Antibiotics Inhibiting Bacterial Cell Wall Or Membrane Integrity (Lecture Notes, PDF)

Joshua L. Hood M.D., Ph.D. Antibiotics Inhibiting Bacterial Cell Wall or Membrane Integrity This course covers antibiotics that inhibit bacterial cell wall and membrane integrity antibiotics (e.g., beta-lactams, vancomycin, daptomycin). The lecture material is focused on the mechanism(s)...

Joshua L. Hood M.D., Ph.D. Antibiotics Inhibiting Bacterial Cell Wall or Membrane Integrity This course covers antibiotics that inhibit bacterial cell wall and membrane integrity antibiotics (e.g., beta-lactams, vancomycin, daptomycin). The lecture material is focused on the mechanism(s) of action and mechanism(s) of antibiotic resistance within antibiotic classes. Example drugs within each antibiotic class and susceptible bacteria within the spectrum of activity of each drug class are listed as a reference. It is not expected for you to memorize the lists of drugs or susceptible bacteria presented throughout, but rather to have a general understanding of the indications of each drug class. Occasionally there may be a unique piece of trivia pertaining to a particular drug that has known to be included on licensing and board exams (e.g., penicillin hypersensitivity). Be sure to keep track of know those kinds of associations. Selecting the correct drug for a particular indication is an evolving process based on experience and drug sensitivity. So, while there are drugs of choice for certain indications, they may not always work given the ever- changing landscape of antibiotic resistance necessitating the use of drugs from alternative classes. Finally, go through the practice exam questions with explanations at the end of the lecture. This serves two purposes. One, they function as a summary to cover the important concepts presented in the lecture you should know, and two, they are very similar to the exam questions. If you can answer the practice questions you should do well on my antibiotic questions on the exam. Antibiotics Inhibiting Bacteria Cell Wall or Membrane Integrity Outline Learning Objectives General Bacterial Cell Wall, Membrane and Beta Lactam Structure Penicillins (β-Lactam Cell Wall Inhibitors) Cephalosporins (β-Lactam Cell Wall Inhibitors) Carbapenems (β-Lactam Cell Wall Inhibitors) Monobactams (β-Lactam Cell Wall Inhibitors) Vancomycin (Unique Cell Wall Inhibitor) Daptomycin (Cell Membrane Inhibitor) Polymyxins (Cell Membrane Inhibitor) Bacitracin (Cell Wall and Membrane Inhibitor) Practice Questions with Explanations References Lesson 1 of 13 Antibiotics Inhibiting Bacteria Cell Wall or Membrane Integrity Outline Joshua L. Hood M.D., Ph.D. Lesson 2 of 13 Learning Objectives Joshua L. Hood M.D., Ph.D. Describe the general mechanism(s) of action for each drug class and how they differ between the drug classes. Compare and contrast mechanism(s) of antibacterial drug resistance between each drug class. List medical conditions typically treated with each drug class and appreciate how the drug used to treat those medical conditions is related to the spectrum of activity of the drug class (e.g., gram + or gram -). See the note below for an example. Note any unique severe adverse drug effects that might be anticipated on board type questions. For example, penicillin can cause hypersensitivity reactions.  For example, if a particular drug class is useful for treating plague, then you know from your microbiology lectures that plague is caused by Yersinia pestis. So, in that case, it’s easy to connect the drug class to the susceptible microorganism. However, if a drug class is particularly good at treating abscesses, then you know there are a myriad of Staphylococcal spp., Streptococcal spp. and other bacterial species that might be responsible. There is no need to track and attempt to memorize all potentially susceptible bacterial strains that could cause an abscess. Lesson 3 of 13 General Bacterial Cell Wall, Membrane and Beta Lactam Structure Joshua L. Hood M.D., Ph.D. Bacterial Cell Wall and Membrane Structure Image adapted from https://www.pinterest.com.au/pin/82331499427600732/ Image adapted from https://cdn1.byjus.com/biology/2018/05/16065016/ Difference-between-Gram-positive-and-Gram-negative-Bacteria.png Beta-lactam Antibiotic Structure Figure adapted from Fig. 1 Major subfamilies of b-lactams. Hamed RB, Gomez- Castellanos JR, Henry L, Ducho C, McDonough MA, Schofield CJ. The enzymes of β- lactam biosynthesis. Nat Prod Rep. 2013;30(1):21-107. Epub 2012/11/09. doi: 10.1039/c2np20065a. PubMed PMID: 23135477. All beta-lactam antibiotic classes include a beta- lactam ring structure. Figure adapted from Figure 2. Penicillins, cephalosporins, monobactams, and carbapenems all contain a beta-lactam ring, the site of inactivating beta-lactamase enzymes. https://www.coursehero.com/study-guides/microbiology/mechanisms- of-antibacterial-drugs C O NT I NU E Lesson 4 of 13 Penicillins (β-Lactam Cell Wall Inhibitors) Joshua L. Hood M.D., Ph.D. Penicillins Penicillin were originally discovered by Alexander Fleming in 1928. Staphylococcus aureus failed to grow when contaminated by the green mold Penicillium notatum.  