Antibacterials Part 1 2009PHM PDF

▪ Describe how peptidoglycan is made and the mechanism of action of the β-lactams ▪ Discuss how resistant bacterial strains have arisen, and what worsens the resistance ▪ Outline how we test for antibiotic sensitivity/resistance ▪ Outline the different penicillins and where/when they are us...

▪ Describe how peptidoglycan is made and the mechanism of action of the β-lactams ▪ Discuss how resistant bacterial strains have arisen, and what worsens the resistance ▪ Outline how we test for antibiotic sensitivity/resistance ▪ Outline the different penicillins and where/when they are used (e.g. dosage form, strain susceptibilities)  Inhibition of synthesis or damage to the -lactams e.g. Penicillins, cephalosporins, peptidoglycan cell wall monobactams, carbepanems, vancomycin Inhibition of synthesis or damage to the Polymyxins and daptomycin cytoplasmic membrane Modification in synthesis or metabolism Quinolones inhibit DNA gyrase, rifampin of nucleic acids inhibits RNA polymerase Inhibition or modification of protein Protein synthesis is inhibited by synthesis aminoglycosides, tetracyclines, erythromycin, chloramphenicol, Modification in energy metabolism clindamycin... Folate antagonists such as sulfonamides and trimethoprim interfere with cell metabolism ▪ many drugs have a high therapeutic index since they are selectively toxic ▪ host immune system assist in the clearance of infection ▪ therefore, use of antibiotics especially important in HIV/neutropenic patients  ▪ Need drugs that show selective toxicity to bacteria ▪ Targets should be unique to bacteria, therefore what are the key differences between bacteria and human cells? Bacteria versus human cells ▪ Cell wall in prokaryotes has peptidoglycan –  unique to prokaryotes. - important for nutrient import and protection against osmotic shock. ▪ DNA and RNA synthesis – some biochemical processes are common to both eukaryotes and prokaryotes, so we need to find key differences between them. ▪ Cytoplasm – contains soluble enzymes and other proteins, ribosomes (for protein synthesis) and small molecule biochemical intermediates, but bacteria do not have nuclei or mitochondria. ▪ The outer membrane in bacteria, which defines them taxonomically as Gram-negative or Gram- positive bacteria, may prevent penetration of antibacterial agents.  Peptidoglycan ▪ Both gram-positive and gram-negative bacteria have peptidoglycan (PG) in their cell wall. PG’s do not occur in eukaryotes. - Gram-positive: much thicker PG layer - Gram-negative: thin PG layer, contains a lipopolysaccharide layer 15-30 PG strands 3-5 PG strands Gram stain of a mixed S. aureus (Gram positive) and E. coli (Gram negative) culture. A Gram stain is a laboratory technique that distinguishes between Gram positive and Gram negative groups of bacteria by colouring these cells with red or violet dyes. Gram positive bacteria stain violet because their thick peptidoglycan holds on to the violet dye; Gram negative bacteria stain red (or pink) due to their thinner peptidoglycan wall which doesn’t retain the crystal violet but retains a red dye. ▪ Peptidoglycans (PGs) are composed of repeating disaccharide units of N-acetylglucosamine (NAG) and N-acetylmuramate (NAM) connected by -1,4-linkages ▪ A pentapeptide is joined to the NAM glycan ▪ A transpeptidase* enzyme does the following reaction: - NAG - NAM - NAG - NAG - NAM - NAG Peptide cross-link * the transpeptidase is also transpeptidase known as a “penicillin binding protein” or PBP, because that is where penicillins act. - NAG - NAM - NAG - NAG - NAM - NAG  Note this peptide cross- link (“bridge”) between the two glycan peptides ▪ The peptide cross-links form a meshwork, thus providing mechanical strength, barrier to osmosis etc.  The discovery of penicillins ▪ Sir Alexander Fleming discovered penicillin when he noticed that a culture of bacteria was contaminated by an airborne fungus (a Penicillium species) which produced a clear zone of inhibition around the fungal colony. The fungus appeared to be secreting a Sir Alexander Fleming substance which killed the bacteria. ▪ The unstable antibiotic was eventually purified and trialed clinically in humans and then used in the armed forces in 1943. ▪ In 1993, approximately half the money spent worldwide on antibiotics was for penicillins. ▪ All penicillins are based on the β-lactam structure (a cyclic amide). The original plate A four-membered lactam (a β-lactam) β-lactam antibiotics ▪ -lactams have a -lactam ring structure (cyclic amide). This small ring is structurally unstable – it is readily hydrolysed in the gut due to high stomach acidity. ▪ They inhibit bacterial cell wall synthesis. How? Recall: the transpeptidase enzyme catalyses the cross-linking reaction: ▪ the -lactams form a covalent bond with the bacterial