Antibiotic Resistance and Antibiotics

Mechanisms of antibiotic resistance

1. Target site insensitivity e.g. changes to ribosome in erythromycin resistance 2. Enzymatic inactivation, e.g. Beta-lactamases in penicillin resistance / phosphotransferases in amino glycoside resistance. 3. Development of cell wall impermeability, e.g. mutant porins in Gm-neg bacteria. 4. Drug export, multi-drug efflux system.

Carbapenem resistance in Klebsiella pneumoniae

Carbapenem resistance in Klebsiella pneumoniae is partially mediated by modification of the non-selective porns OmpK35 and OmpK36. A selected insertion into loop 3 of OmpK36 causes significant constriction of the pore and therefore increased tolerance to carbapenems.

(Degradation) Why is carbapanem resistance of particular concern?

Carbapenem resistance is of particular concern as they are among the most potent antibiotics in use. Carbapenemases like KPC (class A), NDM (class B) and OXA (class D) types have the capacity to hydrolyse penicillins, cephalosporins, and carbapenems, greatly reducing the number of drug treatment options for a given infection.

Competitive inhibition?

Competitive inhibition is also complicated by complexity of the systems and it is therefore possible that this approach will be unsuccessful therefore other strategies may be required like prevention of the formation of efflux complex assembly.

EE: Which technique has allowed us to visualise efflux transporters in bound and unbound states?

Cryo-electron microscopy

Transduction in bacteria

DNA is transferred from a donor cell to a recipient via a bacteriophage

N. gonorrhoeae and RND efflux pumps

MtrR is responsible for the repression of mtrCDE, the only RND pump of N. gonorrhoeae. Point mutations within the the MtrR binding site or its DNA binding domain are common and lead to increased mtrCDE expression and resistance against structurally diverse AM agents, including penicillin, tetracycline, azithromycin and third generation cephalosporins.

Quinolone antibiotics

Quinolone antibiotics inhibit essential topoisomerase enzymes by binding near the active site. Amino acid substitutions in the target proteins which result in lower binding efficiency while still allowing the enzyme to function, confer resistance.

Type 3 secretion system (T3SS)

Similar to T6SS, uses injectisome to insert proteins into host cells. Used by E.coli, yersinia, and salmonella

Why are gram positive bacteria naturally more permeable to many antibiotics?

Since gram positive bacteria lack the outer membrane, this makes them naturally more permeable to many antibiotics.

Two component regulators

Two component regulators. These are simplified but elegant ways in which bacteria sense the environment based on a sensor molecule and a response regulator. Sensor kinase is located in a membrane. It detects an extracellular or gram negative bacteria periplasmic signal, which then leads to phosphorylation through the kinase domain of the response regulator, which has its effects through gene transcription. It is a quick signalling pathway to get from the external environment to a response. It does not require a large cascade of proteins in a large signalling pathway. E. coli has lots of these systems.

Dangers of indiscriminate use of antibiotics

drug resistance elimination of normal microbiota

Rifampicin - bactericidal

inhibition of transcription used to treat TB. Inhibits bacterial DNA-dependent RNA polymerase. Mutations in the enzyme can lead to resistance i.e. in Mycobacterium tuberculosis and E. coli. Broad spectrum against most gram positive and gram negative orgamisms (including Pseudomonas aeruginosa - gram negative bacterium opportunistic pathogen)

Transformation

process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria

integrons

this non-mobile genetic element is unique because it carries genes in circular units called gene cassettes which are freely transmissible between these elements

Quorum sensing

Quorum sensing. Gene regulation in bacteria where bacteria sense in the environment then go through a programme change of gene expression. An example of this is where bacteria sense extracellular iron concentrations and then change their gene response, following detection of that low iron concentration.

What type of proteins are RND transporters?

RND transporters are homotrimeric proteins that recognise ligands and transduce electrochemical energy from the proton gradient across the inner membrane.

transposons

(jumping genes) short strands of DNA capable of moving from one location to another within a cell's genetic material

Trimethoprim

-Inhibits bacterial dihydrofolate reductase. Bacteriostatic. - inhibits folate metabolism and hence DNA synthesis e.g. 2,4-diaminopyridines -Used in combination with sulfonamides (trimethoprim-sulfamethoxazole [TMP- SMX]), causing sequential block of folate synthesis. Combination used for UTIs, Shigella, Salmonella, Pneumocystis jirovecii pneumonia treatment and prophylaxis, toxoplasmosis prophylaxis.

