Lesson 3: How Bacteria Become Resistant to Antibiotics PDF

Lesson 3: How do bacteria become resistant to antibiotics? Prepared by Dr/ Essam KOTB Associate Professor of Microbiology, IAU [email protected]...

Lesson 3: How do bacteria become resistant to antibiotics? Prepared by Dr/ Essam KOTB Associate Professor of Microbiology, IAU [email protected] +2/01121343400 +966/0563533550  Antibiotic resistance is the ability of pathogenic bacteria to resist the action of antibiotics so that they survive exposure that would normally kill or stop their growth. It is either: 1. Natural resistance – This is due to the lack of targets or impermeability to antibiotics. – Intrinsic resistance is the innate ability of a type of bacteria species to resist the action of an antibiotic as a consequence of the bacteria’s structural or functional characteristics i.e bacteria lack the target for a particular antibiotic or because the drug can’t get to its target. – In contrast to acquired resistance, intrinsic resistance is normal and fixed for given type of bacteria. – It is more common in G-ve bacteria because they structurally have an outer membrane which is impermeable to many antibiotics. 1. Acquired resistance – This is not innate to a bacterial type. It is acquired when a bacterium exposed to a particular antibiotic repeatedly by overuse, or misuse. – Therefore, it is only found in some populations of a bacterial type. – This makes acquired resistance harder to track. – Therefore, it is much more significant healthcare concern comparing with innate resistance. – Infections caused by these bacteria can no longer be treated with that antibiotic. Consequently, identifying the type of pathogenic bacteria causing an infection may not always be sufficient to determine which antibiotics will be effective treatments. – Therefore, these pathogenic isolates must be tested for antibiotic sensitivity to determine which antibiotics are effective before treatment can be prescribed.  Acquired resistance occur as a result of: 1. Genetic mutations (vertical gene transfer). 2. Transfer of resistance elements from other bacteria (horizontal gene transfer). The treatment options for these bacteria can be further limited because bacteria can accumulate resistance to a variety of antibiotics over time. This is known as multidrug resistance (MDR). However, it was found that, some bacteria that never interact with humans also have acquired resistance. This may be due to: a. Leakage of antibiotics to the environment or b. Leakage of resistant bacteria to the environment with subsequent horizonal gene transfer or c. Natural exposure to antibiotic producers in their natural habitat.  Antibiotic resistance mechanisms 1) Preventing the antibiotic entry. 2) Increasing the efflux. 3) Modifying the antibiotic. 4) Destroying the antibiotic. 5) Lack of the target. 6) Modifying the target. 7) Protecting the target. 8) Amplification of the target. Bacteria may use multiple resistance mechanisms simultaneously. Figure 1 The mechanisms of antibiotic resistance.  1) Modifying the target Antibiotics are selectively toxic because they target structural features or cellular processes in the bacterial pathogen that are different or lacking in the host’s cells. For example: 1. β-lactam antibiotics work by binding to penicillin-binding proteins (PBPs), preventing them from binding to their normal target, peptidoglycan. 2. Trimethoprim prevents dihydrofolate reductase reacting with dihydrofolic acid. 3. Oxazolidinones disrupt bacterial growth by preventing the initiation of protein synthesis. The target of linezolid is the bacterial large ribosomal subunit (50S). Changes to the structure of the target prevent antibiotic binding but still enable the target to carry out its normal function conferring antibiotic resistance (Figure 2). These structural changes are brought about by genetic mutations or by adding chemical groups. Figure 2 Schematic diagram showing how structural changes in a target enzyme can lead to antibiotic resistance. The substrate is the chemical on which the target enzyme reacts. It binds to the enzyme and is converted into a product or products through the action of the enzyme. 2) Destroying the antibiotic This occurs by the production of bacterial enzymes like β- lactamases which deactivate the β-lactam ring, preventing it from binding to its target. Consequently, production of β-lactamases by pathogenic bacteria reduces the available treatment options. One successful strategy for treating these infections is to combine antibiotic treatment with a β-lactamase inhibitor such as clavulanic acid. The β-lactamase inhibitor blocks the ability of the β-lactamase to deactivate the β-lactam antibiotic so that it can bind to its target molecule. Figure 3 Inactivation of a β-lactam antibiotic 3) Modifying the antibiotic This occurs by adding chemical groups to the antibiotic to prevent it from binding to its target. One group of antibiotics that are particularly susceptible to modification are the aminoglycosides which include streptomycin (Figure). Aminoglycoside-modifying enzymes produced by resistant bacteria add bulky chemical groups to the exposed hydroxyl (- OH) and amino (-NH2) groups of the antibiotic, which prevent it from binding to its target. Figure 4 Structure of streptomycin. An exposed hydroxyl (-OH) group that can be modified by aminoglycoside- modifying enzymes is highlighted in green. 