Pathogen: Any organism that can produce disease.

A pathogen may also be referred to as an infectious agent, or simply a germ.

Virus, bacterium, protozoan, or fungus.
Small animals, such as certain worms or insects, can also cause or transmit disease, these animals are usually referred
to as parasites rather than pathogens.

Pathogenesis: The process by which a disease or disorder develops.

Greek… pathos = suffering or disease, genesis = creation
Internal factors or external factors. Not to be confused with pathogen.

Disorder: A functional abnormality or disturbance.

Medical disorders can be categorized into mental disorders, physical disorders, genetic disorders, emotional and
behavioural disorders, and functional disorders.
Syndrome: The association of several signs and symptoms, or other characteristics that often occur together,
regardless of whether the cause is known.
Down syndrome, Parkinsonian syndrome, Acute coronary syndrome, etc.

• A disease is a pathophysiological response to internal or external factors.

• A disorder is a disruption to regular bodily structure and function.
• A syndrome is a collection of signs and symptoms associated with a specific health-related cause.

Diseases in humans that are caused by infectious agents are known as pathogenic diseases.
Not all diseases are caused by pathogens, other causes are, for example, toxins, genetic disorders and the
host's own immune system.
Antibiotic: A substance active against bacteria. Usually a medicine.
Antibacterial: An antibiotic, a type of antimicrobial agent used mainly against bacteria; it may kill or inhibit bacteria.
Not necessarily a medicine, like antibacterial soap

Inhibit: Don’t let them multiply or reproduce.

Inhibition: A process that inhibit.

Antibacterial agents may also refer to:

Antiseptic: A principal type of antimicrobial agent used mainly against bacteria; it may kill or inhibit them.
Disinfectant: Kill or inhibit microbes in cleaning/sanitation but not taken internally as medicine.
Bactericide: Kills bacteria. Focus on –cide, referring to killing … suicide, insecticide, etc. Stop a process in bacteria
necessary for its survival.
Bacteriostatic: An agent that does not kill bacteria but inhibits their multiplication. Focus on word static. Stop a
process in bacteria necessary for replication.
Types of bacteria
Hans Christian Gram
Gram positive and Gram negative…G … CAPITAL
• Gram staining differentiates bacteria by the chemical and physical properties of their cell walls.
• Gram-positive cells have a thick layer of peptidoglycan (sugars and amino acids) in the cell wall, giving violet or purple
colour.
• Gram-negative cells have a thinner peptidoglycan layer that stain pink or red.

• A broad-spectrum antibiotic is an antibiotic that acts on both Gram-

positive and Gram-negative bacteria, or on multiple bacterial strains.
• Narrow-spectrum antibiotic is an antibiotic that is only able to kill or
inhibit limited species of bacteria.
When broad-spectrum antibiotic is good?
A Gram stain of mixed Staphylococcus
When narrow-spectrum antibiotic is good? aureus (Gram-positive cocci, in purple)
Anaerobic: Don’t need oxygen and Escherichia coli (Gram-negative, in
red)
Aerobic: Need oxygen
Resistance: Act against …

Bacterial resistance:
Bacteria antibiotic resistance:
Could be against any antibacterial agents,
antiseptic, disinfectant, bactericide, or
bacteriostatic.

Natural Selection: Survival for the fittest

Efflux:
Influx:
Inactivating enzyme:
Target amplification:
Target alteration:
SOME DEFINATIONS
In vitro: Enzyme inhibition or cell-based
In vivo: Animal model
In silico: Computer-based

Water: Medium in living systems, inside or outside of cells

Liquids: Volume of solution and amount of solute…. Here comes concentration.
% (w/w, w/v, v/w, v/v), molarity, mg/ml, μg/ml, etc.
IC50: Concentration at which 50% inhibition takes place

IC90: Concentration at which 90% inhibition takes place

MIC: Minimum inhibitory concentration, lowest concentration of an agent that prevents visible growth
 Dose-response Curve.

How to validate if the test was OK

Controls
1- Negative control (no control)
2- Positive control (a standard)

Why 50, 90, or MIC

MIC
Start from very low concentration, keep on
increasing, see at which concentration there is no
growth.
Other way around.
Start form a very high concentration, keep on
decreasing, see at which concentration growth
starts.
The success of antibacterial agents owes much to the fact that they can act selectively against bacterial cells rather than
animal cells.
Bacterial cells and animal cells differ both in their structure and in the biosynthetic pathways which proceed inside them.