(Modification by the transfer of a chemical group) Aminoglycoside modifying enzymes

Aminoglycoside modifying enzymes like ApmA, which is an acetyltransferase capable of inactivating apramycin, an antibiotic that can currently evade other mechanisms of aminoglycoside resistance.

Sulphonamides

Antifolates Now rarely used because of resistance

Antigenic variation

Antigenic variation is a qualitative phenomenon. An example is the Type 4 pili in N. meningitidis, important organelles which mediate DNA uptake during transformation. They mediate bacteria: bacteria interactions, so that bacteria form microcolonies based on type 4 pili interactions and the type 4 pilus is essential for bacteria binding to host cells. It is formed by the multimerization of the pilE protein into a helical array. The conserved region of the type 4 pilus which sits inside the helical array - its hydrophobic alpha helix - these sequences are subject to detection by the immune system and undergo variation through a specific event where there is a single locus for expression of pilE (the major pilus subunit) and there a number of silent loci which are not expressed - pilS. Through specific recombination parts of the pilS sequences can integrate into pilE through genetic exchange and then you can make a new pilE sequence. This shows that there is a huge amount of variation through genetic exchange within a genome that can lead to antigenic variation and immune escape.

Conjugation in bacteria

Bacterial cells can join together and pass plasmid DNA from one bacterial cell to another. This process can take place between bacteria of different species and is of concern in terms of passing plasmid-located genes for antibiotic resistance.

(Degradation) Beta lactamases

Beta lactamases provide resistance to beta lactam drugs by hydrolysing the amide bond of the beta-lactam ring, thus degrading the drug. They have evolved in nature in response to the production of beta-lactam antibiotics by microorganisms and have been studied since the 1940s.

Isoniazid - bactericidal to growing organisms, otherwise bacteriostatic

Blocks long chain mycolic acids (unique to Mycobacteria). It is used to treat tuberculosis. Isoniazid is activated by KatG, a bacterial catalase peroxidase enzyme in Mycobacterium tuberculosis. KatG catalyses the formation of a radical which forms a complex and blocks the substrate and action of fatty acid synthases which inhibits the synthesis of mycolic acids required for components of the mycobacterial cell wall.

Trimethoprim and Sulfamethoxazole (TMP-SMX) used to treat urinary tract infections

Combination of trimethoprim and sulfamethoxazole (TMP-SMX) is commonly used to treat urinary tract infections and prophylaxis for pneumonia caused by Pneumocystis carinii, which occurs in individuals with HIV. These active agents block the biosynthesis of bacterial folic acid, which is essential for nucleic acid and protein synthesis. In general, resistance emerges more slowly to combination therapy, making it an attractive strategy. Yet, resistance can occur when novel alleles of dihydropteroate synthetase (DHPS) are acquired or overproduced, which is the enzyme inhibited by SMX. This causes an increase in target availability and a decrease in the binding activity of TMP-SMX, maintaining folic acid production and ensuring cell survival.

Inhibiting the efflux pump?

Concept of inhibiting the efflux pump is attractive as efflux pumps are also required for virulence and biofilm formation. Several competitive inhibitors of RND efflux pumps have been discovered or developed but none have reached the clinic largely due to host toxicity.

Inactivation of antibiotics occurs by

Degradation and modification by the transfer of a chemical group

Efflux status

Efflux status affects the rate of evolution of resistance in a population as lack of efflux has been shown to decrease the frequency with which antibiotic resistant mutants are selected. Cells with high expression of acrAB had lower levels of expression of the DNA mismatch repair gene mutS and increased mutation rate enabling high level resistance via accumulation of point mutations. In Staph A, amplification of the NorA efflux pump led to more rapid evolution. Positive epistasis was also detected. This is where a mutation can exert a phenotypic effect but only in concert with other genes making the impact conditional on the genetic background of where it occurs. Increased NorA expression interacted positively with mutations conferring ciprofloxacin resistance in S. aureus to further increase resistance.

Pseudomonas aeruginosa

Encapsulated, gram-negative, aerobic-facultatively anaerobic, rod shaped bacterium that can cause disease. Oppurtunistically infects in immunocompromised. It is capable of aggregating into enduring biofilms.

Changes to ribosome in erythromycin resistance (Erythromycin- a macrolide - against Streptococcus, which is gram positive) - bacteriostatic

Erythromycin displays bacteriostatic activity at high concentrations by binding to the 50S subunit of the bacterial rRNA complex so inhibition of protein synthesis and processes critical for life or replication. It interferes with aminoacyl translocation, preventing the transfer of tRNA bound at the A site of the rRNA complex to the P site of the rRNA complex. The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein.