Penicillium Notatum. Image source: https://www.youtube.com/watch?v=z-G-vKTxb9c Structure of Penicillins Image adapted from: https://image.slidesharecdn.com/penicillins-151005180633- lva1-app6892/95/penicillins-13-638.jpg?cb=1444143249 All penicillins have a common general structure. A thiazolidine ring is connected to a β-lactam ring carrying a secondary amino group. The R-group corresponds to the penicillin drug name. Penicillins: Mechanism of Action Bacteria contain a variety of penicillin binding proteins (PBPs). PBPs are transpeptidase enzymes that are necessary for bacterial cell wall synthesis. PBP2 in E. coli maintains their rod shape. PBP3 facilitates septation during bacterial cell division. Penicillins are bactericidal. They work by inhibiting bacterial cell wall synthesis. They bind to the active site of PBPs with transpeptidase activity. This inhibits the bacterial transpeptidation reaction necessary for cell wall synthesis. This results in osmotic instability and autolytic bacterial cell death by activating enzymatic breakdown of peptidoglycan which normally should only be broken down at locations of cell wall remodeling. Image adapted from https://www.dnatube.com/video/2418/Penicllin-mechanism Penicillins bind to the active site of bacterial transpeptidase preventing cell wall synthesis activity. Image adapted from https://image2.slideserve.com/4558522/the-selective-binding- of-penicillin-to-the-active-site-of-transpeptidase-n.jpg  Penicillins and cephalosporins inhibit bacterial transpeptidase synthesis of glycopeptide polymers making of up the bacterial cell wall. Image adapted from https://basicmedicalkey.com/penicillins-cephalosporins-and- other-%CE%B2-lactam-antibiotics-2/ Penicillins: Indications and Spectrum of Activity Penicillin G (im, iv) and V (oral) are natural penicillins. Others are semi-synthetic enabling broader spectrum coverage as shown in the table and other sections below. Natural penicillins are effective against: gram positive staphylococci spp., streptococci spp., actinomyces gram negative meningococci (N. meningitidis), spirochetes (T. Pallidum), Borrelia and Leptospira.  Penicillin is the drug of choice for all clinical stages of syphilis, neurosyphilis, congenital syphilis, or syphilis during pregnancy. Penicillins differ in their spectrum of activity. Figure adapted from Figure 2. Penicillins, cephalosporins, monobactams, and carbapenems all contain a beta-lactam ring, the site of inactivating beta-lactamase enzymes. https://www.coursehero.com/study-guides/microbiology/mechanisms- of-antibacterial-drugs/ Penicillin Drug Resistance Antibiotic resistance encoded on bacterial plasmids can be transferred to other bacteria.  Image adapted from Figure 1. Primary mechanisms of b-lactam resistance in Enterobacteriaceae. Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: Here is the storm! Trends Mol Med. 2012;18:263-72. doi: 10.1016/j.molmed.2012.03.003. Major mechanisms of penicillin drug resistance include: enzymatic inactivation β-lactamases (penicillinase)  decreased permeability Gram (-) bacteria efflux porin modifications penicillin binding protein (PBP) mutations Penicillinase Penicillinase is a beta-lactamase produced by bacteria specific to penicillins. It inactivates penicillins by hydrolyzing the beta-lactam ring to produce penicilloic acid. Penicilloic acid does not have antibacterial activity. Methicillin was developed to be resistant to penicillinase but resistance to methicillin has occurred as well. Methicillin-resistant Staphylococcus aureus (MRSA) is an example. Interestingly, Syphilis, caused by the spirochete Treponema pallidum, has remained sensitive to penicillin. T. pallidum beta lactamase may paradoxically produce penicillin hydrolysis byproducts that inhibit T. pallidum Tp47 PBP rather than eliminating penicillin function. Image adapted from Figure 1. Diagram of the two principal antibiotic resistance mechanisms observed in MRSA bacteria. Murphy J, Walshe R. Modeling Antibiotic Resistance in Bacterial Colonies Using Agent-Based Approach. 2011. p. 131-54.  Image adapted from https://image4.slideserve.com/570544/the-effect-of- penicillinase-on-penicillins-l.jpg Bacteria can produce different, clinically relevant classes of β-lactamases to inactivate beta lactam antibiotics. Penicillin Efflux  Image adapted from Figure 2. Potential Mechanisms of Antimicrobial Resistance in Acinetobacter. Munoz-Price LS, Weinstein RA. Acinetobacter infection. N Engl J Med. 2008;358(12):1271-81. Epub 2008/03/21. doi: 10.1056/NEJMra070741. PubMed PMID: 18354105. Gram (-) negative bacteria have penicillin efflux pumps that transport β-lactam antibiotics from the periplasm back across the outer membrane. Porin Modification Porin resistance occurs in gram (-) bacteria. They have an impermeable outer membrane that is absent in gram (+) bacteria. Beta-lactam antibiotics cross the outer membrane and enter gram (-) organisms via porin protein channels. Porin modification imparts antibiotic resistance. Porin's can be absent, downregulated or differentially expressed. Expression of different porins such as outer membrane protein F (OmpC) vs. OmpF in E. coli results in differences in channel size that restrict penicillin uptake. Image adapted from Figure 1. The major non-selective porins in the outer membrane of E. coli. Ferenci T, Phan K. How Porin Heterogeneity and Trade-Offs Affect the Antibiotic Susceptibility of Gram-Negative Bacteria. Genes. 2015;6(4):1113-24. PubMed PMID: doi:10.3390/genes6041113. Penicillin Binding Protein Mutations Alteration of PBPs causes antibiotic resistance by impairing the the ability of penicillin to bind to and inhibit the PBP transpeptidase active site. As a result, cell wall cross-linking, necessary for bacterial survival and cell division, is not inhibited. An example is methicillin-resistant S. aureus (MRSA) strains expressing the mecA gene. The mecA gene encodes PBP2a that prevents beta- lactam antibiotic function Image adapted from Figure 1. Diagram of the two principal antibiotic resistance mechanisms observed in MRSA bacteria. Murphy J, Walshe R. Modeling Antibiotic Resistance in Bacterial Colonies Using Agent-Based Approach. 