Lecture 3: β-lactams and β-lactamases

Russell E. Lewis
Associate Professor of Infectious Diseases
Department of Molecular Medicine
University of Padua

russelledward.lewis@unipd.it
https://github.com/Russlewisbo

Outline

• Antibiotic classes
• Mechanism of action
• Pharmacokinetics/Pharmacodynamics
• Resistance
• Role in therapy

Discovery of penicillin

Sir Alexander Fleming in his laboratory, Pathé Films

(Fleming, 1929)

Natural penicillins

Isoxazolyl penicillins

Aminopenicillins

Extended-spectrum penicillins

Mechanism of action

Inhibition of transpeptidase (PBP)

Lysis of growing bacterial cells

Spectrum of activity

• Good: Treponema pallidum, most streptococci, including Streptococcus pneumoniae

• Moderate: Enterococci

• Poor: Almost everything else (penicillinases, β-lactamases)

Source: Sanford Guide Web

++ Preferred + activity; ± limited utility; 0 not recommended

Pharmacodynamics of β-lactam antibiotics

• β-lactam antibiotics exhibit relatively concentration-independent killing

  • The rate of killing reaches its maximum very quickly as the drug concentration increases from the MIC to 4–6 times the MIC and falls precipitously when the drug concentration decline below the MIC

  • Related to mechanism of action: acylation of their targets, the β-lactam-binding proteins

  • Once maximal acylation is achieved, the killing rates cannot increase any further. This explains why the killing rates for β-lactam drugs are maximal at a low multiple of the MIC

• % Time > MIC is the pharmacodynamically linked variable
• Clinical implications: Activity of β-lactams with short half-lives is optimized by prolonging drug exposure above the MIC (extended or continuous infusion)

(Drusano, 2004)

Pharmacokinetics: Natural penicillins

Absorption	Penicillin VK (oral); bioavailibility 60-73% Penicillin G (IV) Benzathine penicillin (IM)
Distribution	0.35 L/kg 65% protein bound 500% bile:serum 5-10% CSF:blood (therapeutic for some pathogens)
Metabolism	No metabolism t½ 0.5 hr Substrate of OAT transporters
Elimination	Renal 100%, impaired by probenicid
PK:PD target	≥ 40-50% fTime>MIC
Typical doses	(Pen VK) 250-500 mg TID-QID before meals and at bedtime (Low dose) 600,000-1.2 MU IM/day (High dose) Penicillin G: > 20 MU IV daily

Source: Sanford Guide

CNS Toxicities

The β-lactam ring structure is an important determinant of the epileptogenic properties. Evidence suggests that substitutions occurring at the 7-aminocephalosporanic or 6- aminopenicillanic acid (6-APA) positions may lead to alterations in epileptogenic activity. The thiazolidine ring and side chain length also plays a role in determining the pro-convulsive effects of β-lactams

(Wanleenuwat et al., 2020)

Other adverse effects

• Most serious reaction is immediate IgE-mediated anaphylaxis; incidence only 0.05% but 5-10% fatal. Other IgE-mediated reactions: urticaria, angioedema, laryngeal edema, bronchospasm

  • Morbilliform rash after 72 hours is not IgE-mediated and not serious

• Serious late allergic reactions: Coombs-positive hemolytic anemia, neutropenia, thrombocytopenia, serum sickness, interstitial nephritis, hepatitis, eosinophilia, drug fever

Clinical role

• Poor empiric choice for most infections because of resistance

• Drug of choice for syphilis particularly neurosyphilis (ceftriaxone use is increasing)

Pharmacokinetics:
Anti-staphylococcal penicillins

Absorption	Dicloxacillin (oral- bioavailibility 60-73%) Methicillin (IV-not used); Flucloxacillin (oral and IV, bioavailibility 50%) Nafcillin (IV) Oxacillin (IV)
Distribution	0.1-0.5 L/kg >95% protein bound Limited data on CNS penetration
Metabolism	Not metabolised but some drugs can induce CYP P450 (dicloxacillin, flucloxacillin)
Elimination	Biliary with some renal (no dosing adjustment for renal impairment)
Typical doses	125-500 every 6h (oral); 1-2 gram IV every 4-6h

