Antibiotics Part 1

Dr P Gayo MunthaliConsultant Microbiologist UHCW

Honorary Associate Clinical ProfessorUniversity of Warwick

Objectives• By the end of this lecture you should be able

to:1) Explain the mode of action of beta lactams,

aminoglycosides, fluoroquinolones, macrolides, tetracyclines and glycopeptides

2) Mention the major side effects of the antibiotic groups in (1)

3) Appreciate different types of resistance and in simple terms, explain the mechanisms of resistance to beta lactams

4) Explain some limitations in the use of antibiotics in (1)

5) Understand the general spectrum of activity of antibiotics in (1)

Antibiotics, Point of Action

Cell membranePolymixin, bacitracin,colistin

Folic acid MetabolismTrimethoprim, Sulphonamides

Cell wall SynthesisBeta-lactams,GlycopeptidesDaptomycinFosfomycin

DNA ReplicationQuinolones

DNA Dependent RNA Pol. Rifampicin

Protein Synthesis 30S Tetracyclines,Aminoglycosides50S Chloramphenicol, Clindamycin,Erythromycin, Linezolid,Streptogramin

50S

30S

Important mechanisms of resistance to antibiotics

Mechanism Typical example Antibiotics affectedInactivation ß-Lactamases ß-Lactams

Aminoglycoside modifying enzymes

Aminoglycosides

Changes in target binding

Changes in PBPs/Peptide Terminal

ß-Lactams/

GlycopeptidesRibosomal methylation Macrolides

DNA gyrase mutation Fluoroquinolones

Efflux and permeability changes

Efflux pumps Pump specific

Porin protein loss Most except polymyxins and aminoglycoside

ß-Lactams

Β-Lactam Ring

Thiazolidine Ring

Penicillins and Cephalosporins

s

o

R-CONH

N

COOHs

No

R-CONH

COOH

R

Penicillins 1940-

Cephalosporins 1948-

Carbapenems and Others

S R

COOH

No

CHз

HO

oN

o

HO

COOH

Carbapenems 1976-

Clavulanic acid 1976

Mobactams

No

R

R

R-CONHMonobactam 1981-

Mechanisms of Action

• Inhibit bacterial enzymes involved in cell wall synthesis– Penicillin binding proteins (PBPs) essential for

peptidoglycan synthesis

• Trigger membrane associated autolytic enzymes that destroy cell wall

• Inhibit bacterial endopeptidase and glycosidase enzymes which are involved in cell wall growth

• Time dependent activity

Beta Lactams Against Bacterial Cell Wall

Cell wall

Osmotic Pressure

Antibiotic against cell wall

Osmotic Pressure

Cell membrane

Rapture

Cell Membrane

Spectrum of Activity

• Very wide

• Gram positive and negative bacteria

• Anaerobes

• Spectrum of activity depends on the agent and/or its group

• Aztreonam only active against gram negatives

Pharmacokinetics

• Absorption– PO forms have variable absorption– Food can delay rate and extent of absorption

• Distribution– Widely to tissues & fluids– CSF penetration:

IV – limited unless inflamed meninges IV 3rd & 4th generation cephalosporins, meropenem, & Aztreonam – penetrate well

• Metabolism & Excretion– Primarily renal elimination– Some have a proportion of drug eliminated via the liver– ALL -lactams have short elimination half-lives

Adverse Effects

Penicillin hypersensitivity – 0.4% to 10 %– Mild: rash – Severe: anaphylaxis & death

• There is cross-reactivity among all Penicillins• Penicillins and cephalosporins ~5-15%• Penicillins and carbapenems~1% (may be higher)

– Desensitization is possible for mild hypersensitivity

• Aztreonam does not display cross-reactivity with Penicillins and can be used in penicillin-allergic patients

Resistance to ß-Lactams

•Penicillin-Binding Protein (PBP) mediated Resistance

•ß-Lactamase

•Efflux pumps/loss of porins

• PBP over expression

• Acquisition of Foreign PBPs genes

• Mutation by recombination with foreign DNA

• Point mutation

Penicillin-Binding Protein (PBP) mediated Resistance

PBP over expression

• Rare– The more PBPs are expressed, the more an

organism becomes resistant• S.aureus increased resistance to methicillin by

over expression of PBP4• E.faecium strains that over express PBP5 have

increased resistance to penicillin.