2011. p. 131-54. Penicillins: Pharmacokinetics The pharmacokinetics of penicillins are uncomplicated and not typically associated with major issues. They are eliminated in the urine, mostly unchanged. Penicillins: Adverse Effects and Hypersensitivity The major adverse effects noted for penicillins include hypersensitivity reactions, interstitial nephritis, hemolytic anemia, and Pseudomembranous colitis (C. difficile). Penicillins are generally well-tolerated Allergic, maculopapular rashes can occur. Benzylpenicillin is most likely to result in anaphylaxis. Sensitivity to one penicillin typically results in sensitivity to other classes of penicillins. Penicillins are contraindicated in patients with a history of severe cutaneous reaction (Stevens-Johnson syndrome or toxic epidermal necrolysis), interstitial nephritis or hemolytic anemia. Confirmatory skin testing for a penicillin allergy in such patients is not permitted. Approximately 10% of patients allergic to penicillins will exhibit hypersensitivity reactions to cephalosporins. Penicillins and cephalosporins can cause diarrhea via disruption of gut microbiota. Rarely, pseudomembranous colitis can occur. Penicillinase Sensitive Semi-Synthetic Penicillins  Image adapted from Figure 1. Diagram of the two principal antibiotic resistance mechanisms observed in MRSA bacteria. Murphy J, Walshe R. Modeling Antibiotic Resistance in Bacterial Colonies Using Agent-Based Approach. 2011. p. 131-54. Aminopenicillins Aminopenicillins are semi-synthetic but still susceptible to β-lactam ring cleavage. They are generally useful for broader spectrum coverage against gram (-) species including Hemophilus influenzae, Escherichia coli. Amoxicillin  Dental prophylaxis for heart valve patients. Better oral availability than penicillin. Ampicillin Drug of choice for gram (+) Listeria monocytogenes Image adapted from Figure 1. Diagram of the two principal antibiotic resistance mechanisms observed in MRSA bacteria. Murphy J, Walshe R. Modeling Antibiotic Resistance in Bacterial Colonies Using Agent-Based Approach. 2011. p. 131-54. Anti-pseudomonal Penicillins Anti-pseudomonal penicillins are semi-synthetic carboxypenicillins but still susceptible to β-lactam ring cleavage. Piperacillin, Ticarcillin, Carbenicillin Anti-pseudomonal penicillins are used as extended spectrum coverage against Pseudomonas species such as Pseudomonas aeruginosa and other gram (-) rods. Penicillinase Resistant Penicillins Anti-staphylococcal Penicillins Penicillinase resistant penicillins are semi-synthetic modifications of natural penicillins that inhibit penicillinase-based antibiotic resistance. Steric interference imparted by R-group side chains of different penicillins protects the β- lactam ring from penicillinase cleavage. Generally useful for gram (+) β-lactamase producing staphylococci or streptococci. Dicloxacillin Localized staphylococcal infections Cloxacillin Skin, structure, upper RTI, pneumonia, and bladder infection *Methicillin Not used clinically given adverse effects. Interstitial nephritis Used for antibiotic susceptibility testing.  methicillin resistant S. aureus (MRSA) Nafcillin or Oxacillin Drugs of choice for staphylococcal endocarditis Image adapted from https://image.slidesharecdn.com/penicillin- 170130061717/95/penicillin-34-638.jpg?cb=1485757294 Penicillins incorporating a penicillinase (β-lactamase) inhibitor β-lactamase inhibitors include clavulanic acid, sulbactam, and tazobactam. They are co-administered with β-lactam antibiotics to inhibit microbial resistance. They have no significant antibacterial activity alone. β-lactamase inhibitors differ in their potency against different beta-lactamases.  Clavulanic acid is most potent in general. β-lactamase inhibitors have a similar structure to β-lactam antibiotics. They are hydrolyzed similar to β-lactam antibiotics resulting in the irreversible inactivation of β-lactamases via covalent crosslinking. Augmentin = amoxicillin + clavulanic acid Timentin = ticarcillin + clavulanic acid Unasyn = ampicillin + sulbactam Zosyn = piperacillin + tazobactam Image adapted from Figure 1. Chemical structures of the clinically available β- lactamase inhibitors. Watkins R, Papp-Wallace K, Drawz S, Bonomo R. Novel β- lactamase inhibitors: A therapeutic hope against the scourge of multidrug resistance. Front Microbiol. 2013;4:392. doi: 10.3389/fmicb.2013.00392. Adapted from Figure 1. Scheme illustrating the β-lactamase destruction of amoxycillin and its inhibition by potassium clavulanate. Cole M. Biochemistry and Action of Clavulanic Acid. Scott Med J. 1982;27:S10 - S6. C O NT I NU E Lesson 5 of 13 Cephalosporins (β-Lactam Cell Wall Inhibitors) Joshua L. Hood M.D., Ph.D. Cephalosporins Cephalosporium acremonium. Image source: https://1.bp.blogspot.com/-dAV8Xp- s13s/VRVsuISGHNI/AAAAAAAAJ2U/xmTDbrbDAXE/s1600/11-  %252BAcremonium_1000X.jpg Cephalosporins were discovered in 1948 when crude filtrates from cultures of Cephalosporium acremonium were found to inhibit the in vitro growth of Staphylococcus aureus. Cephalosporins: Structure Cephalosporin R1 and R2 side chains influence the antibacterial spectrum of activity. Image adapted from: Turner J, Connolly K, Aberman K, Fonseca J, Singh A, Jerse A, Nicholas R, Davies C. Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae. ACS infectious diseases. 2021;7. doi: 10.1021/acsinfecdis.0c00400.  Cephalosporin generations are distinguished by their R1 and R2 side chains. Image adapted from Figure 1. Generations of cephalosporin antimicrobials. Representative structures from each generation show new features discovered and adapted during each generation. Turner J, Connolly K, Aberman K, Fonseca J, Singh A, Jerse A, Nicholas R, Davies C. Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae. ACS infectious diseases. 