Differences between bacterial and animal cells

1- The bacterial cell has a cell wall, as well as a cell membrane,
whereas the animal cell has only a cell membrane.
The cell wall is crucial to the bacterial cell's survival. Bacteria have
to survive a wide range of environments and osmotic
pressures, whereas animal cells do not. If a bacterial cell lacking a
cell wall was placed in an aqueous environment containing a low
concentration of salts, water would freely enter the cell due to
osmotic pressure. This would cause the cell to swell and
eventually 'burst'. The cell wall does not stop water flowing into
the cell directly, but it does prevent the cell from swelling and so
indirectly prevents water entering the cell.
• The bacterial cell does not have a defined nucleus, whereas the
animal cell does.
• Animal cells contain a variety of structures called organelles
(e.g. mitochondria, etc.), whereas the bacterial cell is relatively
simple.
 Bacterial Cell Animal Cell
It does not have a well-defined, membrane-enclosed nucleus. It contains a well-defined membrane-enclosed nucleus.

It does not have cell organelles.
It is much smaller in size than an animal cell, e.g. 0.2 to 10
micron in size.
It has a well-defined cell wall.
Membrane-bound cell organelles are present in the cytoplasm.
It is larger than a bacterial cell, e.g. 100 microns or more.
It lacks the cell wall.

They are generally autotrophs and maybe heterotrophs. They are always heterotrophs.
They reproduce asexually by binary fission and sexually by They can reproduce asexually by mitosis and sexually by
conjugation. meiosis.
It lacks the plasmids. It has well-defined linear DNA in the
It has plasmids (circular DNA in the cytosol).
nucleus.
It has a single chromosome. It has many chromosomes.

It does not have mitochondria. It has mitochondria in the cytoplasm.

It has well-defined cell shape. It has irregular shapes as it lacks the cell wall.

It lacks the cytoskeleton. It has cytoskeleton (a network of filaments and tubules in the
cytoplasm).
It lacks histone proteins. Its DNA is wrapped around histone proteins.
The mechanism of action of a drug is the biochemical way in which a drug is pharmacologically effective.
This can be a specific target where the drug binds like an enzyme, as is the case with many antibiotics, or a receptor.
Mechanism of action describes the biochemical process specifically at a molecular level.
What is the molecular level?
How different molecules, small molecules or macromolecules, interact.
By interact we mean, chemically react or attach to each other via different intermolecular forces, e.g., H-bond, vdW.

Antibacterial action or antibiotic mechanism of action

Four types… either enzyme inhibition or cell membrane disruption

a- enzymes involved in cell wall synthesis

b- nucleic acid metabolism and repair
c- protein synthesis
d- Cell membrane disruption

Many of these cellular functions targeted by antibiotics are most active in multiplying cells. Since there is often
overlap in these functions between bacterial cells and mammalian cells, therefore all antibiotics have some degree of
side effect.
Antimetabolites
Compounds that interrupt cell metabolism in bacteria.

Metabolism is a term that is used to describe all chemical reactions involved in maintaining the living state of the
cells and the organism. Metabolism can be conveniently divided into two categories:
Catabolism - the breakdown of molecules to obtain energy
Anabolism - the synthesis of all compounds needed by the cells

Sulphonamides/sulfonamides also known as sulfa drugs were first antimetabolites.

World War 2 and German dye industry

Use of dyes to stain microorganisms for microscopy
Bayer AG discovered prontosil
Antibacterial in in vivo, but inactive in in vitro
Prodrugs
The compounds that are inactive in their original form, but
when metabolised produce fragment having active drug.

Gram-positive streptococcus pneumoniae (pneumococcus), and

neisseria meningitidis (meningococcus)
During those days typhoid (Gram negative, salmonella typhi) was a major concern, and prontosil was inactive
against it.
Triaminobenzene is toxic.
Due to the activity against limited type of bacteria, and side effects, penicillin was preferred over prontosil.

Structure-activity relationships (SAR)

Research and development
Improve activity and reduce toxicity

1- Parts
i- Sulfonamide (red)
ii- Benzene ring (blue)
iii- Amino group (black)
The aromatic ring, sulfonamide, amino all three are necessary

2- Configuration of three parts

The aromatic ring must be para-substituted only.
3- Substitution on amino group

4- The only possibility for R is acyl group to sustain activity .

It forms an amide, which is inactive but can be metabolized in the body to
regenerate the active compound.
 4-Substitution on sulfonamide

The sulfonamide nitrogen must be secondary.

R is the only possible site that can be varied in sulfonamides.

5-Substitution on benzene ring

Substation on benzene ring usually diminishes the activity
Summary
Short acting or long acting drugs
Plasma proteins, such as albumin and globulin bind with the
drugs and carry them throughout the body.
This binding is somewhat similar as detergents work
(nonpolar bind with nonpolar and polar bind with polar).

Greater the binding with plasma greater would be half life of

the drug.

Half life of a drug

After one half-life, the concentration of the drug in the body
will be half of the starting concentration.

