Antibiotics 1 - Beta Lactams and Other Cell Wall Agents PDF

+ The Berlin Wall Antibiotics 1 – Beta lactams and other cell wall agents Dr. Jo Chua Clinical Microbiologist & Senior Clinical Lecturer PathWest Laboratory Medicine and University of Western Australia ...

+ The Berlin Wall Antibiotics 1 – Beta lactams and other cell wall agents Dr. Jo Chua Clinical Microbiologist & Senior Clinical Lecturer PathWest Laboratory Medicine and University of Western Australia  On Noongar land We acknowledge we are situated on Noongar land, and that Noongar people remain the spiritual and cultural custodians of their land, and continue to practise their values, languages, beliefs and knowledge. We pay our respects to the traditional owners of the lands on which we live and work across Western Australia and Australia. Artist: Dr Richard Barry Walley OAM + The battle against infectious agents Antimicrobials are our weapons. Precision, minimizing collateral damage, and preserving their effectiveness for future battles are our core strategies. + Learning outcomes List the key β-lactam and glycopeptide antibiotics. Discuss the β-lactam and glycopeptide antibiotics mode of action and spectrum activity. Explain the mechanism of resistance against β-lactam and glycopeptide antibiotics. Outline the pharmacology, toxicity and potential side effects of β-lactam and glycopeptide antibiotics. Explain the methods for antimicrobial susceptibility testing, including phenotypic and genotypic approaches. + Mode of action Spectrum Resistance Framework of learning antibiotics Clinical Use Pharmacology Toxicity and Side Effects + Definitions Antimicrobials/Anti-infectives: Drugs active against microorganisms, including antibacterials, antivirals, antifungals, and antiparasitic agents. Antibiotics: Natural or synthetic compounds that inhibit bacterial biochemical functions (e.g., cell wall synthesis, protein synthesis, DNA replication, or cellular respiration). Antiseptics/Disinfectants: Antimicrobial substances too toxic for internal use, used externally to kill or inhibit microorganisms. Broad Spectrum Antibiotics: Effective against a wide variety of bacterial species. Narrow Spectrum Antibiotics: Target specific bacterial species while minimising collateral damage to the microbiome and other commensal bacteria that coexist with us. + Cidal kill organisms Definitions Static inhibit organisms’ growth long enough for body defences to remove it + Antibiotic Mechanisms of Action Cell Wall Agents Discussed in Lecture Mechanism of Action: Inhibit bacterial cell wall synthesis, disrupting structural integrity and leading to cell death. β-Lactam Antibiotics: Penicillins Cephalosporins Monobactams Carbapenems Glycopeptide Antibiotics: Vancomycin Teicoplanin + Cell wall + Cell Wall Structure Composed of a peptidoglycan layer made up of repeating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) subunits. NAM subunits have short peptide chains (D-Ala to L-Ala) attached that provide structural integrity by forming cross-links between NAM subunits. Penicillin-binding protein(PBPs) catalyse the cross linking of peptide chains are crucial for the final stages of peptidoglycan assembly. Teichoic acid found in Gram-positive bacteria contribute to cell wall stability and bacteria attachment to mucosal surface. Aryal S, 2023, Cell Wall Synthesis Inhibitors: Examples, Inhibition, Resistance + Beta-Lactam Antibiotics: Mechanism of Action and Structure β-lactam antibiotics bind to PBP( e.g. transpeptidase Chemical structures of the four clinically important β-lactam enzyme) and block formation of peptide cross-links antibiotics. The common b-lactam ring is highlighted in green. Aryal S, 2023, Cell Wall Synthesis Inhibitors: Examples, Inhibition, Resistance DOI: 10.1002/pro.2414 + Which of the following was the first beta- lactam antibiotic to be discovered? Cephalosporin Carbapenem Penicillin Monobactam + Which of the following was the first beta- lactam antibiotic to be discovered? Penicillin + Penicillin Group Classification Example Inhibits transpeptidation of peptidoglycan leading to bacterial cell lysis. 1 Benzylpenicillin Penicillin G 2 Orally absorbed Penicillin V Pharmacokinetics: penicillin Penicillins differ based on their oral absorption and spectrum of activity. 3 Ant staphylococcal Methicillin, Good distribution. Reduced penetration to penicillin Oxacillin bone of CSF unless inflamed. 