2021;7. doi: 10.1021/acsinfecdis.0c00400. Cephalosporins: Mechanism of Action Cephalosporins have the same mechanism of action as penicillins. They work by inhibiting bacterial cell wall synthesis They bind to the active site of penicillin binding proteins (PBPs). This inhibits the bacterial transpeptidation reaction necessary for cell wall synthesis resulting in bacterial cell death.  Image adapted from https://www.dnatube.com/video/2418/Penicllin-mechanism Cephalosporins: Indications and Spectrum of Activity There are now five generations of cephalosporins. They are especially useful for skin infections, penicillin resistant bacteria, and meningitis. Cephalosporins are β-lactams, structurally similar to penicillins. Generations classifications are based on antimicrobial activity. The earlier generations (1st – 2nd), have great activity against Gram (+) bacteria. The later generations (3rd – 5th) have greater activity toward Gram (-) bacteria and increased beta lactamase resistance.  Adapted from Figure 2. Evolution of cephalosporin characteristics over semi- synthetic generations. Ribeiro da Cunha B, Fonseca L, Calado C. Antibiotic Discovery: Where Have We Come from, Where Do We Go? Antibiotics. 2019;8:45. doi: 10.3390/antibiotics8020045. Table adapted from Table 11.3 Cephalosporins classified by generation/spectrum of activity. Gilmore BF, Denyer SP. 9th edition Hugo and Russell’s Pharmaceutical Microbiology. John Wiley & Sons; 2023. Cephalosporins: Resistance  Image adapted from https://www.stabilis.org/images/Molecules/Molecule.997.jpg Similar to penicillins, cephalosporins can be susceptible to β-lactamase hydrolysis by cephalosporinases. To overcome bacterial resistance, avibactam was developed. Avibactam is a non-β-lactam β-lactamase inhibitor typically combined with ceftazidime (3rd generation cephalosporin) to produce AVYCAZ®. Avibactam inactivates cephalosporinases via a reversible covalent interaction. This prevents ceftazidime hydrolysis by cephalosporinases. AVYCAZ® is useful for complicated drug resistant gram (-) infections. Complicated UTIs, including pyelonephritis Hospital acquired bacterial or ventilator-associated bacterial pneumonia (HABP/VABP) Combined with metronidazole, it can be used to treat complicated intra-abdominal bacterial infections.  Image adapted form Figure 3. Mechanism of action of avibactam. Qin W, Panunzio M, Biondi S. β-Lactam Antibiotics Renaissance. Antibiotics. 2014;3:193-215. doi: 10.3390/antibiotics3020193. Cephalosporins: Pharmacokinetics Similar to penicillins, cephalosporin pharmacokinetics are uncomplicated and not associated with major issues. They are excreted in the urine, largely unchanged. Cephalosporins: Adverse Effects Significant adverse effects associated with cephalosporins include hypersensitivity reactions (cross-reactivity with penicillins), rash, autoimmune hemolytic anemia, disulfiram-like reaction to alcohol, and vitamin K deficiency C O NT I NU E Lesson 6 of 13 Carbapenems (β-Lactam Cell Wall Inhibitors) Joshua L. Hood M.D., Ph.D. Carbapenems  Image source https://3.bp.blogspot.com/_yidj06XFPPw/SEAdYp5OQ2I/AAAAAAAAAUI/qqUKME8DE Wc/s200/S_cattleya.png Carbapenems were developed in the 1970s to address the widespread emergence of β- lactamase-based antibiotic resistance. Thienamycin was the first carbapenem discovered in 1976. It is produced by the soil bacterium Streptomyces cattleya. However, it was very unstable and rapidly degraded. Imipenem was subsequently produced as a stable derivative of thienamycin.  Carbapenem Structure Figure adapted from Figure 2. Penicillins, cephalosporins, monobactams, and carbapenems all contain a beta-lactam ring, the site of inactivating beta-lactamase enzymes. https://www.coursehero.com/study-guides/microbiology/mechanisms- of-antibacterial-drugs/ Carbapenems: Mechanism of Action Carbapenems have the same mechanism of action as penicillins but with a broader spectrum of activity. Carbapenems: Indications and Spectrum of Activity Carbapenems are β-lactamase (penicillinase) resistant. The have a broad spectrum of antibacterial activity. However, they are susceptible to carbapenemases, produce by Enterobacteriaceae spp. for example. Cilastatin is co-administered with Imipenem to prevent renal brush border degradation of Imipenem by membrane bound dehydropeptidase I. Carbapenems are used for serious, life-threatening infections when other β-lactams do not work. Carbapenems have a broad spectrum of antibacterial activity. Table adapted form https://www.antiinfectivemeds.com/bronchitis/carbapenems/ Carbapenems: Resistance Carbapenems are resistant to a variety of beta lactamase classes including penicillinases, cephalosporinases (AmpC), and ESBLs. However, bacterial acquisition of metallo-beta- lactamase or carbapenemases can results in carbapenem resistance. Additionally, structural changes in bacterial PBPs or loss of outer membrane porins can result in resistance. MRSA resistance to carbapenems occurs via PBP2a. Enterococcus faecium resistance occurs via PBP5. Pseudomonas aeruginosa exhibits loss of OprD porin activity resulting in reduced influx of carbapenems. Adapted from Figure 5. Mechanism relevant for the inactivation of carbapenem antibiotics and detection of carbapenemases. Pfaendler HR, Schmidt H-U, Freidank H. The Novel CarbaLux Test for Carbapenemases and Carbapenem Deactivating AmpC Beta-Lactamases. Front Microbiol. 