Suppose, at a particular time the concentration of the drug is

10 mg/litter.
The drug would be metabolised and eliminated from the
body, primarily through liver and kidneys.
The time at which the concentration is 5 mg/litter is half life.
If half life is 4 hr …
What would be the concentration of the drug after 3-half-
life.
Solubility
Higher the solubility lesser the toxicity

R’’ = Plasma binding and half life (short or long acting), effect on solubility
R’ = Acyl groups, effect on solubility. Nonpolar acyl groups are more toxic.

Sulfanilamide analogues
1- Sulfathiazole

Greater the pKa,

lesser the ionization,
and lesser the
solubility

2- Sulfadiazine
3- Sulfadoxine and sulfadimethoxine

Applications of sulfonamides
• Before the appearance of penicillin, the sulfa drugs
were the drugs of choice.
• Penicillins largely superseded sulfonamides in the
fight against bacterial infections.
• The interest in sulfa drugs increased after the
discovery of a new 'breed' of sulfonamides that has
exceptionally high half life .
The sulfa drugs presently have the following
applications in medicine:
• treatment of urinary tract infections
• eye lotions
• treatment of infections of mucous membranes
• treatment of gut infections
4- Succinyl sulfathiazole

Intestine pH
The pH gradually increases in the small intestine from pH 6 to about pH 7.4 in the terminal ileum. The pH
drops to 5.7 in the caecum, but again gradually increases, reaching pH 6.7 in the rectum.

Due to these pH conditions succinyl sulfthiazole can not absorbed in intestine, so, it is unable to reach blood.
It stays in the gut and slowly converts into sulfathiazole with the help of enzymes in the gut. Hence, low
amount of active sulfathiazole in the gut is good enough to kill gut bacteria. Its very small amount reaches
blood that has negligible effect.
Mechanism of action

Folic acid is the synthetic form of folate, which is a

naturally occurring B vitamin. Folate helps make DNA
and other genetic material. It is especially important in
prenatal health. Folate, also called vitamin B-9, is a B
vitamin that naturally occurs in certain foods.

Human cells can not make folic acid, it is acquired from

food.
Bacterial cells can not transport dietary folic acid via
their cell membrane.
A biochemical reaction normally
taking place in the body.
Conversion of substrate to products
is necessary for the normal body
function.

The inhibitor (our drug) can occupy

the place on the enzyme where
substrate needs to interact
(commonly called active site of the
enzyme).

The drug binds to the enzyme at a

place other than active site. This
changes the shape of the active site
and now enzyme can not catalyse
the reaction with substrate.
 Trimethoprim

The overall effect, however, is the same as with sulfonamides—

the inhibition of DNA synthesis and cell growth
Just for ref. (no need to prepare)
Fig. 10.15

Trimethoprim

History of penicillin

Reading…..
Penicillin structure
3D structure of penicillin.
Blue = N
Red = O
White = H
Penicillin contains a highly unstable-
looking bicyclic system due to the
presence bond constraints in the ring
system.

Dorothy Hodgkins established the

complex structure of penicillin by
structure by X-ray analysis (1945).
Penicillin analogues
Potency relative
UK US
Name
nomenclature nomenclature
to penicillin G
2-Pentenylpenicillin Penicillin I Penicillin F 70–82%
Benzylpenicillin Penicillin II Penicillin G 100%
p-Hydroxybenzylpenicillin Penicillin III Penicillin X 130–140%
n-Heptylpenicillin Penicillin IV Penicillin K 110–120%
Isolation and production of penicillin Just for reference. No need to prepare.

Unfortunately, it was unstable and Fleming was unable to isolate and purify the
compound. He therefore came to the conclusion that penicillin was too unstable to
be used clinically.

In 1938, Florey and Chain isolated penicillin using freeze-drying under much milder
conditions.

The synthesis of such a highly strained molecule presented a huge challenge.

Sheehan who completed a full chemical synthesis of penicillin by 1957 (1 % yield).

Modification in fermentation media
The acyl side-chain (R) varies, depending on the make up of the fermentation media. For example, corn steep liquor
was used as the medium when penicillin was first mass-produced in the United States and this gave penicillin G
(R=benzyl). This was due to high levels of phenylacetic acid (PhCH2COOH) present in the medium.

Only acids of general formula RCH2COOH can give desired product.

This in turn restricts the variety of analogues which can be obtained.
It is a tedious and time-consuming process due to the involvement of slow fermentation.
Semi-synthetic penicillin
In 1958, Beechams isolated a biosynthetic intermediate of penicillin
called 6-aminopenicillanic acid (6-APA) which provided a readily
accessible biosynthetic intermediate of penicillin. This revolutionized
the field of penicillins by providing the starting material for a huge
range of semisynthetic penicillins.

Allows the synthesis of a huge number of analogues.

In common practice, the fermentation media contain 6-APA and different analogues of penicillin.
Properties of penicillin G

• Active versus Gram-positive bacilli (e.g. staphylococci, meningitis, and gonorrhoea) and many (but not all) Gram-
negative cocci. So, not broad spectrum.

