Antimicrobial Resistance 101: Resistance Mechanisms

Foreword

Antibiotics were a major breakthrough in science. Microorganisms such as viruses, bacteria, fungi, and more can be treated by antibiotics relatively simply. Upon the introduction of antibiotics, previously deadly illnesses could now be treated with incredible efficacy. However, the pathogens targeted by the first antibiotics soon started to become resistant.

Antimicrobial resistance is one of the biggest problems in modern medicine. There is evidence of bacterial infections evolving to become resistant to the complete arsenal of antibiotics. As a result, people are increasingly dying from simple infections that would not have been dangerous a few years ago. Because the situation is only going to get worse in the coming decades, it is time to think about alternatives.

The Antimicrobial Resistance 101 series will be mainly divided into the following chapters:

1. Antimicrobial Resistance 101: Evolution As a Driver of Resistance

2. Antimicrobial Resistance 101: Resistance Mechanisms

3. Antimicrobial Resistance 101: The Global Problem of Antimicrobial Resistance

4. Antimicrobial Resistance 101: The Clinical Burden of Resistant Microorganisms

5. Antimicrobial Resistance 101: Solutions to Antimicrobial Resistance

6. Antimicrobial Resistance 101: Antimicrobial Resistance in the Future

Antimicrobial Resistance 101: Resistance Mechanisms

Antimicrobial resistance is a threat to public health across the globe (McEwen & Collignon, 2018). The problem is enormously complex, and much science has yet to be revealed on potential new solutions to combat resistance in microorganisms. In past decades, much research has been done on pathogens, including viruses, bacteria, and, fungi, which are now understood much better than when antibiotics were discovered about 80 years ago (Aminov, 2010). As a result, antimicrobials have been developed. These are generally categorised into classes. Drugs within the same class function similarly. Therefore, when a microbe develops resistance to a particular drug, it will also be resistant to all of the drugs in this class (Perlin et al., 2017). In this article, an overview will be presented of mechanisms by which microbes may become resistant. As explained in the previous article of this series, the focus lies on the most common microbes; fungi, viruses, and bacteria.

Fungi

Fungi, like other microbes, are becoming more resistant to the available therapeutic arsenal. Pandemics as explored in The Last of Us will not become reality any time soon, however. In healthy patients, fungal infections tend to be minor, without severe complications (Perlin, 2017). This is not the case for immunocompromised patients. These are patients of which the immune system is not working properly. This may be caused by a number of things including HIV or age. Immunocompromised patients may suffer from pneumonia or meningitis, as a result of fungal infections. It is worth noting that the number of available antifungal drugs is very limited, especially when compared to drugs that treat bacterial infections, such as antibiotics. There are several reasons for this. One of which is that scientists have not been actively researching pathogenic fungi for a long time, relative to bacteria and viruses (Perlin, 2017). As a result, the complex interactions between fungi and their host remain somewhat elusive. Because the number of classes of antifungals is so small, when a fungus becomes resistant to a class, this severely limits the therapeutic options.

Figure 1: Fungi continue to surprise researchers. This is a cricket with a severe fungal infection (Buggide, 2019).

As explained in the previous article, resistance is a natural phenomenon that is a direct consequence of evolution, specifically of natural selection. By exposing fungi to a drug, only the individuals that have randomly acquired traits to render themselves less susceptible to the drug will survive and reproduce, leading to a new generation of individuals that are yet more resistant to the drug (Perlin et al., 2017). This applies to all microbes, though fungi are different from bacteria and viruses in an important way. Viruses are not cells. They are biological machines that invade cells, which they then manipulate in order to reproduce. Bacteria are relatively simple organisms called prokaryotes. These organisms do not have a separate nucleus in the cell, meaning the DNA is found “loosely” floating around in the cell. Other forms of life, that evolved much later, are called eukaryotes. These cells do have a compartmentalised nucleus, which governs the cell’s behaviour and acts like a control centre (Sapp, 2005). Eventually, these more complex organisms evolved, which later gave rise to plants, animals, and fungi. In other words, fungi, plants, and animals are evolutionarily much more similar to each other, than they are to bacteria. As a result, their cells function quite similarly, and vastly differently from bacterial cells. Antibiotics are very effective at killing bacteria and tend to not be harmful to humans or animals. This is simply because human cells are much more complex than those of bacteria. While antifungals are very effective at killing fungi, they tend to do serious damage to the patient as well, given that these too, are eukaryotes and therefore share many molecular mechanisms (Campoy & Adrio, 2017).

