β-lactam antibiotics

Sayan Das
Antibiotics

• anti- means fighting, opposing, or killing & bios is the Greek

word for life
• antibiotic literally means life-killing
• But Antibiotics are medicines that fight bacterial infection in
people and animals.
• The Range of bacteria that an anti biotics affects can be divided
into
Narrow spectrum and Broad spectrum
Narrow Spectrum Antibiotics

• A type of antibiotic that targets specific types of Gram positive or

Gram negative bacteria.
• Ex- penicillins (penG), the macrolides and vancomycin.

Broad Spectrum Antibiotics

• Broad-spectrum antibiotics are active against a wider number of
bacterial types and, thus, may be used to treat a variety of
infectious diseases.
• Ex- aminoglycosides, the 2nd and 3rd generation cephalosporins,
the quinolones and some synthetic penicillins.
Based on MOA Classification

• Bactericidal: The agents or chemical compound, which kill the

microorganisms, are called Bactericidal.
• ex-penicillin’s, ampicillins, cefixime, cefadroxil, ciprofloxacin
etc..
• Bacteriostatic: The agents or chemical compound or drugs which
prevent the further growth or multiplication of microorganism,
are called Bacteriostatic.
• Ex- sulphonamide, chloramphenicol tetracyclines.
Antibiotics having β-lactam ring

• Penicillins
• Cephalosporins
• Monobactams and carbapenems
Beta-lactam structure
Beta-lactam ring
• A beta-lactam (β-lactam) ring is a four-
membered lactam.
• A lactam is a cyclic amide
• Beta-lactams are named so because the
nitrogen atom is attached to the β-carbon atom
relative to the carbonyl

Cyclic amide
β-lactam
Bacterial Cell walls

Gram +ve • Gram positive cell wall Gram –ve

Bacteria Bacteria
• Gram negative cell wall

• It is important to note that not all bacteria have a cell wall.

• Having said that though, it is also important to note
that most bacteria (about 90%) have a cell wall.
• The technique used in identifying the different bacteria called
Gram stanning
• After this stain technique is applied the gram positive bacteria will
stain purple, while the gram negative bacteria will stain pink.
Overview of Bacterial Cell Walls

• Outside of cell
• Semi-rigid structure
• Both gram positive and gram negative cell walls
contain an ingredient known as contain an ingredient
known as peptidoglycan (also known as murein)

Functions
• Providing overall strength to the cell
• Maintain the cell shape
• Obtained nutrients and Move
• Protect from Osmotic lysis
Structure of Peptidoglycan

• Peptidoglycan is a polysaccharide made of two glucose derivatives, N-acetylglucosamine

(NAG) and N-acetylmuramic acid (NAM), alternate in long chains.
• The chains are cross-linked to one another by a tetrapeptide
• Tetrapeptide is made up of
• L-alanine
• D-glutamine
• L-lysine or meso-diaminopimelic acid (DPA)
• D-alanine
• The tetrapeptides can be directly cross-linked to one another, with the D-alanine
on one tetrapeptide binding to the L-lysine on another tetrapeptide.
Cont.…

• Typically only the L-isomeric form of amino acids are utilized by

cells but the use of the mirror image D-amino acids provides
protection from proteases that might compromise the integrity of
the cell wall by attacking the peptidoglycan.
• In many gram positive bacteria there is a cross-bridge of five
amino acids such as glycine (peptide interbridge) that serves to
connect one tetrapeptide to another.
• In either case the cross-linking serves to increase the strength of
the overall structure, with more strength derived from complete
cross-linking, where every tetrapeptide is bound in some way to a
tetrapeptide on another NAG-NAM chain.
Structure of Peptidoglycan
Gram +ve cell walls and Gram –ve cell wall
Gram +ve bacteria cell walls

• Contains thick layers of peptidoglycan to support the cell

membrane and provide a place of attachment for other molecules.
The thick layers also enable Gram positive bacteria to retain most
of the crystal violet dye during Gram staining causing them to
appear purple.
• The cell walls also contain chains of teichoic acid that extend from
the plasma membrane through the peptidoglycan cell wall. These
sugar- containing polymers helps in maintaining cell shape and
play a role in proper cell division.
Cont.…