AAC 39:2415-2422, AAC 38:1980-1983, AAC 45: 1480-1486

Acquisition of Foreign PBPs

• Represented best by Methicillin Resistant S.aureus (MRSA)

• S.aureus acquires foreign PBP2a encoded by mecA gene

• PBP2a has low affinity for all ß-lactams• PBP2a can perform all the combined functions

of all the S. aureus PBPs • Almost all MRSAs express ß-Lactamase

Clin. Microbiol. Rev.10:781-791, J.Infect.Dis.162:705-710

Result

• All PBPs in S.aureus become redundant

–MRSA is resistant to all ß-lactams

Mutation by Recombination with Foreign DNA

• Streptococcus pneumoniae and Neisseria are capable of picking up foreign DNA and integrating it with their own DNA– Form mosaic gene

• Pneumococcus picks up resistant genes from alpha haemolytic streps– Reduced affinity to beta lactams

• Seen as penicillin resistant Pneumococci

Beta Lactam Activity Against 100 Penicillin Resistant Pneumococci from Spain

0

1

2

3

4

5

6

7

8

9

MIC

90

Ag

ain

st

10

0 Is

ola

tes

/BS

AC

MIC

90

R

es

ista

nc

e B

rea

kp

oin

ts

Series1Series2Series3

JAC 1992,30(3);279-288

MIC for meningitis

Isolate

BSAC

MICs

Efflux pumps/Loss of Porins

• Important type of resistance in Pseudomonas– A combination of ß-Lactamase production and

porin loss can lead to complex resistance pattern• Can lead to carbapenem resistance without

carbapenemase production

Overexpressed Efflux pumps

Porins

Porins and Pumps

Adapted from Journal of Bacteriology, April 2006, p. 2297-2299, Vol. 188, No. 7

Resistance due to ß-Lactamases

• Mode of action– Classification

ß-Lactamase

ß -pleated sheet-5

ά-helices

AAC 39:2593-2601

Bound ß-lactam by ß-Lactamase

ß-Lactamases-action

s

o

R-CONH

N

COOH

No

R-CONH

COOH

CH3

CH3

Enzyme-Ser-OH

C

C

O

Ser

sCH3

CH3

HEnzyme

HOH

Annu.Rev.Microbiol.45:37-67

Beta Lactam Classification

• You do not need to know the classification or detailed information on ß-Lactamases

• However you need to appreciate the following concepts;– Simple betalactamases– Extended spectrum betalactamases (ESBL)– Betalactamases against the Carbapenems

Simple ß-Lactamases • Many Based on genes called TEM-1 and SHV-1

found on mobile DNA elements– TEM-1 and SHV-1 are simple penicillinases in

Enterobacteriaceae– Inactive against cephalosporins– Confer resistance to Penicillins such as Benzylpenicillin and

amoxicillin– On mobile elements and therefore transmissible

• Staphylococci also produce simple beta lactamases not based on TEM-1 and SHV-1– Flucloxacillin designed to resist betalactamases in

Staphylococcus aureus

AAC 33:1131-1136

Extended Spectrum ß-lactamases

• Based on TEM-1 and SHV-1

• Amino acid mutations in active site progressively increase their activity against cephalosporins– When they hydrolyze extended-spectrum

cephalosporins• They are then called ESBLs

– Also attack a monobactam Aztreonam

-On mobile elements thus transmissible– Carry other resistance genes, Gentamicin,

Ciprofloxacin

ESBLs • Hydrolyze extended-spectrum cephalosporins

with an oxyimino side chain

• These include;– Cefotaxime– Ceftazidime– Ceftriaxone

• Loose term

• Among the beta-lactam, only the Carbapenems are stable against ESBLs– Imipenem, Meropenem, Ertapenem and

Doripenem are in clinical use

Characteristics of ESBLs

• May appear sensitive to some cephalosporins and combinations of piperacillin and tazobactam as well as amoxicillin and clavulanic acid– However, use of these ß-lactam agents will lead to

microbiological and clinical failure– Only carbapenems among the ß-lactams can be

used successfully

AmpC ß-Lactamases

• Produced by almost all gram-negative bacteria

• Chrosomally encoded versions important in Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Morganella morganii, Pseudomonas aeruginosa and Serratia marcescens (not found in Salmonella and Klebsiella)

• AmpC ß-Lactamase genes have been found on transferable plasmids

Class C ß-Lactamases

• All ß- lactams induce AmpC ß-lactamase production– Only carbapenems are resistant to AmpC ß-

lactamases • If there is loss of porins as well, this will lead to

carbapenem resistance

– Other ß- lactams will be hydrolysed

Metallo-ß-Lactamases

• Require Zinc or other heavy metal for activity• Hydrolyse all ß-Lactams including

carbapenems• Most will be associated with resistance to many

antibiotic classes• Currently New Delhi Metallo-ß-Lactamase

(NDM-1) is a new flavour in the UK– Associated with India– Resistant to almost all antibiotics in use in the UK