EE: Horizontal Gene Transfer

Extent of bacterial horizontal gene transfer became evident once whole genome sequencing projects were being undertaken and paper in 2002 (Extensive mosaic structure released by the complete genome sequence of Uropathogenic E. coli). This paper took three isolates of E. coli, a standard lab strain (K12 strain), which is used for cloning in the lab, a Uropathogenic strain (used extensively in the laboratory- actually the strain which was studied to show the involvement of type 1 fimbriae and the Pap Pilus in the ability to cause urinary tract disease) and then another organism of enterohemorrhagic E. coli. The paper looked at the core genes and it turned out that only 40% of the genes in these organisms are actually shared but most of the other genes are genes which are being passed around (accessory genes). This then led to the concept of the pangenome - core genes found in lots of E. coli, but there are lots of other genes which are passed between different strains of E. coli and E. coli then become representations of their genome plus a repertoire of accessory genes which they harbour, and that either means they are pathogenic or non-pathogenic and gives them specific features. Paper showed that there is a mosaic structure in the genome which is so highly conserved as E. coli and this is true for other bacteria as well. The consequence of this is that bacteria are passed around and handing off genes all of the time. This is reflected most obviously nowadays in terms of emergence of antimicrobial resistance.

Fusidic acid-resistant S. aureus

Fusidic acid-resistant S. aureus often expresses FusB-type proteins. Fusidic acid inhibits translation by binding to elongation factor G (EF-G), the ribosome translocase that is involved in processing mRNA and tRNAs and in preventing dissociation of the complex once RNA translocation is complete. FusB proteins contain a zinc finger domain that rescues translation by promoting dissociation of the stalled complex, even though drug binding has occurred.

Gasdermin D leads to cell lysis - proinflammatory form of cell death

Gasdermin D is a promolecule which in its full length state folds back on itself so that its active domain is bound by a sort of immunity portion of the protein. One lobe is cleaved away from the lobe which is the effector domain and that forms pores within the cell membrane, it multimerises and forms pores so gasdermin D is a pore forming toxin and in its full length state its prevented by forming pores by its immunity component. Once this is cleaved by the inflammatory component and that is the active caspase-1 that then releases gasdermin D to insert inside a membrane leading to liberation of IL-1beta (IL-18) and that proinflammatory signalling that goes on with this form of cell death is called pyroptosis. Bacteria can manipulate this pathway as well as being sensed by this pathway.

Aminoglycosides - targets gram negative aerobic and facultative anaerobic bacilli display bactericidal activity - inhibition of bacterial protein synthesis by binding bacterial ribosomes

Gentamicin - pseudomonas, E. coli Tetracycline - bacteriostatic and target Streptomycin bacteria and treat chlamydia Streptomycin - tuberculosis, inhibits both gram-positive and negative bacteria and useful broad spectrum antibiotic

Resistance-nodulation-division efflux affects horizontal gene transfer

In E. coli, the presence of the translation-inhibiting antibiotic tetracycline, the AcrABTolC efflux pump, was required for the acquisition of plasmids carrying TetA, which confers high level tetracycline resistance. Efflux pump reduced intracellular levels of the drug, allowing time to translate proteins encoded on the newly acquired plasmid.

L-form bacteria

In L form bacteria, which are cells that lose their cell wall after stress exposure. L-form bacteria are consequently resistant to cell-wall targeting agents (i.e. beta-lactams) and exposure to these drugs rapidly selects for cell wall-deficient bacteria.

Gram negative bacteria and reduced permeability

In gram negative bacteria it is especially important for antimicrobials to be able to cross the double membrane cell envelope. Alterations to envelope structure, such as porin loss or changes to phospholipid and fatty acid content can affect the ability of a drug to penetrate the cell and can contribute to the emergence of AMR.

Inflammasome is a way of detecting and responding to bacterial invasion.

Inflammasome is a way of detecting and responding to bacterial invasion. Within a few proteins which elicit a proteolytic cascade. Sensor molecules for the inflammasome include examples like NLRP3 which have an ASC adaptor and Caspase-1 effector detecting ATP, ion flux, ROS and pore forming toxins as stimuli. Sensory molecules bind to an adaptor which forms a complex and stimulates an effector which is a protease which cleaves inactive components inside the cell which then lead to effector activities. Gasdermin-D then leads to cell lysis - this is a proinflammatory form of cell death, essentially being a proinflammatory way, alerting neighbouring cells to the fact that there is a pathogen around.