4 Moderate spectrum Amoxicillin Excreted by renal tubular secretary penicillin system. 5 Antipseudomonal Piperacillin Toxicity and side effects: penicillins Allergic reactions 6 β- Methicillin, Gastrointestinal , haematological, elevated lactamase(penicillin Oxacillin transaminases and encephalopathy. ase) resistant + Which of the following penicillins is effective against Penicillinase-producing Pseudomonas aeruginosa Staphylococcus aureus + Which of the following penicillins is effective against Penicillinase-producing Staphylococcus aureus (PSSA) Pseudomonas aeruginosa Methicillin Piperacillin Oxacillin + Cephalosporins Spectrum of activity Generation Example Staphylococci Streptococci Easy Gram Other Gram Pseudomonas Anaerobes β-lactamase Negatives Negatives stability 1st Cephalexin, PSSA, MSSA +/- Cefazolin 2nd Cefuroxime + 3rd Ceftriaxone, ++ Ceftazidime, Cefotaxime 4th Cefepime PSSA, MSSA +++ 5th Ceftaroline MRSA ++  This simplified visualization represents the spectrum of activity for cephalosporins:  Green indicates effective activity.  Red indicates no activity.  Yellow highlights specific antibiotics within a generation that may have activity. “Easy” Gram negative rods.  Key Notes: Escherichia coli  Methicillin resistance renders cephalosporins (1st to 4th generation) ineffective. Proteus mirabilis  Cephalosporins (1st to 5th generation) are intrinsically resistant to enterococci. Klebsiella pneumoniae + Cephalosporins: Pharmacokinetics Oral formulations: Cephalexin & cefuroxime Parenteral formulations: The other cephalosporins from the chart. All cephalosporins, except ceftriaxone, require dose adjustment in severe renal failure. Good distribution: Achieve therapeutic concentrations in pleural, pericardial, peritoneal, synovial fluids, urine, and bile. First- and second-generation cephalosporins have poor CSF penetration and are unsuitable for CNS infections. Third-generation cephalosporins achieve reliable CSF levels in patients with meningeal irritation. + A patient presents with an intra-abdominal infection that is polymicrobial and includes anaerobic organisms. Your senior advises prescribing ceftriaxone. Will this provide adequate coverage? Heye, P., et al. "Perforated sigmoid diverticulitis in the presence of toxic epidermal necrolysis." Case reports in dermatology 6.1 (2014): 49-53. + Monobactam and Carbapenem  Stable and more resistant to beta-lactamase cleavage. (Used in specific circumstances)  Pharmacology:  Most antibiotics in this class are administered intravenously in Australia.  Widely distributed in the body, including inflamed meninges.  Primarily excreted renally; dose adjustment may be required in renal impairment.  Generally well-tolerated, with β-lactam allergy being the most common side effect. Monobactam Carbapenem Example: Aztreonam Examples: Ertapenem, Imipenem & Meropenem Spectrum: Spectrum: Active against Gram-negative bacteria, Broad coverage, including Gram-positive, including Pseudomonas aeruginosa. Gram-negative, Pseudomonas aeruginosa, and anaerobes. No coverage for Gram-positive bacteria Exception: Ertapenem does not cover or anaerobes. Pseudomonas aeruginosa. + Mechanism of β-lactam resistance Mechanism of resistance Two principal β-lactam resistance Low affinity binding of antibiotic to target of PBPs (e.g. MRSA). Destruction of antibiotic with β- lactamase (acquired) Efflux pumps across the outer membrane of Gram-negative bacteria Failure to penetrate outer membrane of Gram-negative bacteria. Lack of cell wall Murphy, James T., and Ray Walshe. "Modeling antibiotic resistance in bacterial colonies using agent- based approach." Understanding the Dynamics of Biological Systems: Lessons Learned from Integrative Systems Biology (2011): 131-154. + Mechanism of β-lactam resistance Altered target Resistance to methicillin for Staphylococcus spp (Diagram): Acquired mecA gene which encodes for low affinity binding protein PBP-2A. Resistance to methicillin and other β-lactam antibiotics. Penicillin and cephalosporin resistance in Streptococcus pneumoniae and Viridans streptococci: Results from a series of stepwise mutations in the penicillin-binding protein (PBP) genes, altering their binding affinity. This leads to a range of resistance levels, spanning from intermediate to complete resistance. + β-lactamases are only produced by Gram-negative bacteria. Which of the following There are more than 1,000 statements is correct different types of β-lactamases produced by both Gram-positive regarding β-lactamases? and Gram-negative bacteria. β-lactamases work by altering the target of penicillin-binding protein. + Mechanism of β-lactam resistance : Destruction of antibiotic with β-lactamase Type Class Characteristics Examples of Enzymes Narrow-spectrum β- Hydrolyze penicillin; primarily produced by Staphylococcal penicillinase, TEM- A lactamases Enterobacteriaceae. 