transpeptidase enzyme, thus inactivating it and preventing cross-linking - Note that penicillins and cephalosporins are actually structural analogs of the D-Ala-D-Ala end of the peptide to be cross-linked ▪ Bacterial cells then undergo autolysis. They burst. ▪ However, many are prone to degradation by bacterial -lactamase enzymes Unfortunately, bacterial resistance arose very quickly (within 15 years) after penicillin was discovered and used clinically. Let’s talk about that type of resistance, and the other types of resistance that have arisen since then. ▪ A serious global issue has arisen whereby bacteria (and other microbes) survive in the presence of drugs that used to treat them. In other words, strains have become resistant. ▪ The way by which resistant organisms develop under selection pressure (i.e. constantly exposed to the drug) is via spontaneous random mutation. e.g. 1 in 10 million will have a spontaneous mutation that makes them relatively less sensitive to any one drug ▪ Drug treatment → kills off the more sensitive bacteria. The more resistant bacteria will grow (i.e. be selected)…. they have a selective growth advantage over the others. - more rounds of mutation and selection → bacteria become progressively more resistant. ▪ The alternative to this, of course, is that bacteria could acquire a plasmid encoding a resistance gene (by conjugation – see next slide). ▪ Intrinsic resistance: Some microbes such as Pseudomonas aeruginosa are already resistant – or are intrinsically resistant – to many antibiotics because the antibiotics are simply unable to cross its outer membrane or bind to target sites. ▪ Acquired resistance: some bacteria acquire new resistant genes (e.g. from another bacterial strain) or mutate their own genetic information. They acquire resistance in some way. How do bacteria acquire resistance? A. Conjugation: two bacteria exchange genetic information, usually from a plasmid containing a gene that encodes an enzyme. B. Transduction: a virus (bacteriophage) that exchanges DNA. C. Transformation: when bacteria pick up exogenous DNA from the environment. Mechanism A is the most common, and resistance-conferring plasmids have been identified in virtually all bacteria. What are the types of resistance mechanisms? A. Altered receptors for the drug B. Enzymatic degradation of the drug C. Bypassing by using another metabolic pathway (resistant metabolic pathways). The bacteria survive inhibition of a pathway by a drug by finding another metabolic pathway to meet its needs D. Decreased entry (reduced entry of a drug) or increased efflux (pumping out) of a drug. What is the bacterial resistance mechanism against the β-lactams? ▪ Unfortunately, bacterial quickly developed resistance to the β-lactams (within 15 years of the discovery of penicillin). Somehow, bacteria could survive in the presence of the drug. ▪ It was soon discovered that a serine protease enzyme, now called β-lactamase*, was responsible for the destruction of β-lactam drugs. Bacteria had evolved this mechanism, and we now find β-lactamase enzymes in all strains of penicillin-resistant bacteria. For Gram positive bacteria, the β-lactamases are secreted into the medium and encounter the drug outside the cell wall. For Gram negative bacteria, the β-lactamases are present between the outer lipopolysaccharide layer and the inner peptidoglycan layer. *N.B. sometimes β-lactamases are called “penicillinases”  β-lactamases enzymes in action ▪ The β-lactamase enzyme comes from a gene within a plasmid inside the bacterial cell. In other words, the resistance is plasmid-encoded which means it can be spread from strain to strain by conjugation. The left half of this plate was spread with a resistant strain of E. coli bacteria, the right half spread with the sensitive (wild-type) bacteria. Two different drug-containing tablets were placed in the centre-line on the plate, and the plate incubated at 37°C overnight for the bacteria to grow. drug still effective against both strains drug ineffective against resistant strain Different classes of -lactamases now exist, depending on their mechanism of action Over 300 different -lactamases have been characterised. -lactam-resistant wild-type E. coli strain  β-lactam resistance: PBP mutation ▪ Other than evolving, or sharing via conjugation, β-lactamase enzymes, bacteria have “learned” another technique in order to escape β-lactams: mutate their own penicillin binding protein (PBP). ▪ Remember that the PBPs are also known as the transpeptidase enzyme that performs the peptidoglycan cross-linking enzyme reaction. There are 4 or 5 different types of PBP, and they are located in the bacterial cytoplasm. e.g. cephalosporin (a β-lactam) bound directly to serine 62 of the bacterial PBP active site → this prevents peptidoglycan crosslinking