Adverse Effects

• Local phlebitis, (problematic with frequent IV dosing),

• Fever, rash (4%), anaphylaxis (rare)

• + Coombs (rare), neutropenia (prolonged treatment), eosinophilia (22%), thrombocytopenia

• C. difficile colitis (rare)

• Increased LFTs, headache (rare), confusion (rare),

• Acute interstitial nephritis (less than methicillin)

• Hepatic dysfunction-more problematic with doses ≥12 gm/day (oxacillin > nafcillin)

  • LFTs usually increase 2–24 days after start of treatment, reversible

Drug interactions

• Flucloxacillin (also dicloxacillin?) activates the pregnane X receptor (PXR), which can induce the expression of CYP450 and UGT enzymes, and P-gP transporters

(Wei et al., 2016)

Mechanisms of Resistance

(De Oliveira et al., 2020)

• Methicillin resistance encoded by mecA, mecC, which code for low-affinity PBP2a and PBP2c

• Genes can be detected by latex agglutination test of PCR (nasal swabs)

Current resistance epidemiology

Clinical role

• Infections caused by MSSA, such as endocarditis, bloodstream infections, and skin and soft-tissue infections

• β-lactams kill staphylococci more quickly than vancomycin, so patients with MSSA infections who lack serious beta-lactam allergies should be switched to beta-lactams, such as antistaphylococcal penicillins or first-generation cephalosporins

Spectrum: Aminopenicillins

Source: Sanford Guide Web

++ Preferred + activity; ± limited utility; 0 not recommended

Ambler classification of beta-lactamases

Clavulanic acid and sulbactam are suicide inhibitors of Ambler class A enzymes

(Noster et al., 2021)

Mechanisms of resistance to
beta-lactamase inhibitors

• Intrinsic (e.g., 1st-Gen BLIs don’t work against AmpC or Carbapenemases)

• BLI-R beta lactamases (point mutation —> less binding of 1st-gen BLIs)

  • Narrow-spectrum: TEM-30, SHV-10

  • ESBL: TEM-50

• Hyperproduction of beta-lactamase (more common)

  • E.g. ESBL E.coli, or Klebsiella SHV-1

Amoxicillin-clavulanate resistance

Pharmacokinetics: Aminopenicillins

Absorption	Ampicillin (IV) Ampicillin-sulbactam (IV) Amoxicillin (oral, 80% bioavail.) Amoxicillin-clavulanate (oral 80% bioavail., IV)
Distribution	0.25-0.36 L/kg 18-22% protein bound 500% bile:serum 5-10% CSF:blood (therapeutic for some pathogens)
Metabolism	No metabolism t½ 1.2-1.5 hr
Elimination	ampicillin, amoxicillin, sulbactam- renal, clavulanate-hepatic, bile and renal
PK:PD target	≥ 40-50% fTime>MIC
Typical adult doses	Ampicillin 1-2 gram IV q4-6h Ampicillin-sulbactam : 3 gram IV q6h; up to 9 gram IV over 4hrs q8h for Acinetobacter spp. VAP Amoxicillin 500-1000 mg TID; ER tab 775 mg once daily; Amoxicillin-clavulanate 500-1000 mg ( 125-200 clav) twice daily Amoxicillin-clavulanate 1000/200 IV q8h IV