• Some Gram positive bacteria have an additional component,

mycolic acid that produce a waxy outer layer for additional
protection for mycobacteria, such as Mycobacterium tuberculosis.
Gram positive bacteria with mycolic acid are also called acid-fast
bacteria because they require a special staining method, known as
acid-fast staining, for microscope observation.
• Pathogenic Gram positive bacteria cause disease by the secretion
of toxic proteins known as exotoxins.
Cont.…
Gram –ve bacteria cell wall

• The Gram negative bacterial cell wall is composed of a single thin layer
peptidoglycan.
• The cell wall of Gram negative bacteria is more complex than that of Gram
positive bacteria. Located between the plasma membrane and the thin
peptidoglycan layer is a gel-like matrix called periplasmic space.
• Gram negative bacteria have an outer membrane layer that is external to
the peptidoglycan cell wall. Membrane proteins, murein lipoproteins,
attach the outer membrane to the cell wall.
• Another unique characteristic of Gram negative bacteria is the presence of
lipopolysaccharide (LPS) molecules on the outer membrane. LPS is a large
glycolipid complex that protects bacteria from harmful substances in their
environment. It is also a bacterial toxin (endotoxin) that can cause
inflammation and septic shock in humans if it enters the blood.
Cont.…

• There are three components of the LPS:

• lipid A
• a core polysaccharide
• and an O antigen
• The lipid A component attaches the LPS to the outer membrane. Attached to
the lipid A is the core polysaccharide. It is located between the lipid A
component and the O antigen. The O antigen component is attached to the core
polysaccharide and differs between bacterial species. It can be used to identify
specific strains of harmful bacteria.
• Some Gram negative bacteria also produce exotoxins or endotoxin.
Cont.…
Ex of gram +ve & gram –ve bacteria

GRAM +VE GRAM -VE

Staphylococcus aureus Staphylococcus aureus

Streptococcus pneumoniae E. coli

Staphylococcus epidermidis Staphylococcus

Mycobacterium tuberculosis Anthrax bacterium

Clostridium botulinum Lactobacillus

Bacterial cell wall synthesis

• The bacteria synthesize UDP-N-acetylmuramic acid pentapeptide,

called ‘Park nucleotide’ and UDP-N-acetyl glucosamine.
• The peptidoglycan residues are linked together forming long
strands and UDP is split off.
• The final step is cleavage of the terminal D-alanine of the peptide
chains by transpeptidases.
• The energy so released is utilized for establishment of cross
linkages between peptide chains of the neighboring strands . This
cross linking provides stability and rigidity to the cell wall.
Steps
Cont.…

• These steps involve the synthesis of the peptidoglycan precursors

lipid I and lipid II, the flipping of lipid II across the cytoplasmic
membrane, and the polymerization of glycan chains from lipid II
and their incorporation into the existing cell wall by peptidoglycan
synthases.
Video of bacterial cell wall synthesis
MOA of Beta lactam antibiotics

• The β-lactam antibiotics inhibit the transpeptidases so that cross

linking (which maintains the close knit structure of the cell wall)
does not take place.
• These enzymes and related proteins constitute the penicillin
binding proteins (PBPs) which have been located in the bacterial
cell membrane.
• Each organism has several PBPs, and PBPs obtained from different
species differ in their affinity towards different β-lactam
antibiotics.
• This fact probably explains their differing sensitivity to the various
β-lactam antibiotics.
Cont..

• When susceptible bacteria divide in the presence of a β-lactam

antibiotic—cell wall deficient (CWD) forms are produced.
• Because the interior of the bacterium is hyperosmotic, the CWD
forms swell and burst → bacterial lysis occurs. This is how β-lactam
antibiotics exert bactericidal action.
• Under certain Conditions and in case of certain organisms, bizarre
shaped or filamentous forms, which are incapable of multiplying,
result. Grown in hyperosmotic medium, globular ‘giant’ forms or
protoplasts are produced. Lytic effect of these antibiotics may also
be due to derepression of some bacterial autolysins which normally
function during cell division.
Cont..