In total there are four classes of antifungals, each of which works differently. The first, the azoles, interfere with the synthesis of a certain chemical called ergosterol (Fisher et al., 2022). This molecule is vital for fungal growth. In other words, when azoles deplete the amount of ergosterol synthesised, the fungus will die. The second class are the echinocandins, which attack a certain molecule in the fungal cell wall and make this wall lose its integrity. As a result, the fungal cells practically explode (Fisher et al., 2022). The third class, polyenes, increase the permeability of the fungal cell, which causes the cell to lose valuable resources. The final class, the pyrimidine analogues, target the fungal DNA. Consequently, breaks appear on the DNA of the fungus which causes the cell to die.

Different fungal species have learned to tolerate these four classes of drugs extremely well, using four different mechanisms (Fisher et al., 2022). One mechanism is that the fungus may evolve to produce enzymes that manipulate the drug, rendering it ineffective. This is how certain yeasts are becoming resistant to polyene drugs, among others. Alternatively, the fungus may start to overexpress the protein that is being targeted by the drug. If the concentration of this protein becomes very high, then there is likely not enough of the antifungal to target the fungus effectively. The third mechanism by which a fungus may become resistant is by mutations occurring in the fungal protein that is targeted by the drug. This may change the protein structure in such a way the fungus is no longer susceptible. Lastly, the fungus may increase the concentration of efflux pumps. These pumps occur in all cells and transport certain chemicals and ions out of the cell (Pfaller, 2012). However, fungi have been shown to create more efflux pumps, leading to an increase in the transportation of specific drugs. When the fungus transports the drug outside of the cell, the effectiveness of the drug is nullified.

Figure 2: An overview of the antifungal classes and the resistance mechanism against them (Fisher et al., 2022).

In recent years the number of antifungal-resistant fungi identified has grown. Not only has the absolute amount increased, but also the amount of classes fungi are resistant to is on the rise. There are now multidrug-resistant fungi, which are not susceptible to at least two out of the aforementioned four classes, making treatment incredibly difficult. Additionally, the use of antifungals in agriculture has caused fungi in the environment to become resistant to antifungals treated in human medicine. New antifungals need to be identified fast, and their use should be very limited to prevent resistance against these new drugs (Perlin et al., 2017).

Viruses

Viruses are a curious class of biological entities. The scientific community has not been able to define whether viruses are living or non-living. Instead, they form a grey area around this distinction. Though scientists' opinions on viruses differ, the majority of biologists agree that living organisms consist of cells. Consequently, viruses would not classify as alive (Villarreal, 2008). As a result, viruses cannot be “killed”. Instead, they are said to be “inactivated” or “destroyed”. Viruses are a truly fascinating class of entities, that are not much more than some genetic information stored in a hull of proteins. Viruses do not have any complex biological processes going on, so they cannot replicate or synthesise proteins by themselves. Instead, they need to hijack a cell to use the cellular machineries. There are many different kinds of viruses, all with vastly different mechanisms of replication, that may infect bacteria, fungi, or complex animals. Viruses are thought to be the most abundant lifeform on Earth, with an unimaginable variety (Villarreal, 2008). One of the contributing factors to this enormous variety is the intrinsic rate of replication and errors of viruses. When a cell becomes infected with a certain virus, depending on the type of virus, the cell may become completely submissive to the will of the virus, in the sense that the cell’s biosynthesis changes such that the maximal number of viruses is produced. When infected with the influenza virus, for example, a single cell may produce 10,000 copies. In other words, within a few days 100 trillion viral particles may be circulating in one’s body (Zimmer, 2013). Many viruses have a relatively high mutation rate (Peck & Lauring, 2018). Say that only 1% of viruses acquire mutations, this small fraction of 10,000 new viruses per cell still accounts for amazing variability. This rapid mutation rate is a direct consequence of the error-prone mechanism by which viruses replicate. Although this too heavily depends on the specific virus, many viruses bring their own enzyme that replicates the virus’ genome (Peck & Lauring, 2018). In contrast to the enzyme that is responsible for the replication of cellular DNA, the virus’ enzymes typically lack a certain ability to correct mistakes; the focus of the virus lies on the quantity not the quality of the new viruses. This is especially evident from infection by the Hepatitis B virus (HBV). When a cell is infected with this virus, erroneous viral particles may be produced, which interfere with the replication of the original virus (Yuan et al., 1998).