Source: Sanford Guide

Beta-lactamase inhibitors

	Clavulanic acid	Sulbactam	Tazobactam
Chemical structure	Clavam, similar to penicillin nucleus	Penicillanic acid sulfone	Penicillanic acid sulfone
Origin	Isolated from Streptomyces clavuligerus in the 1970s	Developed 1978 (synthetic)	Developed 1980 (synthetic)
Drugs	Amoxicillin: Co-amoxiclav (Augmentin) 1988 Ticarcillin: Ticarcillin-clavulanate (Timentin)	Ampicillin/Sulbactam (Unasyn)	Piperacillin/Tazobactam Ceftolozane/Tazobactam
Active against	Penicillinases / narrow-spectrum beta lactamases ESBL , OXA-53	Penicillinases / narrow-spectrum beta lactamases ESBL Intrinsically active against Acinetobacter baumannii (directly inhibits PBP1 & PBP3). - Also against N.gonorrhoea + B.fragilis but not used clinically.	Penicillinases / narrow-spectrum beta lactamases ESBL • OXA-2,32
Not active against	AmpC Cephalosporinases Carbapenemases (KPC, OXA-48, MBLs)	AmpC Cephalosporinases Carbapenemases	AmpC Cephalosporinases Carbapenemases

• These are structurally similar to penicillin, and target class A including ESBL (but not KPC), but have no activity against class B (MBL), C (AmpC) or D (OXA) beta lactamases.

• Act as ‘suicide inhibitors’ —> covalently bind serine residue in binding site (many molecules of BLI per beta-lactamase required to produce inhibition)

Amoxicillin/clavulanate dosing

• Plasma amoxicillin exposures are generally linear and proportional at doses up to 750 mg, after which absorption becomes saturated and bioavailability decreases from approximately 60% at 750 mg to approximately 55% at 875 mg or 1000 mg and to approximately 35% at 2000 mg
  • Standard dose: (oral, e.g., UTI): (0.5 g amoxicillin + 0.125 g clavulanic acid) x TID
  • UTI dose: (0.5 g amoxicillin + 0.125 g clavulanic acid) TID
  • High dose: (0.875 g amoxicillin + 0.125 g clavulanic acid) TID
  • High dose IV: (2 g amoxicillin + 0.2 g clavulanic acid) q8h

(Huttner et al., 2020) and EUCAST Clinical Breakpoint Tables v. 14.0

Empiric considerations for amox/clav use

(Huttner et al., 2020)

Adverse effects

• Persons with infectious mononucleosis, i.e., Epstein-Barr virus (EBV), are likely to develop rash. It is not a permanent allergy

• In patients with true Amoxicillin allergy, increased risk of cross allergenicity with oral cephalosporins that have an identical R1 side chain: i.e., cefadroxil and cefprozil

• Hepatotoxicity linked to clavulanic acid. Amoxicillin-clavulanate is most common cause,13-23%, of drug-induced cholestatic liver injury

  • Onset delayed several days after end of antibiotic therapy

  • Genetic predisposition. Usually mild; rare liver failure

• Treatment stopped due to adverse affects (2-4.4%)

  • fever, rash (3%), anaphylaxis (rare), neutropenia, eosinophilia, thrombocytopenia (rare), nausea/vomiting (3%), diarrhea (9%), C. difficile colitis, increased LFTs, headache, seizures (rare)

• With BID regimen, less clavulanate & less diarrhea

• In patients with immediate allergic reaction to amoxicillin-clavulanate, 1/3 are due to the clavulanate. Diarrhea due to clavulanate

What is the clinical role of aminopenicllins
+/-β lactamase inhibitor?

• Ampicillin is a drug of choice for susceptible enterococci
  • Enterococcus faecalis is almost always susceptible; Enterococcus faecium is usually resistant
• These drugs are often listed as alternative regimens for urinary tract infections (UTIs) in pregnant women because they are pregnancy category B and eliminated renally
• Amoxicillin/clavulanate is used for upper and lower respiratory tract infections when beta-lactamase–producing organisms are found or suspected
  • It can also be useful for UTIs when resistance to other drugs is seen, but it should not be given for a short 3-day course, as with fluoroquinolones or TMP/SMX

Extended-spectrum
(anti-pseudomonal) penicillins

Spectrum of piperacillin-tazobactam

Source: Sanford Guide Web

++ Preferred + activity; ± limited utility; 0 not recommended

Ambler classification of beta-lactamases

Should piperacillin-tazobactam be used for severe ESBL infections?