2020;11. doi: 10.3389/fmicb.2020.588887. Carbapenemases are β-lactamases with a wider spectrum of activity. They can hydrolyze penicillins as well as cephalosporins, carbapenems and monobactams. They can also be resistant to β-lactamase inhibitors. Carbapenems: Pharmacokinetics Similar to penicillins, carbapenem pharmacokinetics are uncomplicated and not typically associated with major issues.. They are excreted in the urine, largely unchanged. Carbapenems: Adverse Effects Major side effects of carbapenem use include neurotoxicity (seizures), drug-induced immune thrombocytopenia (DITP), rash and GI upset. C O NT I NU E Lesson 7 of 13 Monobactams (β-Lactam Cell Wall Inhibitors) Joshua L. Hood M.D., Ph.D. Monobactams  Nocardia spp. Imagesource: https://cdn.lecturio.com/assets/Stain-nocardia-species.jpg Monobactams were discovered in the 1970s to address the widespread emergence of β- lactamase-based antibiotic resistance. Nocardicins, natural monobactams isolated from Nocardia uniformis in 1976, demonstrated very high resistance to most known β-lactamases at the time. However, antimicrobial activity was lacking. To address this, synthetic aztreonam was created in 1986. Monobactams: Structure  Figure adapted from Figure 2. Penicillins, cephalosporins, monobactams, and carbapenems all contain a beta-lactam ring, the site of inactivating beta-lactamase enzymes. https://www.coursehero.com/study-guides/microbiology/mechanisms- of-antibacterial-drugs/ Monobactams: Mechanism of Action Aztreonam is the only FDA approved monobactam in the USA. It has the same mechanism of action as penicillins. Monobactams: Indications and Spectrum of Activity Aztreonam has little to no activity against gram (+) or anaerobic bacteria. It is structurally similar structure to ceftazidime, a 3rd generation cephalosporin. Aztreonam's spectrum of activity is limited mainly to gram (-) aerobic rods via high affinity for PBP3. This includes H. influenzae, N. meningitides, and Pseudomonas aeruginosa. Aztreonam exhibits synergistic activity with aminoglycoside antibiotics for the treatment of cystic fibrosis infections with P. aeruginosa. Aztreonam is used as an alternative for patients with penicillin allergies. Image adapted from Figure 5. Structure-activity relationships of aztreonam. Decuyper L, Jukič M, Sosič I, Žula A, D'Hooghe M, Gobec S. Antibacterial and β- Lactamase Inhibitory Activity of Monocyclic β-Lactams. Med Res Rev. 2017;38. doi: 10.1002/med.21443. Aztreonam's unique structural components are responsible for its gram (-) spectrum of activity and β-lactamase resistance. Monobactams: Resistance Aztreonam is penicillinase resistant, but can be susceptible to cephalosporinases (AmpC) and carbapenemases. Monobactams: Pharmacokinetics Similar to penicillins, monobactam pharmacokinetics are uncomplicated and not typically associated with major issues.. They are excreted in the urine, largely unchanged. Monobactams: Adverse Effects There are minimal adverse effects associated with monobactam use. GI upset has been reported. C O NT I NU E Lesson 8 of 13 Vancomycin (Unique Cell Wall Inhibitor) Joshua L. Hood M.D., Ph.D. Vancomycin Vancomycin is a glycopeptide antibiotic discovered in 1952 while searching for new antibiotics to treat penicillin-resistant staphylococci. It is produced by the soil bacterium Streptomyces orientalis and is active against penicillin-resistant staphylococci, some anaerobes (Clostridia), and Neisseria gonorrhea. Streptomyces Spp. Vancomycin Structure Vancomycin Mechanism of Action Vancomycin is a glycopeptide antibiotic that inhibits bacterial cell wall synthesis via a unique mechanism. It binds to D-alanyl-D-alanine dipeptides of newly generated peptidoglycan subunits which prevents their incorporation into the cell wall by penicillin binding proteins (PBPs).  Image adapted from https://i2.wp.com/slideplayer.com/8575829/26/images/3/Mechanism+of+Action+of +Vancomycin.jpg Vancomycin Indications and Spectrum of Activity Vancomycin is administered IV to treat serious gram (+) bacterial infections. It is particularly effective against Staphylococcus aureus infection including MRSA. Other FDA approved vancomycin indications include endocarditis (Diphtheroid, Enterococcal, Staphylococcal, and Streptococcal species), and staphylococcal infections (septicemia, skin and soft tissue infections, bone infections, and lower respiratory tract infections). Oral vancomycin is only FDA approved to treat intestinal infections given its low systemic absorption. This includes Staphylococcal enterocolitis, Clostridioides difficile-associated diarrhea (CDAD), and pseudomembranous colitis. Vancomycin Drug Resistance Vancomycin resistant enterococci (VRE) or vancomycin resisistant Staphylococcus aureus (VRSA) replace D-alanyl-D-alanine with D-alanyl-D-lactate or D-alanyl-D-serine which are unrecognizable to vancomycin. As a result, construction of the bacterial cell wall continues. Image adapted from https://onlineacademiccommunity.uvic.ca/myuviclife/wp- content/uploads/sites/743/2016/03/vancomycin.jpg  Image adapted from https://i2.wp.com/slideplayer.com/8575829/26/images/3/Mechanism+of+Action+of +Vancomycin.jpg Vancomycin Pharmacokinetics Vancomyin is administered intravenously, orally, or rectally (off-label). Oral bioavailability is < 10%. Vancomycin has a large volume of distribution in tissue and body fluids and CSF under conditions of bacterial meningitis. The half-life is 4-6 hours. IV and oral routes of adminisntrateion are secreted unchanged in the urine and feces, respectively. Vancomycin Adverse Effects Phlebitis, rash (vancomycin flushing syndrome) Ototoxicity, acute kidney injury or nephrotoxicity, especially in the geriatric population can occur as a result of declining renal function with age. Caution is necessary when administering vancomycin with other nephrotoxic agents such as aminoglycosides, amphotericin products, and IV contrast. Vancomyin use is contraindicated in patients with vancomycin hypersensitivity. Avoid using vancomycin during pregnancy if possible given the risk of fetal ototoxicity and nephrotoxicity. C O NT I NU E Lesson 9 of 13 Daptomycin (Cell Membrane Inhibitor) Joshua L. Hood M.D., Ph.D. Daptomycin Daptomycin is a cyclic lipopeptide antibiotic (antimicrobial peptide). It is a fermentation product produced by the bacterium Streptomyces roseosporus.  