Quinolones - Ciprofloxacin

Inhibition of DNA synthesis by binding to DNA gyrase Ciprofloxacin - against gram negative bacteria, including Pseudomonas aeruginosa - bactericidal

Intergenomic vs. Intragenomic events

Intergenomic: 1. Transformation 2. Transduction 3. Conjugation Intragenomic: Phase variation Antigenic variation

EE: Transformation by Griffiths

Investigating pneumonia - Two strains of pneumococcus (rough non-virulent and smooth virulent) - Smooth strain had a polysaccharide capsule that provided immune evasion, but the rough strain did not - Smooth strain caused the death of the host - Bacteria from the smooth strain were killed by heat, and their remains were added to the rough strain - whilst neither alone harmed the mice, the combination was able to kill the host - isolating the strains from the dead mice Griffith concluded that the rough strain had been 'transformed' by the transforming principle - now we know that the DNA in the smooth strain (killed by heat), had been taken up by the rough strain bacteria containing genes that form the smooth protective polysaccharide capsule

Vancomycin used for enterococcal and MRSA infections - MOA?

It inhibits cell wall synthesis by binding to the terminal D-Alanine-D-Alanine from the pentapeptide precursors, inhibiting peptidoglycan crosslinking, which is essential for the synthesis of the bacterial cell wall. Vancomycin resistance in enterococci is mediated by the acquisition of the van cluster, with the vanA gene cluster being the most prevalent in clinical vancomycin-resistant strains. Instead of binding to D-Alanine-D-Alanine, vancomycin now binds with reduced affinity to terminal D-Alanine-D-lactate or D-Alanine-D-serine. This bypass strategy confers resistance to vancomycin in enterococcus species and other gram-positive bacteria.

Examples of Gram Positive Bacteria - Listeria and Shigella (Intracellular Bacteria)

Listeria causes food poisoning and more serious diseases in immunocompromised. Does not encode T3SS and does not cause this trigger mechanism of entry. Instead it enters by a zipper mechanism in which the bacteria gently adhere to or land on the surface of cells and then just sink below the surface and enter. This is mediated by inernalin A and B in Listeria. Once inside, Listeria take the same route as Shigella in that they escape the phagosome and undergo cell actin mediated motility. Vascular exit mechanism of Listeria is through a pore forming toxin called listeriolysin. The mechanism of Shigella exit are unclear, but has been proposed to be IpaB - translocon protein of the T3SS and both organisms can undergo actin based motility inside a cell but they do this through distinct mechanisms. A protein on one pole of shigella IcsA recruits N-WASP, while listeria Act A recruits and activated ARP 2/3 complex. Here, bacteria have evolved to pick up different parts of the same pathway to lead to the same phenotype and ability to move within cells.

Target bypass - development of MRSA

Methicillin (beta lactam) binds to PBPs and inhibits the transpeptidase domain, causing disruption of the cell wall synthesis. S.aureus can acquire an exogenous PBP (PBP2a) that is homologous to the original target but with lower affinity for beta-lactam antibiotics. This is encoded by the mecA gene located in the staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element that confers methicillin resistance. When methicillin binds to this alternative target site, inhibition of cell wall synthesis is prevented as transpeptidase activity of PBP2a is maintained.

Mycobacteria outer layers

Mycobacteria produce an extensive outer lipid layer and a polysaccharide capsule coat, thereby preventing entry of hydrophilic molecules into the cell.

Biofilms

One common lifestyle for bacteria is growth in biofilms, which are aggregated communities of cells producing a protective extracellular matrix. Biofilms are inherently tolerant to the action of antibiotics and some exhibit resistance due to multiple factors. During the planktonic lifestyle, bacteria are inhibited by the drug. Yet, cells within a biofilm exhibit a large degree of heterogeneity of metabolic states and gene expression which results in individual cells being resistant within a biofilm due to reduced permeability, low metabolic activity resulting in reduced target expression, and production of large numbers of persister cells. Biofilms can experience high rates of genetic exchange, allowing movement of antimicrobial resistance genes.

Beta-lactams (Bactericidal)

Penicillin Amoxicillin Benzylpenicillin Flucloxacillin Block peptide cross linking in growing cell wall

Persisters

Persisters are dormant cells that are often growth arrested but viability is maintained. Persister cells are highly resistant to the killing action of bactericidal antibiotics but regain normal sensitivity once replication resumes. Mechanisms for persister cell formation have been suggested, including toxin-antitoxin systems and production of (p)ppGpp, although there remains much debate about the importance of each. Cells with low levels of ATP are likely to become persisters.

