What are the Mechanisms of Antimicrobial Resistance?

Overview

In addition to the intrinsic mechanisms of resistance, bacterial pathogens can acquire genes and mutations that mediate resistance to antibiotics. In some cases, bacteria may acquire multiple mechanisms of resistance to the same antibiotic, and in multidrug resistant bacteria, they acquire resistance to multiple classes of antibiotics.

The mechanisms of resistance can be broken down into the following:

1. Enzyme inactivation and modification
2. Modification of the antibiotics target site
3. Overproduction of the target
4. Replacement of the target site
5. Efflux and reduced permeability

1.i Enzyme inactivation.

One of the first mechanisms of resistance to be discovered was resistance to penicillin (a β-lactam antibiotic). Penicillin resistant strains of Staphylococcus aureus were found to have acquired an enzyme known as a β-lactamase (originally known as a penicillinase).β-lactamase enzymes target a part of β-lactam antibiotics known as the β-lactam ring, this is found in all β-lactam antibiotics. The β-lactamase enzyme breaks this ring open, preventing the antibiotic from binding to their target. See Figure 1 below.Figure 1. Schematic of a β-lactamase enzyme degrading penicillin into fragments; chemical structure of penicillin with the β-lactam ring highlightedβ-lactamases are a family of enzymes (there are thousands of different versions) found in many bacterial pathogens. They have different activities, meaning some will work against specific members of the β-lactam family, while others will not. Certain members of the β-lactamase family, known as Carbapenemases, are the most problematic because they break down all members of the β-lactam family of antibiotics, including carbapenems, severely limiting treatment options.

1.ii Enzyme modification.

Two other mechanisms of resistance are mediated by bacteria acquiring enzymes. Firstly, bacteria can acquire enzymes that chemically modify the target of the antibiotic in the bacteria by adding additional chemical groups. An example of this is the erm (erythromycin ribosomal methylation) gene that provides resistance against macrolide antibiotics like erythromycin. This enzyme methylates (adds a methyl group: CH₃) to part of the ribosome, which is the target of erythromycin. This means that erythromycin can no longer bind to the target, as shown in Figure 2 below, meaning the bacteria can continue to thrive in the presence of the antibiotic.Figure 2. Schematic of a ribosome with an antibiotic fitting into a groove; the same ribosome methylated blocking the groove so the antibiotic can no longer fitThe second type of enzyme acts by chemically modifying the antibiotic itself, which prevents the antibiotic binding to its target site. An example of this is aminoglycoside-modifying enzymes such as N-acetyltransferases, which add an additional acetyl group (CH₃CO) to aminoglycoside antibiotics such as kanamycin. As illustrated in Figure 3 below, this stops it binding to the ribosome, meaning the bacteria becomes resistant. There are many different types of these enzymes which have different activities against antibiotics from many different classes of antibiotics including aminoglycosides, tetracyclines, phenicols and lincosamides.Figure 3. Schematic of kanamycin fitting into a groove on an enzyme; in the upper section the unmodified antibiotic fits into a groove on a ribosome, and in the lower section the antibiotic is modified with methyl groups and can no longer bind to the ribosome

2. Modification of the antibiotic target site.

A common mechanism that bacteria use to become resistant to antibiotics is by modifying the target of the antibiotic. As bacteria grow and replicate they copy their genetic material (the genome). When they do this, occasionally mistakes in the DNA sequences get included (e.g. an A gets replaced with a C). These mistakes only happen very rarely, but the very large population sizes (billions and trillions) of bacteria, means that this happens frequently enough that occasionally these mutations are present in bacterial populations in the presence of antibiotics. If one of these mutations happens to be at a location of a gene that encodes for a protein that is the target of an antibiotic, then sometimes these mutations mean that the antibiotic can no longer bind to the target. This means that the bacteria with the mutation will have a growth advantage and will survive the antibiotic while the rest of the population will die.This is a common mechanism for resistance to penicillin in Streptococcus pneumoniae, where the acquisition of mutations in the penicillin binding proteins (PBP) which are the target of penicillin. The presence of the mutations in the PBPs mean that penicillin can no longer bind and kill the bacteria.Figure 4. Normal and mutated PBP against a background of the bacterial cell membrane and cell wall; Penicillin fits into a groove on normal PBP but cannot bind to mutated PBPSimilarly resistance in many bacterial pathogens to fluoroquinolone antibiotics such as ciprofloxacin is mediated by mutations in the DNA gyrase and DNA topoisomerase IV genes, which are the target of ciprofloxacin.

3. Replacement of the target site.

While bacteria like Streptococcus pneumoniae mutate the targets of the antibiotics, another similar mechanism of resistance is to gain an additional copy of the gene that encodes a protein that still retains activity (e.g. the antibiotic can’t bind to it) in the presence of the antibiotic. This is how the pathogen Staphylococcus aureus becomes resistant to most β-lactam antibiotics such as penicillin. Methicillin-resistant Staphylococcus aureus (MRSA), which is the name given to S. aureus that is resistant to β-lactam antibiotics, becomes resistant by gaining an extra copy of penicillin binding protein 2, which is the target of β-lactam antibiotics. This additional version known as penicillin binding protein 2a (PBP2a) can still function in the presence of β-lactam antibiotics.Figure 5. PBP and PBP2a against a background of the bacterial cell membrane and cell wall; Penicillin fits into a groove on normal PBP but PBP2a has an elongated shape with no groove

4. Overproduction of the target.

Bacteria can also overproduce the target of the antibiotics, meaning there is an excess of the protein target of the antibiotics compared to the antibiotic itself. This means that there is enough of the target protein for it to continue its role in the cell in presence of antibiotics; this is a mechanism of resistance to trimethoprim in Escherichia coli and Haemophilus influenzae. The overexpression is sometimes found in combination with mutations that lower the ability of the antibiotic to bind to its target. (Note: trimethoprim is typically used with sulfamethoxazole, a combination known as co-trimoxazole or SXT).Figure 6. Normal scenario: SXT antibiotic fits into the grooves of DHPS enzymes, with some excess SXT; Overproduction of DHPS enzymes, only some of which bind SXT leaving some free

5. Efflux and reduced permeability.

In the previous section, we discussed that some bacterial species are intrinsically resistant to some antibiotics via reduced permeability and efflux pumps. In addition, bacteria can acquire additional efflux pumps that specifically pump a single type of antibiotic, for example TetA efflux pumps that specifically pump tetracycline from the cell. Equally the permeability of the cell can be altered by the acquisition of mutations in porins (protein channels through cell membrane). These mutations can include porin loss, a modification of the size or conductance of the porin channel, or a lower expression level of a porin. Ultimately both mechanisms, efflux pumps and reduced permeability, lower the intracellular antibiotic concentration in the bacterial cell by either exporting the antibiotic or by not allowing its importation, respectively.Figure 7. Outer membrane, peptidoglycan cell wall, and plasma membrane. A porin channel sits in the outer membrane, and an antibiotic cannot enter the cell from the channel labelled reduced permeability. A large multi-subunit efflux pump crosses all three layers and an arrow shows the antibiotic being removed from the cell