Acquired resistance
When a microorganism is initially susceptible to an antimicrobial, but then develops resistance, the resistance is described as acquired. Whilst intrinsic resistance is a feature of all members of a particular bacterial species, acquired resistance is only present in a subpopulation of a species.
Acquired resistance is possible because of the impressive genetic plasticity of bacteria, that is, the high variance phenotypic variance within individuals of the same strain. This allows them to quickly respond to potentially threatening environmental conditions, such as antibiotic treatment, by mutations that can take place in their DNA. Bacteria can use two main genetic strategies to respond to antibiotics or other stressful conditions.
The first is spontaneous mutations, something you are probably quite familiar with as they can occur in eukaryotes too. These occur randomly when a cell’s DNA polymerase makes a mistake during DNA replication. These mutations are often neutral, meaning they have no effect on the cell’s survival. However, occasionally a mutation can be deleterious or beneficial. In the presence of an antimicrobial compound, a mutation that reduces susceptibility to the compound would be advantageous.
The second strategy is horizontal gene transfer, a process that is much more common in prokaryotes than eukaryotes. Horizontal gene transfer occurs when a bacterium acquires foreign DNA from another bacterium. This foreign DNA can encode useful traits, such as resistance to antibiotics.
Whichever method is used, resistant bacteria will be selected under the selective pressure of an antibiotic, just as Darwin predicted in his Theory of Natural Selection. Imagine a population of susceptible bacteria which will be killed in the presence of an antibiotic like penicillin. When antibiotics are administered, they act as a selection pressure. Now, if a subset of the bacteria acquires resistance via mutations or horizontal gene transfer, they will be more likely to survive and reproduce, passing their resistance onto offspring. These ‘fitter’ bacteria are described as having a selective advantage over the susceptible members. As long as the selection pressure remains (antibiotic usage continues), resistant bacteria will continue to be selected for and their frequency in the population will increase. Check out Figure 1 for an illustration of this process.
Figure 1: How natural selection leads to the rise of antibiotic resistance. This figure was adapted from the CDC’s ‘How Antibiotic Resistance Happens’ and made using BioRender.
3. Adaptive resistance
The final type of resistance is adaptive resistance, which, unlike intrinsic and acquired resistance, is a transient state, that is, it is temporarily acquired by changes in gene expression and not mutations at the genetic level. It occurs when a bacterium is exposed to an environmental stimulus such as antibiotics or altered nutrient availability and changes so it is temporarily better able to deal with the condition. This type of resistance is poorly explored compared to the first two.
How bacteria fight back
So now you are familiar with the different types of resistance, let’s look at the main strategies (illustrated in Figure 2) used by bacteria to evade an antibiotic’s mechanism of action. These strategies can be responsible for intrinsic, acquired or adaptive resistance.
It’s important to note that in almost all cases, it is not one single strategy that confers resistance to a microorganism but rather a combination of several of them.
1. Preventing the antimicrobial compound from accessing its target
There are several ways different antibiotic compounds can enter bacterial cells, which is essential for drugs to carry out their functions as most have intracellular targets. For example, antibiotics can diffuse through the cell’s membrane bilayer. This membrane is selectively permeable, meaning it can prevent harmful molecules from entering the cell. However, the membrane needs to allow nutrients to enter the cell if it is to survive. For example, gram-negative bacteria have porins (small protein channels) embedded in their outer membrane which nutrients can diffuse through. These channels have size-exclusion limits so large antibiotic molecules can’t enter through them. This mechanism is responsible for the intrinsic resistance of gram-negative bacteria to certain antibiotics such as vancomycin.
However, some smaller antibiotics, such as beta-lactams, can still diffuse through porins and kill gram-negative pathogens. In this case, mutations to genes that encode porins can result in reduced susceptibility and even acquired resistance in the bacteria. Mutations can result in complete loss of certain porin type from the outer membrane, alter the porin size or reduce the expression of the porin. Any of these will either slow or prevent the drug from diffusing into the cell, subsequently decreasing bacterial killing. An additional resistance mechanism, such as those we will discuss later, can act on the reduced antibiotic concentration to enable the bacterium to survive in the presence of the antibiotic.
2. Expulsion of antimicrobial compound from the cell
Antibiotics need to be present in a bacterial cell at a high enough concentration to be able to kill it. By ensuring this concentration is not reached, bacteria can be resistant to the drug. This is either done by preventing entry, as we’ve just discussed, or by pumping the molecule back out as soon as it enters before it has had a chance to act on its target. Efflux pumps, which are present in the cell membrane of various bacteria, are capable of this. These energy-dependent pumps can be specific to certain antibiotics or can export a range of antibiotic classes (these are known as multidrug efflux pumps).
A susceptible bacterium can acquire resistance to an antimicrobial when certain mutations which lead to overexpression of an efflux pump arise. Alternatively, some bacteria have regulatory mechanisms to increase efflux pump expression in response to antibiotics or other toxic molecules.