Aminoglycosides•Highly positively charged compounds, concentration dependent activity

•Inhibit bacterial protein synthesis by irreversibly binding to 30S ribosomal unit

•Naturally occurring:

•Streptomycin

•Neomycin

•Kanamycin

•Tobramycin

•Gentamicin

•Semisynthetic derivatives:

•Amikacin (from Kanamycin)

•Netilmicin (from Sisomicin)

30S Ribosomal Unit Blockage by Aminoglycosides

•Causes mRNA decoding errors

•Block mRNA and transfer RNA translocation•Inhibit ribosome recycling

Ribosome recycling follows the termination of protein synthesis

Spectrum of Activity• Gram-Negative Aerobes

– Enterobacteriaceae;E. coli, K. pneumoniae, Proteus sp.Citrobacter, Enterobacter sp.Morganella, Providencia, Serratia

– Pseudomonas aeruginosa– Acinetobacter

• Gram-Positive Aerobes (Usually in combination with ß-lactams)S. aureus and coagulase-negative staphylococciViridans streptococciEnterococcus sp. (gentamicin)

Mechanisms of Resistance

• Ribosome changes– Prevents binding

• Loss of cell permeability

• Expulsion by efflux pumps

• Enzyme inactivation by Aminoglycoside modifying enzymes– This is the most important mechanism

Pharmacokinetics• All have similar pharmacologic properties• Gastrointestinal absorption: unpredictable but always

negligible• Distribution

– Hydrophilic: widely distributes into body fluids but very poorly into;• CSF• Vitreous fluid of the eye• Biliary tract• Prostate • Tracheobronchial secretions• Adipose tissue

• Elimination– 85-95% eliminated unchanged via kidney– t1/2 dependent on renal function– In normal renal function t1/2 is 2-3 hours

Adverse Effects• Nephrotoxicity

– Direct proximal tubular damage - reversible if caught early– Risk factors: High troughs, prolonged duration of therapy,

underlying renal dysfunction, concomitant nephrotoxins

• Ototoxicity– 8th cranial nerve damage – irreversible vestibular and

auditory toxicity• Vestibular: dizziness, vertigo, ataxia• Auditory: tinnitus, decreased hearing

– Risk factors: as for nephrotoxicity

• Neuromuscular paralysis– Can occur after rapid IV infusion especially with;

• Myasthenia gravis• Concurrent use of succinylcholine during anaesthesia

Macrolides

• Erythromycin is the prototype antibiotic for this group

• Bacteriostatic- usually• Inhibit bacterial RNA-dependent protein

synthesis• Bind reversibly to the 23S ribosomal RNA

of the 50S ribosomal subunits– Block translocation reaction of the polypeptide

chain elongation

Macrolides

Erythromycin Telithromycin

Clarithromycin

Lactone Ring

Azithromycin

15

14

14

14

Mechanisms of Resistance

• Altered target sites– Methylation of ribosomes preventing antibiotic binding

• Resistance to macrolides , lincosamides (Clindamycin) and streptogramin B

• Can be induced by macrolides

• Efflux pumps– Resistance to macrolides only

• Cross-resistance occurs between all macrolides

Spectrum of Activity

• Gram-Positive Aerobes: – Activity: Clarithromycin>Erythromycin>Azithromycin

• MSSA• S. pneumoniae• Beta haemolytic streptococci and viridans streptococci

• Gram-Negative Aerobes:– Activity: Azithromycin>Clarithromycin>Erythromycin• H. influenzae, M. catarrhalis, Neisseria sp.• NO activity against any Enterobacteriaceae

• Anaerobes: upper airway anaerobes• Atypical Bacteria• Other Bacteria: Mycobacterium avium complex

Pharmacokinetics 1• Erythromycin ( Oral: absorption 15% - 45%)• Short t1/2 (1.4 hr)

• Acid labile• Absorption (Oral)

– Erythromycin: variable absorption of 15% - 45%– Clarithromycin: 55%– Azithromycin: 38%

• Half Life (T1/2)– Erythromycin 1.4 Hours– Clarithromycin (250mg and 500mg 12hrly) 3-4 & 5-7 hours respectively– Azithromycin 68hours – Improved tolerability

• Excellent tissue and intracellular concentrations– Tissue levels can be 10-100 times higher than those in serum

• Poor penetration into brain and CSF• Cross the placenta and excreted in breast milk