Streptomyces Spp. Daptomycin Structure  Image adapted from Figure 1. Chemical structure of daptomycin. Fouad A, Marzouk A, Shaykoon M, Ibrahim S, El-Adl S, Ghanem A. Daptomycin: A Novel Macrocyclic Antibiotic as a Chiral Selector in an Organic Polymer Monolithic Capillary for the Enantioselective Analysis of a Set of Pharmaceuticals. Molecules. 2021;26:3527. doi: 10.3390/molecules26123527. Daptomycin Mechanism of Action Daptomycin binds to the bacterial cytoplasmic membrane of gram (+) bacteria. It forms complexes with calcium resulting in pore formation. This results in membrane depolarization through the rapid efflux of cellular potassium. DNA, RNA, and protein synthesis are prevented causing bacterial cell death. Daptomycin mechanism of action. “C” represents daptomycin (CUBICIN®). Image adapted from https://www.cubicin.com/static/images/cubicin-moa.jpg  Image adapted from Figure 43-10 Proposed mechanism of action of daptomycin. Katzung BG, Vanderah TW. eds. Basic & Clinical Pharmacology, 15e. McGraw Hill; 2021. Daptomycin Indications and Spectrum of Activity Daptomycin is used to treat gram (+) S. aureus bacteremia or endocarditis and complex skin structure infections. It is FDA approved to treat methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE). Daptomycin’s spectrum of activity includes Streptococcus spp. Cornebacterium spp., Leuconostoc spp., and Pediococcus spp. It also effectively treats gram (+) anaerobes such as Clostridium spp. and Cutibacterium acnes. Daptomycin Resistance Daptomycin resistance mechanisms are not fully understood. In S. aureus, there is an association between bacterial membrane remodeling and cross- resistance between vancomycin and daptomycin. In enterococci, gain of function mutations in membrane biosynthesis genes (mprF) result in charge repulsion and membrane fluidity modifications. As a result, the bacterial membrane repels daptomycin, similar to repelling cationic host defense peptides, and simultaneously impedes bacterial recognition by the innate immune system. Daptomycin Pharmacokinetics Daptomycin is administered intravenously. Onset of action is rapid with a large volume of distribution. Half-life is 8-9 hours. Decreased frequency of dosing is required in patients with renal impairment (creatinine clearance < 30 mL/min). Daptomycin is primarily excreted unchanged in the urine (~80%) and < 6% in the feces. Daptomycin Adverse Effects Daptomycin is not recommended for children under one year of age given the potential to cause muscular, nervous, or neuromuscular issues. peripheral neuropathy myopathy and rhabdomyolysis Temporarily discontinue HMG-CoA reductase inhibitors (statins) with use. Associated with eosinophilic pneumonia Discontinue treatment. Clostridium difficile diarrhea and pseudomembranous colitis C O NT I NU E Lesson 10 of 13 Polymyxins (Cell Membrane Inhibitor) Joshua L. Hood M.D., Ph.D. Polymyxins Polymyxins are a class of antibiotics that specifically target gram(-) organisms. They are naturally produced by Paenibacillus polymyxa, gram (+) bacteria. Clinically prescribed members of the polymyxin drug class include polymyxin B and polymyxin E (Colistin).  Paenibacillus polymyxa Image adapted from Fig. 1. Scanning electron microscope observation of Paenibacillus polymyxa OSY-DF. He Z, Kisla D, Zhang L, Yuan C, Green-Church KB, Yousef AE. Isolation and Identification of a Paenibacillus polymyxa Strain That Coproduces a Novel Lantibiotic and Polymyxin. Applied and Environmental Microbiology. 2007;73(1):168-78. doi: doi:10.1128/AEM.02023-06. Polymyxins: Structure  Image adapted from Fig.1 Chemical structures and mechanism of action of polymyxins. Chemical structures of colistin (polymyxin E) and polymyxin B. The amino acid residue that is different between colistin and polymyxin B is labeled red. Sun Z, Palzkill T. Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1. mBio. 2021;12. doi: 10.1128/mBio.02776-21. Polymyxins: Mechanism of Action Polymyxins are positively charged. They are electrostatically attracted to the negatively charged lipopolysaccharide (LPS) lipid A phosphate groups in gram (-) bacterial membranes. The association of polymyxins with lipid A phosphates displaces divalent calcium and magnesium cations bound to the lipid A phosphate groups. This causes increased membrane permeability and efflux of bacterial intracellular contents resulting in bacterial cell death. Polymyxin complex formation with LPS also neutralizes the systemic endotoxin effects of LPS released by dying bacteria.  Image adapted from Fig.1 Chemical structures and mechanism of action of polymyxins. Diagram depicting bactericidal action of polymyxin by targeting lipopolysaccharide on outer membrane and inner membrane Gram-negative bacteria. Sun Z, Palzkill T. Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1. mBio. 2021;12. doi: 10.1128/mBio.02776-21. Polymyxins: Indications and Spectrum of Activity The spectrum of activity of polymyxins is limited to gram-negative bacilli. They are last resort antibiotics, used to treat serious systemic infections (bloodstream, meninges, UTIs) of multidrug-resistant bacteria. Carbapenem-resistant Enterobacteriaceae Pseudomonas aeruginosa Chronic infections in cystic fibrosis patients, nosocomial or ventilator associated pneumonia Acinetobacter baumannii Polymyxin B specific indications.  