3. Modification of the antimicrobial’s target
Antibiotics require a specific interaction with their target in order to inhibit or kill a bacterial cell. Small alterations to the target can interfere with this specific interaction, preventing the drug from binding and acting. This is one of the most common mechanisms of resistance, used by bacteria to escape the activity of almost all antibiotic classes.
To acquire resistance, different bacterial structures are altered depending on the antibiotic’s target. We’ll cover some important examples here, but remember this list is not exhaustive- there are a number of other cases of target modification seen in resistant bacteria.
As you’ll know from the previous article on antimicrobials (“Antimicrobials: From Ancient Mouldy Bread to Modern Antibiotics”), bacterial ribosomes are a common target of various antibiotics classes and are particularly useful as they are structurally different from our eukaryotic ribosomes. In case you forgot or haven’t read that article yet, prokaryotes have 70S ribosomes made up of a 30S and 50S subunit. Depending on the type, certain antibiotics can bind and inhibit the 30S or 50S subunit to inhibit protein synthesis. To escape this, bacteria can acquire resistance by altering the 30S or 50S subunit’s structure, depending on the drug they are exposed to. The resulting structure will remain functional but can't be bound by antibiotics that normally target them.
Gram-positive bacteria also change the structure of one of the antibiotics’ targets involved in peptidoglycan layer formation. Penicillin-binding proteins (PBPs) are important enzymes involved in the synthesis of the peptidoglycan layer of bacterial cell walls. The beta-lactam class of antibiotics, which includes penicillin, inhibit these PBPs to block cell wall synthesis. A common strategy seen in Gram-positive bacteria which have acquired resistance to some beta-lactam antibiotics is mutations conferring modifications to PBPs. Interestingly, Staphylococcus aureus took a more unusual route by acquiring a new PBP which had a much lower affinity to several beta-lactams and could continue its role in cell wall synthesis, even in the presence of these antibiotics.
4. Enzymatic modification or degradation of antimicrobial compounds
Bacteria can protect themselves from antibiotics by simply modifying or degrading the drug so it can’t act on its target.
Beta-lactamases are a well-known example of antibiotic-degrading enzymes found in beta-lactam resistant bacteria. These enzymes catalyse the hydrolysis of the beta-lactam ring, a structure common to all beta-lactam antibiotics. The first beta-lactamase was reported in the 1940s in S. aureus which was acquiring resistance to penicillin at an alarming rate. This enzyme degrades penicillin and therefore is called penicillinase. However, we now know that there are many other types of beta-lactamases capable of hydrolysing different beta-lactams.
Bacteria can also be resistant to antibiotics using enzymes that modify the drugs, rather than degrade them. One example of this is aminoglycoside-modifying enzymes which, as the name suggests, alter aminoglycosides (a class of antibiotics) in some way. These modified antibiotics are ineffective as they are unable to bind and inhibit the bacterial 30S ribosomal subunit. Similarly, chloramphenicol-acetyltransferases modify the antibiotic chloramphenicol so it can’t bind to the 50S subunit.
Figure 2: The main mechanisms of antibiotic resistance. These mechanisms can be responsible for intrinsic, acquired or adaptive resistance. Bacteria can acquire resistant traits by spontaneous mutations or horizontal gene transfer. In most cases, resistant bacteria will use several of these strategies to survive in the presence of an antibiotic drug. This figure was adapted from ‘Antibiotic Resistance Mechanisms in Bacteria: Relationships Between Resistance Determinants of Antibiotic Producers, Environmental Bacteria, and Clinical Pathogens’, Peterson and Kaur (2018) and made using BioRender.
Why is this important?
As I have mentioned before, antibiotics are a mainstay of modern medicine. When bacteria are resistant to them, conditions that were previously treatable, such as urinary tract infections or tooth infections, can become killers. These drugs are also vital for preventing infection, for example before and after operations or during cancer treatments. If they can’t be used effectively, operations, chemotherapy, radiotherapy and many other treatments will become much riskier.
Scientists record where resistant strains are present and research the mechanisms behind antibiotic resistance so measures can be implemented to reduce or prevent resistance from evolving. But, in many cases, it is too late to prevent resistance. Lots of research is being done to find new ways to target MDR, XDR and DDR bacterial pathogens.
Perhaps the most obvious solution is to develop novel antibiotics as the bacteria won’t be resistant to them. Studying bacteria in natural ecosystems, such as soil, has led to the discovery of many antimicrobial molecules which are naturally produced to aid survival in a highly competitive, resource-limited environment. A significant limitation to developing new antibiotics using this strategy is that these compounds are often highly toxic to human cells. They may kill bacteria but could also end up killing the patient.
Another approach being investigated is bacteriophage therapy. This method relies on the ability of bacteriophages (viruses that infect bacteria) to kill their bacterial host.
These new strategies currently aren’t anywhere as effective as antibiotics, so better control of antibiotic usage is critical if we are to avoid the health and economic consequences of antimicrobial resistance in the future.
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. This article’s cover photo was obtained from the CDC PHIL.