Figure 3: Hepatitis B virus (blue) creates defective particles (red), which interfere with the replication of HBV (Ziegler & Botten, 2020).

The fact that viruses mutate so rapidly, resulting in an immense variability in the new generation of progeny, makes treatments sometimes very ineffective. Often, antivirals target very specific proteins. When these enzymes change, as a result of the high mutation rate of viruses, the virus becomes resistant to the drug. Therefore, instead of treating viral infections with a single drug, a more effective treatment is often the combination of multiple antivirals. The efficacy of this is can be seen in HIV patients. When HIV was discovered in the United States in 1982, four drugs were found within six years (Myhre & Sifris, 2022). Though effective at the start, doctors witnessed a very rapid evolution of the virus, which quickly became resistant to these drugs. Later it became evident that these drugs targeted the same protein of the virus, the reverse transcriptase (Myhre & Sifris, 2022). In 1995, a new class of antivirals was found; protease inhibitors. These, in combination with the earlier class of drugs, proved much more effective, because new viruses needed two mutated proteins rather than one, which is significantly less likely to occur. Since this discovery, HIV is no longer treated with only one class of antivirals. Instead, such combined therapy, referred to as highly active antiretroviral therapy (or HAART), has become the golden standard (Myhre & Sifris, 2022).

Every virus is different, which means that not every virus can be treated with the same drug. There are, however, very similar stages in all viruses' lifecycles. These steps require the virus to carry out very specific tasks, for which very specific proteins are responsible. The first step is viral attachment to the cell. Often, viruses bind to specific receptors on the cell they infect. Afterwards, the virus can enter the cell through specific rearranging mechanisms of the cellular membrane, effectively leaving a pore. The virus uncoats itself, disintegrating its coat of proteins, and shooting its genome into the cell which is then replicated (Kausar et al., 2021). This step is followed by the synthesis of proteins from the genome, after which new viral particles emerge. These then exit the infected cell. All these steps may significantly differ per viral species, which is not surprising, given the vast number of viruses. Nonetheless, this is the general lifecycle of a virus, as shown in the figure below (Kausar et al., 2021).

Figure 4: The general viral lifecycle (Khanacademy n.d.).

Antiviral resistance, much like antibiotic and antifungal resistance, is an increasing problem. Viruses, fungi and bacteria are, however, completely different biological entities that may cause vastly different diseases. Because they are so different, treatments for one type of infection might not work for another. For example, antibiotics cannot be used to treat viral or fungal infections, in contrast to popular thought. In other words, when suffering from a cold (influenzavirus, a viral infection), taking antibiotics will not help. Instead, the misuse of antibiotics in this case only contributes to the growing issue of antimicrobial resistance. There are a vast number of viruses, which can each infect a vast number of hosts. Influenza, for example, infects wild birds, which can transmit the virus to massive amounts of poultry, with disastrous consequences. Recently it has been shown that patients taking antivirals secrete relatively large amounts of them in their urine, which then end up in the environment, making more viruses develop resistance. In the clinical setting it is becoming increasingly evident that for viral infections too, new treatments are becoming necessary (Kumar et al., 2020).

Bacteria

When talking about antimicrobial resistance, the focus generally lies on antibiotic resistance and therefore bacteria. This is because the most common resistant infections are bacterial infections, as indicated by the ominously long list of resistant bacteria causing international concern, according to the WHO (World Health Organization, 2021). These bacteria constitute the ESKAPE group, which are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (Mancuso et al., 2021). Not only is this specific group of bacteria often found resistant to the available drugs, but these bacteria are also capable of causing severe infections. Much like fungi, bacteria grow resistant to drugs by inhibiting drug uptake, actively changing the drug, and overexpressing the efflux pump (Mancuso et al., 2021). Every class of drugs targets a different mechanism or type of protein in a bacterial cell, and resistance mechanisms therefore heavily differ, depending on the class of antibiotics. In contrast to antifungals, however, the palette of antibiotics is larger. Though the classification is surprisingly different per organisation or institution, the CDC classifies antibiotics into nine groups (Centers for Disease Control and Prevention, n.d.). Fundamentally, they work similarly to how antiviral and antifungals work, in the sense that antibiotics actively inhibit the processes required for bacterial cells to stay alive. Penicillins, for example, inhibit the synthesis of a cell wall, while fluoroquinolones inhibit DNA synthesis. Another important difference between fungi and bacteria is that the latter are prokaryotes, and therefore much less similar to animals in terms of cellular organisation and structure. That means that antibiotics generally come with significantly fewer side effects.