• Rapid cell wall synthesis occurs when the organisms are actively
multiplying; β-lactam antibiotics are more lethal in this phase.
• The peptidoglycan cell wall is unique to bacteria. No such substance
is synthesized (particularly,D-alanine is not utilized) by higher
animals. This is why penicillin is practically nontoxic to man.
• In gram-positive bacteria, the cell wall is almost entirely made of
peptidoglycan, which is >50 layers thick and extensively cross linked,
so that it may be regarded as a single giant Mucopeptide molecule.
• In gram-negative bacteria,the cell wall consists of alternating layers
of lipoprotein and peptidoglycan (each layer 1–2 molecule thick with
little cross linking). This may be the reason for higher susceptibility of
the gram-positive bacteria to PnG.
MOA figure
1. The bacterial cell wall consists of strands of repeating N-
acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
subunits. The NAM subunits have short peptide chains attached to
them. (The exact composition of these can vary. The proximal
alanine is usually L-ala and the distal two are usually D-ala.)
2. The PBP binds the peptide side chains and forms the cross-link
with the expulsion of one D-Alanine from one peptide side chain.
(See PBP_catalysis.svg for details.)
3. The PBP dissociates from the wall once the cross-link has been
formed.
4. Penicillin is added to the system. It enters the active site of the
PBP and reacts with the serine group that is important in its
enzymatic activity.
5. The beta-lactam ring of penicillin (represented here as the top
of the "P") is irreversibly opened during the reaction with the PBP.
Penicillin remains covalently linked to the PBP and permanently
blocks the active site.
Video
PENICILLINES

• Penicillin was the first

antibiotic to be used
clinically in 1941
• Penicillin was originally
obtained from the fungus
Penicillium notatum, but
the present source is a
high yielding mutant of P.
chrysogenum.
Penicillin discover

• In 1928 Dr Alexander Fleming returned from a holiday to find mould

growing on a Petri dish of Staphylococcus bacteria. He noticed the
mould seemed to be preventing the bacteria around it from growing.
He soon identified that the mould produced a self-defense chemical
that could kill bacteria.
The Real Story Behind Penicillin ?

https://www.pbs.org/newshour/health/the-real-story-behind-
the-worlds-first-antibiotic
PENICILLIN-G (BENZYL PENICILLIN)

Effective against:
• gram-positive bacteria
• few gram negative
Ttreatment for:
• Streptococcal infections: Like pharyngitis, otitis media, scarlet fever, rheumatic
fever
• Pneumococcal infections : pneumonia and meningitis
• Meningococcal infections : meningitis
• Gonorrhoea
• Syphilis
• Diphtheria
Characteristics:
• Narrow spectrum antibiotic
• Should be given I.V or I.M
• Resistance to B lactamase
 How Do Bacteria Develop Resistance
Against β-Lactam Penicillins?
• The mode of action of penicillins depends greatly on the presence of the β-lactam ring.
• This structure produces a molecular ‘key’ that is able to interact with the active site ‘lock’
of the transpeptidase enzyme; disrupting its ability to create peptidoglycan cross-links in
the bacterial cell wall.
I. Bacteria Can Use β-Lactamases to Break Open the β-Lactam Ring
• Just as β-lactam rings can interfere with the activity of enzymes, they can also be cleaved
through enzymatic activity.
• Enzymes called β-lactamases can catalase the opening of the β-lactam ring through
reaction with water, in a hydrolysis reaction
• hydrolysis gives a penicilloic acid, which is not antibacterially active and is readily
excreted from the body once produced.
Cont..

II. Designing Penicillins That Resist Being Inactivated by β-lactamases

• Many bacterial species can synthesise their own β-lactamases which will
target and disable penicillins.
• Penicillins themselves can be modified to provide some protection against
these enzymes.
• For example, in methicillin, bulky substituted benzene (at the variable R site
of the penicillin molecule) provides a steric hindrance to any enzymes that try
to interact with the β-lactam ring of the penicillin
III. Bacteria Can Become Resistant to Penicillin by Modifying Enzymes That
Make the Cell Wall
• Some bacteria, including Streptococcus phenominae, have developed
resistance to β-lactams through modification of their penicillin binding
proteins (or PBPs), which make up the active site of transpeptidase enzymes.
Cont..