4) Preventing the entry (influx) or increasing exit (efflux) of antibiotics To reach their targets inside the cell, antibiotics must cross the cell wall (Video 1). Bacteria can develop resistance by preventing antibiotics from reaching their target by decreasing the permeability of their outer membrane or by actively transporting antibiotics out of the cell by efflux pumps.  How altering the entry of antibiotics across the membrane result in antibiotic resistance? The cell walls of gram-positive bacteria are permeable to most antibiotics. Therefore, easily reach their targets. However, the outer membrane of gram-negative bacteria, like E. coli, forms a permeability barrier that prevents antibiotics from entering the bacterial cell and reaching their target. Embedded in the outer membrane of gram-negative bacteria are protein channels known as porins. Antibiotics cross the outer membrane of gram-negative bacteria by diffusing through these porin channels. Porin channels are non-specific and can transport many antibiotics across the outer membrane.  Some antibiotics can be transported out of the bacterial cell by efflux pumps in the membrane to be prevented from binding to their targets, so bacteria expressing efflux pumps are resistant to antibiotics. Some efflux pumps are specific and only transport one class of antibiotics, but many transport a wide range of molecules. These efflux pumps are known as multi-drug- resistant efflux pumps. Porins and efflux pumps have opposite effects on the concentration of antibiotic inside the cell.  Efflux pumps are membrane proteins that are involved in the export of a wide array of substrates, including antibiotics from within the bacterial cell into the external environment. They are found in all species of bacteria. Efflux pump genes can be found in bacterial chromosomes or mobile genetic elements, such as plasmids. There are five superfamilies of efflux pumps (Figure 4): 1. Multidrug and toxin extrusion (MATE) 2. Small multidrug resistance (SMR) 3. major facilitator superfamily (MFS) 4. ATP-binding cassette (ABC) 5. Resistance-nodulation division (RND). Figure 4. Schematic diagram showing the five superfamilies of efflux pumps found in bacteria and their energy-coupling mechanisms. The efflux pumps typically drive the transport of substrates across the cytoplasmic membrane out into the extracellular environment. RND-type efflux pumps are organized into tripartite systems and can transport substrates from within the periplasm and the cytoplasm across the outer membrane to the outside of the cell. OM, outer membrane; IM, inner membrane. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.  To date, RND efflux pumps have only been found in Gram-negative bacteria and are organized as tripartite systems consisting of a cytoplasmic membrane pump, a periplasmic adaptor protein and an outer membrane protein channel. All the efflux pump superfamilies utilize energy from the proton/sodium motive force, except for the ABC superfamily, which are primary transporters that utilize energy from ATP hydrolysis to mediate the efflux of substances from within the cell. In Gram-positive bacteria, the MFS superfamily of efflux pumps is the most widely studied and includes clinically relevant examples, such as NorA of Staphylococcus aureus, which exports fluoroquinolones and quaternary ammonium compounds.  The most clinically significant efflux pumps in Gram-negative bacteria belong to the RND superfamily, which includes AcrAB- TolC of Escherichia coli and Salmonella enterica, and MexAB- OprM of P. aeruginosa and AdeABC of Acinetobacter baumannii. All species of bacteria can express efflux pumps from more than one superfamily and/or more than one type of efflux pump from the same superfamily. In addition, efflux pumps also exhibit different substrate profiles, which vary within and between the superfamilies. Although efflux pumps are widely implicated in antibiotic resistance, there is growing evidence from numerous studies to suggest that they may play a role in a range of bacterial behaviour, including biofilm formation, QS, pathogenicity and virulence. Figure 3. General bacterial QS systems. (a) In Gram-negative bacteria, one of the more common examples of QS involves the synthesis of AHLs by AHL synthases. AHLs are detected by intracellular receptors that function as transcription factors to drive the transcription of quorum-specific genes. (b) In Gram-positive bacteria, QS is mediated by AIPs, which are translated from an AIP signal precursor locus to produce an AIP precursor, which is then processed to form mature AIPs. They are secreted out of the cell and detected by a sensor kinase that phosphorylates a response regulator, which  5) Target protection as a key antibiotic resistance mechanism Target protection proteins (TPPs) produced by bacteria can mediate antibiotic resistance by: 1. Direct antibiotic displacement (type I). 