(Noster et al., 2021)

MERINO trial

(Harris et al., 2018)

Pharmacokinetics of piperacillin-tazobactam

Absorption	IV only
Distribution	0.4 L/kg 16% piperacillin; 48% tazobactam >100% bile:serum CSF:blood pentration- limited data
Metabolism	No metabolism t½ 1 hr
Elimination	Renal, may interfere with SeCr excretion
PK:PD target	≥ 40-50% fTime>MIC
Typical adult doses	Typical dosing: Piperacillin/tazobactam 3.375 gram IV q6h of 4.5 gram IV q8h (severe infections) P. aeruginosa pneumonia: 3.375 g IV q4h or 4.5 gram IV q6h combined with antipseudomnal aminoglycosides or fluoroquinolone Prolonged infusion: 4.5 gram IV over 30 minutes followed by 3.375 gram IV infused over 4 hours

BLING III

(Dulhunty et al., 2024)

BLING III- Enrolment criteria

(Dulhunty et al., 2024)

BLING III results

Effect size= 2% reduction. NNT of 50 patients to prevent 1 death

(Dulhunty et al., 2024)

BLING III

Meta-analysis: The bigger picture

(Abdul-Aziz et al., 2024)

Adverse effects

• Platelet dysfunction can occur in patients with renal failure, due to impaired platelet aggregation
• Drug-induced immune thrombocytopenia
• Allergic reactions can occur with any of the beta-lactam antibiotics.
• Treatment stopped due to adverse effects: (3.2%):
  • Local phlebitis (1%), fever (2%), rash (4%) - Serum sickness, positive Coombs, neutropenia, eosinophilia, thrombocytopenia, increased PT/PTT, - nausea/vomiting (7%), diarrhea (11%), C. difficile colitis, - Increased LFTs, increased BUN/Creatinine, headache (8%), confusion (rare), seizures (rare). - Hypokalemia

Increased risk of renal failure with vancomycin?

• Does combining vancomycin with piperacillin-tazobactam amplify the risk of vancomycin nephrotoxicity?
  • Many observational retrospective studies have reported an increased risk of acute kidney injury in association with the combination of piperacillin-tazobactam and vancomycin as compared to either drug alone
  • No reported increased risk of AKI if vancomycin is combined with cefepime or meropenem
• The attributable nephrotoxicity due to vancomycin due in part to multiple con founders
  • Animal studies suggest “injury” is a consequence of competetion for tubular secretion of creatinine

(Pais et al., 2020)

Monobactams

Aztreonam: Spectrum of activity

• Only gram-negative with some P. aeruginosa coverage
• No useful activity against gram-positive bacteria or anaerobes
• However, degraded by Class A and C beta-lactamases

Safe drug for penicillin allergic patients?

Exception: Aztreonam has R1 side-chain identical to that of ceftazidime and cefiderocol, so in those with allergy to that side-chain, there may be cross-sensitivity

(Castells et al., 2019)

Coming soon…

Aztreonam-avibactam!

• Why combine?- Aztreonam is not hydrolyzed by metallo-lactamases (class B), but is degraded by some Ambler class A and C β-lactamases
• Avibactam is an inhibitor of serine β-lactamases (Class A and C) and some Class D (OXA-48)
• The combination restores activity against carbapenase-producing organisms
• Avoids use of ceftazime-avibactam + aztreonam

Pharmacokinetics of aztreonam

Absorption	IV only
Distribution	Aztreonam 0.28 L/kg, 38% protein binding Avibactam 0.55 L/kg, 3% protein binding
Metabolism	No metabolism t½ 2-3 hours for both aztreonam and avibactam Bile penetration: 115-400% aztroenam CSF penetration: 3-52%
Elimination	Renal for both aztreonam and avibactam
PK:PD target	≥ 40-50% fTime>MIC
Typical adult doses	2 grams IV q6-8h 2 gm/0.67 gm (aztreonam-avibactam) IV load, then 1.5 gm/0.5 gm IV q6h Infuse maintenance doses over 3 hours