• Where normally β-lactams react with PBPs to

form a relatively stable ring-opened product,
mutations in the genetic coding for proteins
making up the transpeptidase enzyme can
result in multiple PBPs that have a low binding
affinity for β-lactams.
• As such, our β-lactam ‘key’ no longer fits into
the transpeptidase active site ‘lock’. Penicillins
do not bind as effectively to their target PBPs
and the transpeptidase enzymes continue to
function, binding to their original substrate
rather than the antibiotic.
Classification

Oxacillin
Nafcillin
Flucloxacillin
Acid-resistant alternative to (Penicillin G)
Phenoxymethyl penicillin (Penicillin V)
• It differs from PnG only in that it is acid stable.
• Oral absorption is better.
Effective against:
• Gram positive
• Less in gram negative
Treatment for:
• Tonsillitis
• Anthrax
• Rheumatic fever
• Streptococcal skin infection
• The antibacterial spectrum of penicillin V is identical to PnG, but it
is about 1/5 as active against Neisseria, other gram negative
bacteria and anaerobes.
Characteristics:
• Narrow spectrum
• Should be given orally
• Prone to b-lactamase
Penicillinase resistance Penicillins
Methicillin
Effective against:
• Gram positive Bacteria
Treatment for:
• It is use to treat infections caused by susceptible Gram-
positive bacteria, Particularly Beta lactamase producing
Organism such as Staphylococcus auras
Characteristics:
• Very Narrow spectrum
• Should be given parenterally
Side effects
• It is staphylococcal penicillinase resistant but not acid
resistant.
• Because MRSA (methicillin resistant Staph. Aureus) have
emerged in many areas, and because methicillin caused
hematuria, albuminuria and interstitial nephritis, it is no
longer used.
Cloxacillin/Dicloxacillin

Effective against:
• activity against penicillinase producing Staphylococcus
Characteristics:
• Very Narrow spectrum
• Should be given orally
Side effects:
• Allergic reaction
Dicloxacillin

Effective against:
• activity against penicillinase producing
Staphylococcus gram +ve bacteria
Characteristics:
• Very Narrow spectrum
• Should be given orally
Side effects:
• Allergic reaction
• Diarrhea ,nausea, rash , pain in site of
injection
Oxacillin

Effective against:
• activity against penicillinase producing Staphylococcus
Characteristics:
• Very Narrow spectrum
• Should be given parentally
Side effects:
• Hypersensitivity reaction
Nafcillin

Effective against:
• Gram +ve bacteria
Characteristics:
• Very Narrow spectrum
• Should be given parentally
Side effects:
• Allergic reaction
• Nausea and vomiting
• Abdominal pain
Flucloxacillin

Effective against:
• activity against penicillinase producing
Staphylococcus gram +ve bacteria
Characteristics:
• Very Narrow spectrum
• Should be given orally
Side effects:
• Allergic reaction
• Diarrhea ,nausea, rash , pain in site of
injection
EXTENDED SPECTRUM PENICILLINS

• active against a variety of gram-negative

bacilli
• improved ability to penetrate through their
cell membrane
Aminopenicillins:

• This group includes ampicillin, its prodrug

bacampicillin, and amoxicillin.
Ampicillin

Effective against:
• All organisms sensitive to PnG.
• Gram +ve & Gram –ve
Treatment for:
• Urinary tract infections (UTI)
• Respiratory tract infections
• Meningitis
• Gonorrhoea
• Typhoid fever
• Cholecystitis
• Ear infection
Side effects:
• Diarrhoea is frequent after oral administration.
• It produces a high incidence of rashes, especially in patients with
AIDS,EB virus infections or lymphatic leukaemia.
Characteristics:
• Broad spectrum
• Can be given orally and parentally
• Prone to B-lactamase
Amoxicillin