Figure 5: Basic information about the ESKAPE pathogens (Pulgar, 2019).

The severity of a pathogen’s resistance is classified into three groups, which are sometimes used interchangeably. Generally, however, multidrug-resistant (MDR) means resistant to a drug from at least three classes. Bacteria that are resistant to all but two (or fewer) classes are said to be extensively drug-resistant (XDR). Pan drug-resistant (PDR) means that the microbe is resistant to all classes (Magiorakos et al., 2012). PDR bacteria are particularly worrisome, as PDR bacteria require alternative treatment to antibiotics. Luckily, however, they are the rarest. The most worrisome aspect of antibiotic resistance is, however, that internationally the overuse and misuse of antibiotics is not being halted. This means that bacteria that are already classified as MDR or XDR may be exposed to antibiotics to which they are not yet resistant, to which they then become resistant. Misuse of antibiotics, therefore, contributes to an increase in PDR microbes (Mancuso et al., 2021).

Microbes can be resistant naturally, or they may acquire resistance. Naturally resistant pathogens are further classified into intrinsic or induced resistance. The first signifies that the bacterium was resistant before it even was in contact with the antibiotic in question. This may be because its metabolism is so different from that of other bacteria, that the drug just does not work on this species. Induced-resistance bacteria have developed through the turning on or off of certain genes. This type of resistance specifically is not caused by changes in the DNA. Instead, the genome of bacteria that develop induced resistance remains intact. Acquired resistance, in contrast, is caused by changes in DNA. Mutations may arise, or alternatively, the bacterium may pick up genetic information from its environment. These new genes could then make the bacterium resistant (Mancuso et al., 2021).

Figure 6: Few bacteria are intrinsically resistant. These will reproduce, and possibly share this DNA with other bacteria which acquire resistance (Adjusted from Johnson, 2007).

This sharing of DNA in bacteria is fundamentally different from that seen in other organisms. Typically, parents give their genetic traits to their progeny, which is termed vertical gene transfer. Bacteria can exchange DNA with each other, between individuals of the same generation, which is called horizontal gene transfer (Bello-López et al., 2019). This may occur in three separate ways. First, bacteria can simply take up genetic information from their environment, if it finds itself unprotected. After a bacterium dies, for example, the cell disintegrates causing the DNA to be released into the environment, which may be taken up by other bacteria. Alternatively, through a tube called a pilus, bacteria can actively exchange DNA with each other, through a process termed conjugation. Interestingly, the genetic information that is shared in this way is a particular type of DNA, called plasmids. Bacteria have two types of DNA. First, is the cellular genome. Every living cell has this kind of DNA, but bacteria have an additional molecule of DNA. These plasmids can be thought of as mini-chromosomes that are floating around in the cell. These plasmids specifically, can be very easily transmitted between bacteria, partially because these molecules are quite small. These often code for antibiotic traits, allowing more rapid horizontal transfer (Bello-López et al., 2019). The third mechanism through which horizontal transfer may occur is through transduction. In this mechanism, a bacteriophage infects a bacterium, which will then produce many more phages. Sometimes, the produced phages also contain a small fragment of the bacterial DNA, which may code for antibiotic traits. The newly produced phages can afterwards infect more bacteria, causing more widespread resistance (Bello-López et al., 2019). These three mechanisms together allow for a complex network of transferral of DNA between bacteria that may find themselves in a shared space.

In a rather simplified way, this article lists the most widespread mechanisms by which bacteria, viruses and fungi may become resistant to treatment. Though these three classes are not the only ones that might infect humans and other animals, nor are they the only ones that can become resistant to treatment, these three are the most common sources of infection, and of resistance. These three classes each have worrying characteristics that present fundamentally different problems: The current science on fungi is lacking, viruses mutate incredibly rapidly, leading to enormous variety and bacteria may share DNA between different species in the same environment. One thing they do have in common is that their emergence and especially the increasing frequency of their resistance, are troubling. In the next few articles, more emphasis will be put on the international epidemiology of resistant microbes and what resistance means in the clinical setting. After that, alternatives to antimicrobials will be discussed eventually leading to information on future perspectives.

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