Pharmacokinetics 2

• Metabolism & Elimination– Clarithromycin partially eliminated by the

kidney– ALL hepatic elimination

Adverse Effects

• Gastrointestinal (up to 33 %) (especially Erythromycin) • Nausea• Vomiting• Diarrhoea• Dyspepsia

• Thrombophlebitis: IV Erythromycin & Azithromycin

• QTc prolongation, ventricular arrhythmias• Other: ototoxicity with high dose erythromycin in

renal impairment

Fluoroquinolones

Quinolone pharmacore

Fluoroquinolones• Synthetic antibiotics

• Concentration-dependent bactericidal activity• Broad spectrum of activity• Excellent pharmacokinetics

• bioavailability, tissue penetration, prolonged half-lives

• In common use• Ciprofloxacin• Levofloxacin• Moxifloxacin

Mechanism of Action

• Inhibit bacterial topoisomerases which is used by bacteria to;• Relax supercoiled DNA before replication

• DNA recombination

• DNA repair

• DNA gyrase – Primary target for gram-negatives

• Topoisomerase IV – Primary target for gram-positives

Resistance

• Altered target sites due to point mutations.• The more mutations, the higher the resistance

to Fluoroquinolones

• Most important and most common

• Altered cell wall permeability

• Efflux pumps

• Cross-resistance occurs between fluoroquinolones

Spectrum of Activity

• Gram-positive (MSSA Streptococcus pneumoniae )– Moxifloxacin is most active

• Gram-Negative (Enterobacteriaceae H. influenzae, M. catarrhalis, Neisseria sp. Pseudomonas aeruginosa)– Ciprofloxacin is most active

• Atypical bacteria: all have excellent activity

Pharmacokinetics• Absorption

• Good bioavailability

• Oral bioavailability 60-95%

• Divalent and trivalent cations (Zinc, Iron, Calcium, Aluminum, Magnesium) and antacids reduce GI absorption

• Distribution• Extensive tissue distribution but poor CSF penetration

• Metabolism and Elimination• Combination of renal and hepatic routes

Adverse Effects

• Cardiac• Prolongation QTc interval• Assumed to be class effect

• Articular Damage• Cartilage damage

• Induced in animals with large doses

Tetracyclines

•Hydronaphthacene nucleus containing four fused rings

•Tetracycline

•Short acting

•Doxycycline

•Long acting

Mechanism of Action

• Inhibit protein synthesis• Bind reversibly to bacterial 30S ribosomal subunits

• Prevents polypeptide synthesis

• Bacteriostatic

Resistance

• Efflux

• Alteration of ribosomal target site

• Production of drug modifying enzymes

Spectrum of Activity

• All have similar activities• Gram positives aerobic cocci and rods

– Staphylococci– Streptococci

• Gram negative aerobic bacteria• Atypical organisms

– Mycoplasmas– Chlamydiae– Rickettsiae– Protozoa

Pharmacokinetics• Incompletely absorbed from GI, improved by

fasting• Metabolised by the liver and concentrated in bile

(3-5X higher than serum levels)• Excretion primarily in the urine except

doxycycline ( 60% biliary tract into faeces,40% in urine)

• Tissue penetration is excellent but poor CSF penetration– Incorporate into foetal and children bone and teeth

Avoid in pregnancy and children

Adverse Effects

• Oesophageal ulceration

• Photosensitivity reaction

Glycopeptides

• Vancomycin

• Teicoplanin

Vancomycin

Mechanism of Action

• Inhibit peptidoglycan synthesis in the bacterial cell wall• Complex with D-alanyl-D-alanine portion of the cell

wall precursor

Resistance

• Modification of D-alanyl-D-alanine binding site of peptidoglycan

• D-alanyl-D-alanine terminal then ends in D-alanyl-D-lactate

• Leads to lower glycopeptide affinity

• Complex reactions to achieve this

Spectrum of Activity

• Gram positive bacteria only including MRSA

Pharmacokinetics

• Absorption• oral is negligible• IV required therapy for systemic infections

• Distribution– Distributes widely into body tissues and fluids,

including adipose tissue– Variable penetration into CSF, even with inflamed

meninges

• Elimination– Primarily eliminated unchanged by the kidney

Adverse Effects• Red-Man Syndrome

– Erythema multiforme-like reaction with intense pruritus, tachycardia, hypotension, rash involving face, neck, upper trunk, back and upper arms

• Related to infusion rate• Resolves spontaneously after discontinuation• Lengthen infusion (over 2 - 3 hr)

• Hematological– Neutropaenia– Eosinophilia