phase variation

Phase variation is a quantitative phenomenon. This is the on / off switching of genes. An E. coli system has a lacZ reporter beta galactosidase that cleaves a chromogenic substrate and so LacZ expressing strains and those without are different colours (blue and white respectively). Type 1 fimbriae is the same mechanism of variation in uropathogenic E. coli. This on / off switching in phase variation is mediated by an invertible switch called the FIM switch. This is a short sequence which is bound by perfect repeat sequences which occur in an inverted orientation. You have repeat sequences which face each other and can undergo recombination. This is mediated by the FimB invertase enzyme. This is a highly evolved system and there are different mechanisms by which on / off switching can occur. Another mechanism common for Neisseria meningitidis is through homopolymeric tracts. Taking the PorA gene encoding the major surface protein can undergo on / off switching. Homopolymeric tracts - tracts of repeated nucleotides either in the promoter or in the ORF of PorA. Due to the low fidelity of DNA polymerase, particularly over repeated sequences, these tract lengths can expand or contract within the ORF. The rest of the gene can go either in or out of the reading frame that leads and can lead to a stop in translation and that lack of PorA expression and you can see that this is a reversible switch as a subsequent replication, which puts things back in frame, switches the gene back on. A switch in the promoter can lead to reduced PorA expression by affecting the orientation of important promoter sequences so this can be reverted by changes in the tract length during DNA replication. The two mechanisms or two sites within the gene which are affected by homopolymeric tracts of sequence. Furthermore, PorA can be interrupted by insertion of mobile DNA within bacteria, which jumps inside the ORF and stops translation. PorA is very subject to variation as it is a target of the immune system that could also undergo antigenic variation. So aside from phase variation, the actual sequence of PorA can change either through point mutation or uptake of DNA through horizontal gene transfer via transformation. This on / off switching affects mostly surface structures of bacteria such as capsule biosynthetic genes, LPS biosynthetic genes etc. but also affects restriction modification systems necessary for bacteria against horizontal gene transfer.

(Modification by the transfer of a chemical group) Phosphotransferases and esterases

Phosphotransferases and esterases can modify and confer resistance to macrolides because the modified macrolies cannot bind as efficiently to the 50S ribosome.

Mobile genetic elements

Plasmids Bacteriophages Transposons Integrons

Porins in E. coli

Porins allow the influx of hydrophilic compounds into the cell, including many antibiotics. OmpF and OmpC found in E coli are trimeric beta-barrel structures through which different antibiotic classes are able to pass. Multi-drug resistant E. coli isolates from patients found to carry multiple mutations within OmpC that alter the electric charge within the pore and this affects the permeability of antibiotics like cefotaxime, gentamicin, or imipenem.

Resistance breakers

Resistance breakers are compounds that can disrupt or inhibit a specific mechanism of antibiotic resistance to restore the clinical efficacy of a specific antibiotic. This could be used to potentiate the use of existing antibiotics, and there is extensive proof of principle that this strategy works clinically as inhibitors of beta lactamase enzymes have been used successfully since the introduction of clavulanic acid in 1981.

Emergence of MRSA

Resistance is due to a resistance gene, macA, which stops Beta-lactam antibiotics from inactivating the enzymes (transpeptidases) critical for cell wall synthesis. MRSA is any strain of S. aureus that has developed (through natural selection) or acquired (through horizontal gene transfer) a MDR to beta-lactam antibiotics.

Resistance to macrolides

Resistance to macrolides can occur through target modification when the 16S rRNA target can be methylated by ribosomal methyltransferases, thereby preventing binding of macrolides.

RND pumps

Resistance-nodulation-division family of efflux transporters confer the most clinically relevant levels of resistance in gram-negative bacteria. RND pumps can export a broad range of structurally and chemically dissimilar antibiotics, and the overexpression of these pumps contributes to MDR in clinical isolates i.e. E. coli.

Phage therapy

Started using in the early 1900s but fell out of favour as a result of antibiotic introduction in the 1940s Bacteria can be treated with a sample of water that naturally contains phages, if the bacteria die the sample can be centrifuged leaving the phages at the top to be collected and tested to see which ones killed the bacteria. Either the phage or products such as endolysins can then be used as antibacterial agents. Highly specific so unlikely to change the bacterial flora of a patient and cause GI side effects. Mutant phages that are not recognised by the immune system - to not activate the immune response against the phages. At the University of Tokyo, it was shown that silkworm larvae and mice provide comparable results regarding the effectiveness of experimental antibiotic therapies. This makes the cost of drug discovery cheaper as no rodents are required.

E. coli mutation for decreased susceptibility to beta-lactam antibiotics

mutation of PBP3 in E. coli has been identified to be ubiquitous in clinical isolates from India resistant to aztreonam and avibactam.