Use topically for ophthalmic and otic treatment of bacterial conjunctivitis and otitis externa, respectively. Polymyxin B combination therapy Polymyxin B + neomycin is used to treat otitis externa caused by Pseudomonas aeruginosa Polymyxin B + trimethoprim is used to treat bacterial conjunctivitis. Topical formulations of Polymyxin B are used to treat minor skin injuries. Polysporin® is a topical mixture of bacitracin zinc and polymyxin B sulfate. Neosporin® (triple antibiotic ointment) includes Polymyxin B + bacitracin + neomycin. Polymyxin E (Colistin) specific indications. Colistin is more easily administered via inhalation to treat penumonia than polymyxin B. Colistin achieves higher concentrations in the urine for treating UTIs than polymyxin B. Polymyxins Resistance  Image adapted form Fig. 1 Structure of lipid A of E. coli showing reaction catalyzed by MCR-1. Stojanoski V, Sankaran B, Prasad BVV, Poirel L, Nordmann P, Palzkill T. Structure of the catalytic domain of the colistin resistance enzyme MCR-1. BMC Biol. 2016;14(1):81. doi: 10.1186/s12915-016-0303-0. Polymyxin resistance remains rare. Although, carbapenem-resistant gram (-) bacilli have been isolated that are resistant to colistin (polymyxin E). In E. coli a plasmid-mediated colistin resistance-1 (mcr-1) gene has been discovered that codes for a lipid A phosphoethanolamine (PEA) transferase. Lipid A PEA transferase links the bacterial membrane LPS lipid A component to phosphoethanolamine. This decreases the affinity of colistin for LPS lipid A, effectively preventing colistin’s function. Other polymyxin resistance mechanisms include decreased LPS production, or activation of drug efflux pumps. Polymyxins Pharmacokinetics Little pharmacokinetic data is available for polymyxin B. IV administered polymyxin B is administered in the active form and over 95% is cleared non-renally. Polymyxin B is not absorbed by the GI tract. Polymyxin B levels are not influenced by renal function. It is preferred for systemic therapy over colistin since it achieves more rapid and reliable drug levels. Colistin is administered IV as colistimethate sodium (CMS), an inactive pro-drug. Colistin is not absorbed by the GI tract. Colistin easily associates with the lipid membranes of body tissues and organs. Colistin has a half life of 251 minutes. CMS is excreted in the urine, and colistin is not cleared by the kidney. Colistin is preferred over polymyxin B for UTIs since it is concentrated in the urine. Polymyxins Adverse Effects neurotoxicity nephrotoxicity Polymyxin B is less nephrotoxic than colistin since it is not affected by renal function. Contraindications to polymyxin use include hypersensitivity to polymyxin B or polymyxin E (colistin). C O NT I NU E Lesson 11 of 13 Bacitracin (Cell Wall and Membrane Inhibitor) Joshua L. Hood M.D., Ph.D. Bacitracin Bacitracin is a cyclic polypeptide antibiotic. It was originally isolated from Bacillus subtilis bacteria growing in the leg wound of a 7-year old girl in 1945.  Bacillus subtilis. Image adpated from https://images.fineartamerica.com/images-medium- large/bacillus-subtilis-bacteria-sem-steve-gschmeissner.jpg Bacitracin Structure  https://www.apexbt.com/bacitracin.html Bacitracin Mechanism of Action  Bacitracin Mechanism of Action. Image adapted from https://www.biologyexams4u.com/2018/09/5-cell-wall-synthesis-inhibitors- mode-of-action.html Bacitracin inhibits the integrity of the bacterial cell wall and membrane. It sequesters and prevents dephosphorylation of bactoprenol (undecaprenyl pyrophosphate) that is required for the transportation of bacterial cell wall peptidoglycan subunits to the outside (periplasm side) of the inner bacterial cell membrane. This results in bacterial cell lysis and death.  Bacitracin also inhibits proteases and other enzymes that modify bacterial cell membrane function. Bacitracin Indications and Spectrum of Activity Bacitracin is a mixture of cyclic polypeptides that exhibit dose dependent bacteriostatic and bactericidal activity. Efficacy is organism dependent. Bacitracin is FDA approved for acute and chronic localized skin infections. It is available OTC and is used as monotherapy or in combination with polymyxin B +/- neomycin to treat minor skin cuts, scrapes and burns. Bacitracin can be used as an ophthalmic ointment to treat bacterial conjunctivitis and superficial corneal infections. Infrequently, IM administration of bacitracin can be used to treat infantile streptococcal pneumonia and empyema. Bacitracin might be used off-label orally to eradicate vancomycin-resistant enterococci (VRE). Bacitracin’s spectrum of activity is predominantly restricted to gram (+) bacteria including Actinomyces spp., Clostridium spp., Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. Gram (-) Neisseria spp. are also susceptible to bacitracin. Bacitracin Resistance  Image modified from https://www.slideserve.com/baina/streptococcus Bacitracin susceptibility testing on sheep blood agar is traditionally used to distinguish Streptococcus pyogenes, which causes pharyngitis in children, from bacitracin resistant β-hemolytic streptococci. Common resistance mechanisms include: bacterial cellular efflux of bacitracin by ATP-binding cassette (ABC) transporters overexpression of the bacA gene. BacA is a phosphatase enzyme that can rapidly increase the dephosphorylation of bactoprenol (undecaprenyl pyrophosphate) to resist the activity of bacitracin. Bacitracin Pharmacokinetics Bacitracin is predominantly used as a topical or ophthalmic agent which minimizes systemic absorption. Bacitracin is not absorbed by the GI tract and is excreted in the feces if used orally to treat VRE. IM administration of bacitracin results in rapid and wide distribution in all body organs and fluids, and the CSF, but only with meningeal inflammation. Bacitracin is excreted by glomerular filtration. Monitor renal function if used systemically. Bacitracin Adverse Effects Topical use can result in allergic contact dermatitis, anaphylactoid reactions or anaphylaxis. Bacitracin is contraindicated in patients with a history of bacitracin hypersensitivity. IM administered bacitracin has been associated with nephrotoxicity and renal failure caused by renal tubular and glomerular necrosis. Monitor renal function if administered IM. Lesson 12 of 13 Practice Questions with Explanations Joshua L. Hood M.D., Ph.D. Which of these drug classes are beta-lactam antibiotics? Penicillins Cephalosporins Carbapenems Monobactams All choices are beta-lactam antibiotics SUBMIT What bacterial enzyme do penicillins inhibit to prevent bacterial cell wall synthesis? matrix metalloproteinase carbapenemase beta-lactamase transpeptidase penicillinase SUBMIT Which of the following are beta-lactam antibiotic resistance mechanisms used by bacteria?  