reaction from occurring, and the bacteria will die. → BUT: if bacteria mutate this enzyme e.g. by replacing Ser62 with a different amino acid, the drug won’t bind and the bacteria are now resistant to the drug! This is called a PBP mutation. ▪ So: some PBPs within bacteria have acquired PBP mutations that have changed the enzyme’s affinity for the antibiotic. Example: Methicillin Resistant Staphylococcus aureus (MRSA) a serious problem of a staphylococci that is resistant to all available -lactams This strain has acquired a PBP called “PBP-2a” that has a very poor affinity with all -lactams. ▪ Resistance is aggravated by: ❖ trivial use of antibiotics ❖ poor compliance ❖ patients not completing courses or using insufficient doses, or ❖ sharing prescriptions with others. ▪ Using broad spectrum drugs is a problem, as it encourages resistance. ❖ broad spectrum drugs apply selection pressure to many populations of bacteria, providing multiple opportunities for resistance to arise. ❖ use narrow spectrum drugs instead: “narrow the therapy, narrow the chances of resistance.” ▪ Bacterial “bystanders” are non-target strains of bacteria e.g. in the gut flora, that may develop resistant genes by being co-exposed to a drug. Such bystanders can become reservoirs for resistance genes (e.g. on plasmids) and transfer them on to other organisms by conjugation.  Antibiotic sensitivity testing ▪ Let’s look at some examples.  β-lactamase-producing bacteria ▪ Antibiotic susceptibility versus resistance can be detected on agar plates covered with a lawn of bacteria, and with drug “disks” placed on top of the lawn. ▪ The extent of the zone of inhibition around the disk determines the level of susceptibility or resistance, as well as the boundary around the zone. AMP = ampicillin ▪ Below: note the loss of penicillin (P) susceptibility in a Staphylococcus aureus strain that produces β-lactamase. MRSA is even worse!  β-lactamases: ESBLs ▪ Some β-lactamases can confer resistance to most β-lactam antibiotics, including penicillins, cephalosporins and monobactams. These special β-lactamases are called extended-spectrum β-lactamases, or ESBL. ▪ We can test antibiotics on a plate covered with ESBL-producing bacteria, and look at zones of inhibition around the drugs. People who are infected with ESBL-producing organisms have been associated with poor clinical outcomes. A good example of this is community and hospital-acquired ESBL-producing Enterobacteriaceae. This infectious organism is prevalent worldwide. Also, infections with hospital-acquired ESBL-producing Klebsiella pneumonia are on the rise. This species infect the GI tract, eyes, respiratory tract and cause UTIs (urinary tract infections). Infections are often treated with carbapenams, or sometimes with piperacillin/tazobactam. However, strains that destroy carbapenams (they now produce carbapenamases) have arrived.  β Now let’s talk about the various types of β-lactam drugs.  β-lactams: penicillins ▪ Now let’s focus on the drugs themselves. Penicillins ▪ Simple/standard penicillins benzylpenicillin (or penicillin G - this is acid labile) phenoxymethylpenicillin (or penicillin V - this is acid stable) ▪ Phenoxymethypenicillin is less active than benzylpenicillin - reserved for situations where high tissue concentrations are not required (e.g. sore throat) - food has little effect on absorption  ▪ Repository* forms of standard/simple penicillins mix of benzylpenicillin and the stabiliser benzathine benzathine penicillin procaine penicillin mix of benzylpenicillin and the local anaesthetic procaine ▪ Intramuscular (IM) administration of these drugs – NOT intravenous (IV). IV administration can cause death! ▪ The dosage form is via a suspension, NOT a solution. This makes it available in the body for prolonged periods at low concentrations, thus giving long-term antibiotic action for 2-4 weeks after a single IM dose (e.g. useful for syphilis, diphtheria). ▪ Once they has slowly absorbed into the bloodstream, these drugs are hydrolysed to benzylpenicillin. Therefore, they are useful for infections where prolonged, low doses of benzylpenicillin are required (as stated above). *repository form: in pharmacology referring to the injection, usually intramuscularly, of a long-acting drug, which is slowly absorbed and is therefore prolonging its action.  ▪ Anti-staphylococcus penicillins [floo-klox-a-SILL-in] flucloxacillin [dye-klox-a-SILL-in] dicloxacillin [ox-a-SILL-in] oxacillin ▪ These special penicillins were designed by modifying the penicillin molecule so that β- lactamases could no longer break them down. ▪ This made them extremely useful against resistant Staphylococcus strains. Methicillin also belongs to this class, but it has been withdrawn due to MRSA. ▪ BAD NEWS: MRSA has developed resistance against flucloxacillin, dicloxacillin AND oxacillin. So now we have MRSA strains that may be resistant to one, some, or all of these drugs.  ▪ Aminopenicillins [a-mox-a-SILL-in] amoxicillin [am-pi-SILL-in] ampicillin ▪ These are broad spectrum penicillins. ▪ In order to counter β-lactamases, amoxicillin can be combined with a β-lactamase inhibitor called clavulanic acid (or clavulanate). This combination formula is known as “Augmentin”. Amoxicillin is degraded by β-lactamases, and is often given in combination with clavulanate. The clavulanate itself it not an antimicrobial, but it’s similarity in structure to a –lactam allows it to competitively inhibit –lactamase. ▪ Anti-pseudomonal penicillins [pip-er-a-SILL-in] piperacillin [tye-kar-SILL-in] ticarcillin Nail infection with Pseudomonas aeruginosa. ▪ These have the widest spectrum of all penicillins and are sometimes known as extended-spectrum penicillins. They are especially effective at treating Pseudomonas aeruginosa infections. ▪ Piperacillin is combined with the β-lactamase inhibitor tazobactam. ▪ Ticarcillin is combined with the β-lactamase inhibitor clavulanic acid. ▪ Can name example drugs from the 4 different generations of cephalosporin drugs, the strains/diseases they treat and their basic therapeutics ▪ State drugs from the carbapenems and monobactams, and any challenges or advantages in using these medicines ▪ Can outline the mechanism of action of vancomycin and how it is different to a β-lactam drug, and state how vancomycin is used therapeutically ▪ Can show how resistance has arisen to vancomycin Cephalosporins ▪ Unlike penicillin G, the first cephalosporin discovered (cephalosporin C) had very poor antibiotic activity (about 1/1000th the activity of penicillin C). ▪ Cephalosporins (especially 1st generation compounds) are generally not orally active. ▪ They have similarly poor activity against Gram positive and Gram negative bacteria – this feature was observed early in their development, and is key to their performance. ▪ They have their β-lactam ring fused to a 6-membered ring (unlike the 5-membered ring in penicillins). ▪ The cephalosporins have the same mechanism of action as the penicillins, and they are affected by the same resistance mechanisms. - However, they tend to be less vulnerable to β-lactamases compared to penicillins (although that is becoming less so). - They are classified as first, second, third and fourth spectrum, and now “advanced generation”, based mostly on their bacterial susceptibility as well as their vulnerability level to β-lactamases. Each newer generation has greater Gram-negative properties than the preceding, except fourth generation drugs which are truly broad spectrum. First generation cefazolin, cefalexin These can be used as a substitute for benzylpenicillin (penicillin G). This is because they are not destroyed by penicillinase (i.e. the β-lactamase that destroys penicillin). They have activity against Proteus mirabalis, E. coli and K. pneumoniae (i.e. “PEcK”), which are all Gram negative strains.  Second generation cefaclor, cefoxitin H. influenza on blood agar These have greater activity against Gram negative organisms e.g. Haemophilus influenza (which causes many types of infections), but weaker against Gram positive bacteria. Third generation ceftriaxone, cefotaxime Extremely good penetration of the BBB (blood-brain barrier), and are therefore the drugs of choice for treating bacterial meningitis caused by susceptible organisms.  Fourth generation cefepime This drug must be administered parenterally. It has a wide antibacterial spectrum, with activity against streptococci and staphylococci (but only those that are not MRSA!). They are effective against aerobic Gram negative organisms, such as Enterobacter species, E. coli, K. pneumoniae, P. miribalis, and P. aeruginosa. Mechanisms of resistance The chemical mechanism of destruction is similar to penicillin degradation by β- lactamases.. BUT The β-lactamases in Staphylococcus that destroys penicillins does not degrade cephalosporins. Instead, cephalosporins are vulnerable to ESBLs, especially in ESBL-producing bacteria such as E. coli and K. pneumoniae. ▪ Route of administration: Many of them must be administered IV or IM, because of their poor oral absorption. ▪ Distribution: All distribute very well into body fluids. But in order to get into the brain, only a few cephalosporins can do this. This is required in treating bacterial meningitis. Interesting point: cefazolin penetrates bone. Therefore, it is effective in orthopaedic surgery to prevent infection. OA = osteoarthritis; Fx = femoral neck fracture ▪ Elimination: most are excreted via the kidneys, which means you will need to reduce the dosages in patient who also suffer from renal dysfunction (otherwise it will accumulate, leading to kidney toxicity). ▪ Like