2. Inducing conformational changes within the target that allosterically dissociate the drug from the target (type II). 3. Inducing conformational changes within the target that restore functionality despite the presence of the bound antibiotic (type III) 6) Resistance by amplification of the target Here the bacteria become resistant by overproduction of the antibiotic target through gene amplification. Whilst antibiotic resistance genes can be present in the bacterial chromosome, they are also present in mobile genetic elements, such as plasmids. Case study: resistance to cephalosporins Several bacteria are intrinsically resistant to 1st, 2nd and 3rd generation cephalosporins by different strategies. Type Infection Resistance mechanism to cephalosporin MRSA Skin infections Expresses a modified antibiotic target (PBP2a) with which β- Enterococci spp. Urinary tract lactams poorly bind infections Listeria Listeriosis monocytogenes Pseudomonas Nosocomial Expresses cephalosporinase aeruginosa infections (ESBL) Klebsiella Pneumonia ESBL a) By modification of the target site The massive use of cephalosporins to treat infections has led to the emergence of cephalosporin resistant MRSA. This resistance results from the expression of structurally different penicillin binding protein called penicillin- binding protein 2a (PBP2a) with which β-lactams bind more poorly than PBPs (Figure 5). Therefore, cell wall biosynthesis can occur in the presence β-lactams including cephalosporins. Fortunately, the recently developed cephalosporins, including ceftaroline and ceftobiprole can bind to and inhibit the activity of PBP2a. b) By extended spectrum β-lactamases (ESBL) Bacteria also can gain resistance against β-lactams including cephalosporins by production of a special type of degrading β-lactamases called cephalosporinases. The first β-lactamase to be identified was penicillinase. As its name suggests, penicillinase can hydrolyse penicillin but not cephalosporins. Cephalosporinases have the ability to hydrolyse a wider range of β-lactams, therefore are called extended spectrum β- lactamases (ESBL). Since cephalosporins were first described in the early 1980s, the frequency of infections caused by ESBL-producing bacteria has been increasing representing an ever-growing healthcare challenge (Figure 7). Several ESBL classes have been identified worldwide. CTX- M class is the most common. c) Porin expression and cephalosporin resistance in K. pneumoniae K. pneumoniae expresses two porins called OmpK35 and OmpK36 for the transport of β-lactam antibiotics across the outer membrane. Loss of expression of OmpK35 and OmpK36 in K. pneumoniae is a secondary mechanism of resistance after ESBLs. For example, the cephalosporin cefoxitin could be used to treat ESBL-producing K. pneumoniae because it is a poor substrate for ESBLs. Therefore, cross porins reaching its target. However, if these K. pneumoniae strains do not express OmpK35 or OmpK36, cefoxitin will not be able to cross the outer membrane to reach its target. This example illustrates how bacteria rely on multiple resistance mechanisms to protect themselves. Figure 6 shows the percentage of ESBL- and non-ESBL-producing E. coli isolates that were resistant to the cephalosporin cefepime between 2004 and 2016.  Activity : Now answer the following questions based on the data in Figure 6. 1. How has the proportion of ESBLs and non-ESBLs resistant to cefepime changed over time? 1. Explain the difference in resistance between ESBL- and non-ESBL-producing E. coli? 2. Do you think the expression of ESBLs is a major determinant of resistance to cephalosporins in E. coli? Summary This lesson introduced the mechanisms of antibiotic resistance. You should now be able to explain how antibiotic resistance protects bacteria from both natural and synthetic antibiotics and give examples of the main mechanisms of antibiotic resistance.  You should now be able to: State what is meant by the term ‘antibiotic resistance’ Recognise that antibiotic resistance evolved to protect bacteria Describe the three main mechanisms of resistance that bacteria have developed to counteract the action of antibiotics Give examples of these resistance mechanisms Distinguish between intrinsic and acquired antibiotic resistance. Having seen how antibiotic resistance can be either intrinsic or acquired, next lesson you will look in more detail at the processes of mutation and gene transfer that lead to acquired resistance. Thanks for your attention Questions?