1, TEM-2, SHV-1 Extended-spectrum β- Hydrolyze narrow and extended-spectrum β-lactam A SHV-2, CTX-M-15, PER-1, VEB-1 lactamases (ESBLs) antibiotics. Carbapenemase (serine) A Hydrolyze carbapenems. KPC-1, IMI-1, SME-1 Carbapenemase (Metallo-β- B Hydrolyze carbapenems. VIM-1, IMP-1, NDM-1 lactamases) Hydrolyze cephamycins and some oxime β-lactams; AmpC, P99, ACT-1, CMY-2, FOX-1, Cephalosporinases C inducible and chromosomally mediated. MIR-1 Hydrolyze oxacillin, oxime β-lactams, and carbapenems; Carbapenemase: OXA-type D produced by Pseudomonas aeruginosa and Acinetobacter OXA enzymes enzymes baumannii. Spectrum of Activity: β-lactamases vary in their activity spectrum, often encoded by acquired genes. Important Types: AmpC: Intrinsic and acquired. ESBL (Extended-spectrum β-lactamases): Acquired. Carbapenemase: Acquired. Toussaint, Kimberly & Gallagher, Jason. (2014). -Lactam/ -Lactamase Inhibitor Combinations: From Then to Now. The Annals of pharmacotherapy. 49. 10.1177/1060028014556652. + β-lactamase inhibitors β-lactamase inhibitors represent a major strategy to combat β-lactamase-mediated antibiotic resistance. Contain a β-Lactam rings Have a weak antibacterial activity on their own. Hence, they are always used in combination with a β-lactam antibiotic. Enhance the efficacy of β-lactam antibiotics by protecting them from enzymatic degradation. Name Partner β- Common Administration route Spectrum lactam name Clavulanic Amoxicillin Augmentin Oral/ IV Narrow-spectrum β-lactamases (e.g penicillinase) acid Tazobactam Piperacillin Tazocin IV Narrow-spectrum β-lactamases Low Level Amp C Ceftolozane + Which of the following is NOT a beta-lactam antibiotic? Augmentin Cefazolin Aztreonam Vancomycin + Glycopeptide Interfere with peptidoglycan polymerization by binding to the D-alanyl- D-alanine tail of peptidoglycan precursors, thereby inhibiting transpeptidase enzymes required for bacterial cell wall synthesis. Examples: Vancomycin and Teicoplanin Spectrum: Gram positive bacteria, including Methicillin Resistant Staphylococcus aureus Aryal S, 2023, Cell Wall Synthesis Inhibitors: Examples, Inhibition, Resistance + Glycopeptide Pharmacokinetics: Typically given intravenously. Oral administration is reserved for Clostridioides difficile colitis treatment. Widely distributed but has poor CSF penetration in the absence of meningeal inflammation. Requires therapeutic drug monitoring for efficacy and safety. Dose according to weight and renal function. Toxicity and Side Effects: Ototoxicity (hearing loss), particularly in patients with renal impairment. Nephrotoxicity (kidney damage). Infusion-related reactions, including "Red Man Syndrome" caused by histamine release. + Glycopeptide resistance mechanism Example: Vancomycin-resistant Enterococci (VRE). Common Resistance Genes: VanA: High-level resistance to both vancomycin and teicoplanin. VanB: High-level resistance to vancomycin but remains susceptible to teicoplanin. Mechanism of Resistance: Substitution of D-alanyl-D-alanine (D-Ala-D- Ala) with D-alanyl-D-lactate (D-Ala-D-Lac) in peptidoglycan precursors. This reduces binding affinity of vancomycin to its target, preventing inhibition of cell wall synthesis. Aryal S, 2023, Cell Wall Synthesis Inhibitors: Examples, Inhibition, Resistance + Other cell wall & cell membrane agents Usually restricted agents. Fosfomycin Used mainly to treat urinary tract infections Polymyxin Example: Colistin Used to treat Mult resistant Gram negative rods. Adverse reaction: nephrotoxicity Isoniazid and Ethambutol First line drugs used in the treatment of tuberculosis. Interfere with mycolic acid synthesis. + Serology PCR Which laboratory test should be ordered to determine the susceptibility of an organism to antibiotics? MC&S stands for Microscopy, MC & S Culture, and Sensitivity. Cytology + Susceptibility testing Assess how microorganisms respond to specific antibiotics. Methods: Disk Diffusion Test (Kirby-Bauer method): Measures zones of inhibition around antibiotic disks. Gradient test (E-test): Uses a strip with a gradient of antibiotic concentrations to determine the minimum inhibitory concentration (MIC). Automated Systems: Advanced tools to analyse susceptibility patterns.  + The Minimum Inhibitory Concentration (MIC) The lowest concentration at which STANDARDISED BACTERIAL INOCULUM TO EACH TUBE the agent inhibits the growth of the microorganism DECREASING AMOUNT OF ANTIBIOTIC IN EACH TUBE A series of test tubes each containing: 2.0 1.0 0.5 0.25 0.12 0.06 0.0 mg/L broth culture medium decreasing amounts (doubling dilutions) of the antibiotic being tested. an inoculum of the bacterium being tested (same inoculum to each tube) O’connor L, Introduction to antibiotics.  + The Minimum Inhibitory Concentration (MIC) STANDARDISED BACTERIAL INOCULUM TO EACH TUBE DECREASING AMOUNT OF ANTIBIOTIC IN EACH TUBE The tubes are incubated overnight to allow bacterial 2.0 1.0 0.5 0.25 0.12 0.06 0.0 mg/L growth. What is the MIC of this bacterium to the antibiotic being tested? O’connor L, Introduction to antibiotics.  + The Minimum Inhibitory Concentration (MIC) STANDARDISED BACTERIAL INOCULUM TO EACH TUBE DECREASING AMOUNT OF ANTIBIOTIC IN EACH TUBE The MIC of this isolate to this 2.0 1.0 0.5 0.25 0.12 0.06 0.0 mg/L antibiotic is: 0.25 mg/l. The lowest concentration at which the agent inhibits the growth of this bacteria is: 0.25 mg/l. O’connor L, Introduction to antibiotics. + Susceptibility testing Phenotypic detection of enzymes Beta-lactamase detection with a chromogenic cephalosporinase test (Cefinase disk). Carbapenemase Testing (e.g. Carba NP, lateral flow assay): Identifies production of carbapenem-hydrolysing enzymes. Synergy observation with specific antibiotics. (e.g. Keyhole effect with clavulanic acid to detect presence of ESBL) Pros: The methods are rapid and effectively detect the presence of a functioning β-lactamase. | Cons: They do not identify the specific gene responsible for β-lactamase production. + Genotypic detection Identifying genetic determinants of resistance using molecular techniques. Applicable for acquired genes. Real-Time PCR: Amplifies and detects specific resistance genes (e.g., mecA for MRSA). Highly specific and sensitive and enable precision of genetic determination of antimicrobial resistance. + What genes are commonly detected to confirm vancomycin resistance in enterococci? Van A Van B + Whole genome sequencing  Advantages of WGS:  Provides comprehensive data on AMR determinants, including context (mobile elements or mutations).  Enables precise strain characterisation and detailed outbreak tracking.  Supports targeted surveillance for specific resistance genes like IMP-4, NDM carbapenemases.  Limitations of WGS:  High cost, specialised expertise, and longer turnaround times limit routine use.  Genotypic detection does not Argimón, Silvia, et al. "Integrating whole-genome sequencing within the National Antimicrobial Resistance Surveillance Program in the guarantee phenotypic resistance Philippines." Nature communications 11.1 (2020): 2719. expression. + Recap β-Lactam Antibiotics: Mechanism of Action: Bind to penicillin-binding proteins (PBPs), such as transpeptidase enzymes, and block the formation of peptide cross-links in the bacterial cell wall. Spectrum: Variable, depending on the specific β-lactam antibiotic. Key Mechanisms of Resistance: Destruction of the antibiotic by β-lactamase enzymes (acquired resistance).Low-affinity binding of the antibiotic to PBPs (e.g., in MRSA). Glycopeptides: Mechanism of Action: Bind to the D-alanyl-D-alanine tail of peptidoglycan precursors, thereby interfering with peptidoglycan polymerization and bacterial cell wall synthesis. Spectrum: Effective against Gram-positive bacteria. Mechanisms of Resistance: Substitution of D-alanyl-D-alanine (D-Ala-D-Ala) with D-alanyl-D-lactate (D- Ala-D-Lac) in peptidoglycan precursors, reducing the binding affinity of vancomycin to its target. Adverse Effects: May cause nephrotoxicity and ototoxicity. Methods for Antimicrobial Susceptibility Testing: Phenotypic methods: Examples include the Kirby-Bauer (KB) disc diffusion method and E-tests. Genotypic methods: Techniques such as PCR or whole-genome sequencing can be used to detect resistance genes. + Break