Acquired resistance

Aquired antibiotic resistance

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

There are several ways in which resistance can be acquired:

1.Gain of enzymes with antimicrobial inactivation and modification capabilities

1.i Anitmicrobial inactivation by enzymes. Antimicrobials must reach and interact with their target in the cell in order to perform their desctrutive function. One way that bacteria can prevent this is through enzymes which bind to and inactivate the antimicrobial.

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 highlighted.

β-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 fit.

The 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, causing the bacteria becomes resistant. There are many different types of these enzymes, which have different activities against antibiotics from many different classes, 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 antibiotics target site by mutation

Bacteria can become resistant to antibiotics by modifying their target sites. As they grow and replicate, they occasionally make errors when copying their genetic material, known as Single Nucleotide Polymorphisms (SNPs) or Single Nucleotide Variants (SNVs). Although these errors are rare, the large populations sizes of bacteria mean that they occur frequently enough for some to arise in the presence of antibiotics. If a mutation affects a gene encoding a protein that is the antibiotic’s target, the antibiotic may no longer bind, giving mutated bacteria a growth advantage while the rest of the population dies.

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 PBP.

Similarly, resistance to fluoroquinolone antibiotics, such as ciprofloxacin, in many bacterial pathogens is mediated by mutations in the genes encoding DNA gyrase and DNA topoisomerase IV, which are the targets of fluoroquinolone.

3.Replacement of the antibiotic’s target site

Although bacteria such as Streptococcus pneumoniae mutate the targets of antibiotics, another mechanism of resistance involves acquiring an additional copy of a gene that encodes a protein that remains active despite the presence of the antibiotic (meaning the antibiotic cannot bind to it). This is how the pathogen Staphylococcus aureus becomes resistant to most β-lactam antibiotics such as penicillin. The term Methicillin-resistant Staphylococcus aureus (MRSA) refers to S. aureus that is resistant to β-lactam antibiotics. MRSA develops resistance by acquiring an extra copy of penicillin-binding protein 2, which is the target of these 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.Epigenetics: overproduction of the target or efflux pump

The term epigenetics refers to how gene expression is altered without direct changes to the underlying genome. However, in AMR research it is often used to refer to any resistance mechanism where the effect is on the gene expression level and not via a different route as outlined above. This is the definition we will use in this course.

In addition to the mechanisms listed above, bacteria can overproduce the target proteins that antibiotics aim to inhibit, resulting in an excess of these proteins compared to the amount of antibiotic present. This overproduction allows the target protein to continue functioning within the cell despite the presence of the antibiotic. This mechanism of resistance to trimethoprim observed in Escherichia coli and Haemophilus influenzae. Overexpression of the target protein can occur alongside mutations that reduce the antibiotic’s ability to bind effectively. (Note: Trimethoprim is often 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.

Similarly, over expression of efflux pump creation or activity can also result in increased antibiotic resistance. For example, the efflux pump Mmpl5 of Mycobacterium tuberculosis can remove the drug Bedaquiline from the cell. Mutations in its regulator gene, mmpR5, reduce its inhibitory effect on Mmpl5, resulting in more of the drug being pumped out of the cell.

5.Antibiotic efflux and reduced permeability

In the previous step, we discussed that some bacterial species are intrinsically resistant to some antibiotics via reduced permeability and efflux pumps. Bacteria can also acquire additional efflux pumps that expel a particular type of antibiotic from the cell. For example, TetA efflux pumps remove tetracycline. Equally the permeability of the cell can be altered by the acquisition of mutations in porins (protein channels through cell membrane). These mutations can result in 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.

This step is an updated version of a step from Wellcome Connecting Science course Bacterial Genomes: Antimicrobial Resistance in Bacterial Pathogens. Illustrations by Laura Olivares Boldú.