Aztreonam (avibactam) adverse effects

• GI: nausea, vomiting, diarrhea, C. difficile infection, abdominal pain

• Skin: rash, anaphylaxis

• Liver: increased AST, ALT

• Hematologic: anemia, thrombocytopenia

• Confusion, dizziness

Summary-Pencillins

• For select pathogens, still some of the rapidly killing agents
• Short t½ = not the most practical agents
• Time > MIC pharmacodynamics, + short t½= activity maximized with prolonged infusions
• Resistance, particularly ESBL, Amp-C have blunted broad-spectrum
• New β-lactamase inhibitors can revive niche agents

References

Sir Alexander Fleming in his laboratory, Pathé Films Inhibition of transpeptidase (PBP) Lysis of growing bacterial cells

Abdul-Aziz MH, Hammond NE, Brett SJ, Cotta MO, De Waele JJ, Devaux A, et al. Prolonged vs Intermittent Infusions of β-Lactam Antibiotics in Adults With Sepsis or Septic Shock: A Systematic Review and Meta-Analysis. JAMA 2024;332:638. https://doi.org/10.1001/jama.2024.9803.

Castells M, Khan DA, Phillips EJ. Penicillin allergy. New England Journal of Medicine 2019;381:2338–51. https://doi.org/10.1056/NEJMra1807761.

De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial resistance in ESKAPE pathogens. Clinical Microbiology Reviews 2020;33:e00181–19. https://doi.org/10.1128/CMR.00181-19.

Drusano GL. Antimicrobial pharmacodynamics: critical interactions of ’bug and drug’. Nature Reviews Microbiology 2004;2:289–300. https://doi.org/10.1038/nrmicro862.

Dulhunty JM, Brett SJ, De Waele JJ, Rajbhandari D, Billot L, Cotta MO, et al. Continuous vs Intermittent β-Lactam Antibiotic Infusions in Critically Ill Patients With Sepsis: The BLING III Randomized Clinical Trial. JAMA 2024;332:629. https://doi.org/10.1001/jama.2024.9779.

Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of b. influenzæ. British Journal of Experimental Pathology 1929;10:226–36.

Harris PNA, Tambyah PA, Lye DC, Mo Y, Lee TH, Yilmaz M, et al. Effect of Piperacillin-Tazobactam vs Meropenem on 30-Day Mortality for Patients With E coli or Klebsiella pneumoniae Bloodstream Infection and Ceftriaxone Resistance: A Randomized Clinical Trial. JAMA 2018;320:984–94. https://doi.org/10.1001/jama.2018.12163.

Huttner A, Bielicki J, Clements MN, Frimodt-Møller N, Muller AE, Paccaud J-P, et al. Oral amoxicillin and amoxicillinclavulanic acid: Properties, indications and usage. Clinical Microbiology and Infection 2020;26:871–9. https://doi.org/10.1016/j.cmi.2019.11.028.

Noster J, Thelen P, Hamprecht A. Detection of Multidrug-Resistant EnterobacteralesFrom ESBLs to Carbapenemases. Antibiotics 2021;10:1140. https://doi.org/10.3390/antibiotics10091140.

Pais GM, Liu J, Avedissian SN, Hiner D, Xanthos T, Chalkias A, et al. Lack of synergistic nephrotoxicity between vancomycin and piperacillin/tazobactam in a rat model and a confirmatory cellular model. The Journal of Antimicrobial Chemotherapy 2020;75:1228–36. https://doi.org/10.1093/jac/dkz563.

Wanleenuwat P, Suntharampillai N, Iwanowski P. Antibiotic-induced epileptic seizures: mechanisms of action and clinical considerations. Seizure 2020;81:167–74. https://doi.org/10.1016/j.seizure.2020.08.012.

Wei Y, Tang C, Sant V, Li S, Poloyac SM, Xie W. A Molecular Aspect in the Regulation of Drug Metabolism: Does PXR-Induced Enzyme Expression Always Lead to Functional Changes in Drug Metabolism? Current Pharmacology Reports 2016;2:187–92. https://doi.org/10.1007/s40495-016-0062-1.