decreased peptidoglycan production penicillin binding protein mutations production of beta-lactamases penicillin binding protein mutations and/or production of beta-lactamases decreased peptidoglycan production and/or production of beta-lactamases SUBMIT Bacterial produce difference clinically relevant classes of β-lactamases to inactivate beta lactam antibiotics. Which class of beta-lactamase is most likely to be resistant to a beta-lactamase inhibitor? penicillinase  extended spectrum beta-lactamases carbapenemases transpeptidase topisomerases SUBMIT Which statement best describes the mechanism of action of beta lactamase inhibitors? Beta lactamase inhibitors (e.g. clavulanic acid) have a similar structure to beta-lactamases which impairs beta-lactamase function. Beta lactamase inhibitors are structurally similar to beta-lactam antibiotics and inactivate beta lactamases via covalent crosslinking following beta- lactam hydrolysis.  Beta lactamase inhibitors prevent penicillin binding (PBP) protein mutations. Beta lactamase inhibitors prevent bacterial beta- lactam drug efflux by blocking the bacterial drug efflux pump. Beta lactamase inhibitors inhibit beta-lactam drug influx by blocking bacterial porin channels. SUBMIT Match the cephalosporin generation with its common clinicial indication(s). treatment of uncomplicated 1st generation (cefazolin) skin and soft tissue infections 2nd generation treatment of respiratory tract (cefuroxime) infections (e.g. bronchitis)  3rd generation bacterial meningitis (ceftriaxone) reserved for serious systemic 4th generation (cefepime) infections, particularly gram (-) bacilli 5th generation broad spectrum gram (+) and (-) (ceftaroline) coverage including MRSA SUBMIT Which antibiotic does not inhibit bacterial transpeptidase activity to impair bacterial cell wall production ? penicillin vancomycin imipenem  aztreonam cefotaxime SUBMIT Which antibiotic is predominantly restricted to treatment of gram (+) bacteria? Aztreonam Polymyxin B Polymyxin E (Colistin) Ceftriaxone Daptomycin  SUBMIT Which antibiotic is most likely to disrupt the integrity of both the bacterial cell wall and membrane? Penicillin Daptomycin Polymyxin B Bacitracin Vancomycin SUBMIT Which of the following statements concerning the spectrum of activity of antibiotics that inhibit bacterial cell membrane integrity is correct? The spectrum of activity of daptomycin is predominantly restricted to gram (-) bacteria, whereas polymyxins are predominantly restricted to gram (+) bacteria. The spectrum of activity of daptomycin is predominantly restricted to gram (+) bacteria, whereas polymyxins are predominantly restricted to gram (-) bacteria. The spectrum of activity of daptomycin and polymyxins are predominantly restricted to gram (+) bacteria. The spectrum of activity of daptomycin and polymyxins are predominantly restricted to gram (-) bacteria. None of these statements are correct. SUBMIT Match the antibiotic with its mechanism of action. Inhibits the integrity of the Bacitracin bacterial cell wall and membrane. Inhibits the integrity of the cell Polymyxins membrane via associations with bacterial LPS. Forms complexes with calcium Daptomycin resulting in bacterial membrane pore formation. Binds to D-alanyl-D-alanine peptidoglycan subunits to Vancomycin prevent bacterial cell wall growth. SUBMIT A 20-year-old female college student presents to the University clinic with fever, chills, shortness of breath and productive cough. She has a past medical history of sinus infections but is otherwise healthy. You suspect she may be developing an acute bronchitis. A chest X-ray does not demonstrate pneumonia, but sputum culture confirms infection with H. influenzae. You want to treat her bronchitis with Augmentin (amoxicillin + clavulanate), but she quickly mentions that she has had allergic reactions antibiotics in the past. You are unable to confirm the antibiotic identity or severity of her previous allergic reactions, but her descriptions don’t suggest IgE-mediated reactions. You decide to be cautious and try a course of which class of medication? Semi-synthetic penicillin (ampicillin) 1st generation cephalosporin (cefazolin) 2nd generation cephalosporin (cefuroxime) Penicillin V Monobactam (aztreonam)  SUBMIT A 50-year male patient presents to your outpatient clinic with a chief complaint of back pain. His physical exam reveals neck stiffness and balance problems. His past medical history reveals urinary incontinence, memory issues, BPH, hypertension and a sexually transmitted infection (STI) twenty years prior. He cannot recall how the STI was treated. Given your concern for his neck stiffness and neurological issues, you order a lumbar punction and CSF examination to help rule out bacterial meningitis. The CSF examination includes a VDRL test that is positive. The patient has no known drug allergies. Based on this finding, which antibiotic is the recommended drug of choice to treat his condition? neomycin bleomycin quinupristin dalfopristin penicillin SUBMIT Lesson 13 of 13 References Joshua L. 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