penicillins, the cephalosporins are generally well tolerated. The common ADEs (diarrhoea, nausea, rash, electrolyte disturbances, pain and inflammation at injection site) are similar. And, once again, allergic reactions are a concern. anaphylactic reactions Stevens-Johnson syndrome toxic epidermal necrolysis If patients experience any of these during the use of penicillin, then they must not be prescribed cephalosporins. ▪ Avoid cephalosporin use in people with penicillin allergy – remember the cross- reactivity between penicillin and cephalosporins is 5-10% (highest in first generation cephalosporins). Note that this does not have anything to do with the β-lactam ring, but instead due to the similarity in the side chain. ▪ Carbapenams are synthetic β-lactams. They include: imipenam meropenam ertapenam ▪ They are effective against many β-lactamase-producing Gram positive and Gram negative bacteria. ▪ They are able to resist hydrolysis by most β-lactamase enzymes. However, they are vulnerable to another type of β-lactamase enzyme called the metallo-β-lactamases. Note that imipenam is metabolised to a toxic metabolite by the kidney enzyme dehydropeptidase. Therefore, it is co-administered with cilastatin which inactivates this The hydrolysis of imipenam by a metallo-β- lactamase. The enzyme contains a zinc ion. enzyme. ▪ These β-lactam drugs are unique because the β-lactam ring is not fused to another ring. An example drug is aztreonam. ▪ It is able to resist the action of most β-lactamases, but NOT the ESBLs produced by some strains of Klebsiella, E. coli and Enterobacter spp. ▪ It is administered either IM or IV - i.e. a parenteral dose only – and is relatively non-toxic. However, it can still accumulate in patients with renal failure. Allergy – advantage over other β-lactams ▪ The drug has a low immunogenic potential – it shows little cross-reactivity with antibodies induced by other β-lactams. This means that this drug may offer a safe alternative for treating patients who are allergic to other penicillins, cephalosporins, or carbapenams. Now let’s talk about a cell wall inhibitor that is NOT a β-lactam drug.  β-lactam vancomycin ▪ This drug is structurally different from the β-lactams and functions by a different mechanism of action. ▪ It is a glycopeptide, and it binds to the free carboxy end of the pentapeptide that stems from the NAM residue in peptidoglycan. → this sterically interferes with the cross-linking of the peptidoglycan backbone → it actually binds the D-Ala-D-Ala. Cross-linking between the terminal D-Ala and the pentaglycine chain is now not possible. ▪ Incorporation of NAG-NAM-Peptide units into polysaccharide chains during peptidoglycan synthesis is thus prevented. ▪ Typically restricted to the treatment of serious infections caused by resistant, Gram-positive infections (e.g. MRSA) OR ▪ Patients who have these Gram-positive infections but are allergic to β-lactams. ▪ In hospitals with high rates of MRSA, IV vancomycin is used in individuals with prosthetic heart valves (or in patients receiving implants with prosthetic devices). C. difficile ▪ The oral absorption of vancomycin is 0%. This means it can be used orally for the treatment of severe antibiotic associated C. difficile colitis infections. Special note: do not use an excessive rate of infusion of vancomycin, otherwise an infusion reaction called “red neck” or “red man syndrome” occurs within 10 minutes. It is flushing, or an erythematous rash, on the face and upper body. RESISTANCE to vancomycin is caused by: - bacteria which produce a new pentapeptide that ends in a terminal D-Ala-D-Lac (instead of the D-Ala-D-Ala), which does not bind vancomycin → An example is Vancomycin Resistant Enterococci (VRE), which increase the expression of an enzyme that modifies the D-Ala-D-Ala to produce D-Ala-D-Lac ends. VRE bacteria Patient with VRE infection ▪ Widespread use of cephalosporin and vancomycin antibiotics, for example, has promoted the proliferation of the once benign intestinal bacterium Enterococcus faecalis, which is not killed by these drugs but is heavily exposed to them. E. faecalis bacterium in a blood culture. These gut bacteria become a “reservoir” for drug resistance genes as they develop resistance. ▪ The E. faecalis evolves drug resistant genes, enabling it to metabolise antibiotics such as vancomycin. ▪ Fear that vancomycin-resistant E. faecalis will transfer those resistance traits to S. aureus pathogens. Big problem, as some S. aureus strains (like MRSA) are multi-drug resistant and only respond to vancomycin → patients will be incurable! Thus, the normally harmless E. faecalis is becoming a dangerous reservoir of antibiotic-resistance traits. Bacteria will only be selected if they are affected: narrow down the therapy, narrow the chances of resistance spreading