• It is a close congener of ampicillin (but not a prodrug)

except
• Oral absorption is better; food does not interfere with
absorption
• Amoxicillin given 3 times a day is equivalent to ampicillin
given 4 times a day.
• Incidence of diarrhea is lower.
• It is less active against Shigella and H.influenzae.
• It is more active against relatively penicillin resistant
Strep. pneumoniae.
• Many physicians now prefer amoxicillin over ampicillin for
bronchitis, urinary infections, and gonorrhoea.
Bacampicillin

• It is an ester prodrug of ampicillin

• It is nearly completely absorbed from the g.i.t
• and is largely hydrolysed during absorption.
• higher plasma levels are attained.
Carboxypenicillins
Carbenicillin

Effective against:
• Gram –ve & imitated gram +ve bacteria
Treatment for:
• UTI
Characteristics:
• Highly soluble in water and acid liable
• It is inactive orally and is excreted rapidly
in urine
Side effects:
• High doses caused bleeding
Ticarcillin

• It is the second carboxypenicillin,

similar in properties to carbenicillin,
but is more active and produces
fewer adverse effects
• Ticarcillin has therefore mostly
displaced carbenicillin for treating
pseudomonas and proteus infections.
• Its combination with clavulanic acid
extends efficacy to cover b-lactamase
producing strains.
• For the treatment of serious
Pseudomonas infections it is often
used along with gentamicin.
Ureidopenicillins
Piperacillin

• This antipseudomonal penicillin is about 8

times more active than carbenicillin.
• It is combined with tazobactam to cover b-
lactamase producing strains
Effective against:
• Gram –ve & gram +ve bacteria
Characteristics:
• Extended spectrum
• Should be given IV or IM
Mezlocillin

• Another antipseudomonas penicillin, not marketed in USA and in

India.
BETA-LACTAMASE INHIBITORS

• β-lactamases are a family of enzymes produced by many gram-

positive and gram-negative bacteria that inactivate β-lactam
antibiotics by opening the β-lactam ring.
• Three inhibitors of
• this enzyme clavulanic acid, sulbactam and tazobactam are
available for clinical use only in combination with specific
penicillins or cephalosporins.
Clavulanic acid

• Obtained from Streptomyces clavuligerus, it has a β-lactam ring but

no/weak antibacterial activity of its own.
• It inhibits a wide variety of β-lactamases produced by both gram-positive
and gram-negative bacteria.
• Clavulanic acid is a ‘progressive’ inhibitor, because binding with β-
lactamase is reversible initially, but becomes covalent later—inhibition
Increases with time.
• Called a ‘suicide’ inhibitor,it gets inactivated after binding to the enzyme.
• Clavulanate permeates the outer layers of the cell wall of gram-negative
bacteria and inhibitsthe periplasmically located β-lactamase.
Cont..

• AUGMENTIN, ENHANCIN, AMONATE :

Amoxicillin 250mg + clavulanic acid 125
mg tab
Adverse effects:
• tolerance is poorer—especially in children.
• Candida
• stomatitis
• Vaginitis
• Rashes
• hepatic injury
Sulbactam

• It is a semisynthetic β-lactamase inhibitor, related chemically as

well as in activity to clavulanic acid.
• On weight basis, it is 2–3 times less potent than clavulanic acid for
most types of the enzyme, but the same level of inhibition can be
obtained at the higher concentrations achieved clinically.
• Oral absorption of sulbactam is inconsistent.
• Therefore, it is preferably given parenterally.
• It has been combined with ampicillin for use against β-lactamase
producing resistant strains.
Cont..

• SULBACIN, AMPITUM: Ampicillin 1 g +

sulbactam 0.5 g per vial inj;
• Sulbactam has been combined with
cefoperazone and ceftriaxone also
Adverse effects:
• Pain at site of injection,
• thrombophlebitis of injected vein,
• rash and diarrhea
Tazobactam

• Another β-lactamase inhibitor similar to

sulbactam whose pharmacokinetics
matches with piperacillin
• Tazobactam is combined with piperacillin
for use in severe infections like
peritonitis, pelvic/urinary/respiratory
infections caused by β-lactamase
producing bacilli
• Pybactum, tazact , tazobid , TAZAR: 4 g +
0.5 g vial for inj.,