• Non-toxic! This point is worth emphasizing. The penicillins are amongst the safest drugs known to medicine.

• Ineffective when taken orally. Penicillin G can only be administered by injection. It is ineffective orally since it
breaks down in the acid conditions of the stomach.

• Sensitive to all known β-lactamases. These are enzymes produced by penicillin resistant bacteria which catalyse
the degradation of penicillins.

• Allergic reactions are suffered by some individuals. Test dose is used if penicillin is used first time.

Clearly, there are several problems associated with the use of penicillin G, the most serious being acid sensitivity,
sensitivity to penicillinase, and a narrow spectrum of activity. The purpose of making semisynthetic penicillin
analogues is therefore to find compounds which do not suffer from these disadvantages.
Structure-activity relationships of penicillins
A large number of penicillin analogues have been synthesized and
studied. The results of these studies led to the following conclusions.

• The strained β-lactam ring is essential.

• The free carboxylic acid is essential.

• The bicyclic system is important (confers strain on the β -lactam ring

—the greater the strain, the greater the activity, but the greater the
instability of the molecule to other factors).

• The acylamino side-chain is essential.

• Sulfur is usual but not essential.

• The stereochemistry of the bicyclic ring with respect to the

acylamino side-chain is important.

variation which can be made is restricted to the acylamino side-chain

(R group).
Problems with penicillins

Acid sensitivity
There are three reasons for the acid sensitivity of penicillin G.

1- Ring strain
Penicillin suffers large angle and torsional strains

2- β-Lactam carbonyl group is very reactive because it is not Normal amides

resonance stabilized. The delocalization of electrons is not
possible due to the distorted angles.
3- Self dissociation

Influence of the acyl side-

chain (neighbouring group
participation).
Tackling the problem of acid sensitivity
β-Lactam is essential for activity, so any change
in it of ring strain is not possible.

Effect of EWG on side chain

Electron withdrawing group on amide side chain pulls electrons of the amide group
towards itself.

Disubstitution on the alpha-carbon is very good in terms of

activity and stability.
 Sensitivity to β-lactamases

Recall the topic of antibacterial resistance…

β-Lactamases are enzymes that can cleave β-lactam ring in the same fashion as acid hydrolysis.
Bacteria produce β-Lactamases.
Staph. aureus is very efficient in producing β-Lactamases.
In 1960 a strain of penicillin-resistant Staph. aureus emerged that was not sensitive to penicillin G.
If a bacteria is sensitive to a drug, the bacteria is killed by the drug.

Tackling the problem of β-lactamase

Penicillin can bind to both β-lactamase and DD-

transpeptidase (two proteins present in bacteria).

If penicillin binds to β-lactamase, penicillin is decomposed.

If penicillin binds to DD-transpeptidase, bacteria is killed.
A change is needed in penicillin, after which it can lose its
property to bind with β-lactamase but keep its property to
bind with DD-transpeptidase.
Penicillins competitively inhibit penicillin-binding proteins such
as the enzyme DD-transpeptidase. This catalyses the cross-
linking of peptidoglycans in bacterial cell walls.
Inhibition of this process weakens the cell walls, allowing an
influx of water into the cell, which leads to cell swelling and
then cell lysis.
A bulky group on the side-chain
If the drug bigger and going out of the pocket, the binding becomes weaker.

So, there is an optimum size of the side chain that binds very well with DD-transpeptidase
but can not bind with β-lactamase.
drug
drug
drug
drug
Methicilin binds with DD-transpeptidase
but not with β-lactamase.
No electron withdrawing group on the DD-Transpeptidase β-Lactamase
side-chain, it is acid sensitive, and so has
to be injected.

These three compounds are acid-resistant and penicillinase-resistant, and are also useful against
Staph. aureus infections.
The only difference between the above three compounds is the type of halogen substitution on the aromatic ring. The
influence of these groups is found to be pharmacodynamic, that is, they influence such factors as absorption of the
drug and plasma protein binding. For example, cloxacillin is better absorbed through the gut wall than oxacillin,
whereas flucloxacillin is less bound to plasma protein, resulting in higher levels of the free drug in the blood supply.

These three penicillins have inferior activity to the original penicillins (V or G) when they are used against bacteria
without the penicillinase enzyme.
They also prove to be inactive against Gram-negative bacteria.

Acid-resistant penicillins would be the first choice of drug against an infection (oral administration).
If the bacteria proved resistant due to the presence of a penicillinase enzyme, then the therapy would be changed to
a penicillinase-resistant penicillin.
Narrow spectrum of activity

Poor activity against Gram-negative bacteria.

1- Low membrane permeability in Gram-

negative bacteria.
Gram-negative bacteria have a coating on the
outside of their cell wall which consists of a
mixture of fats, sugars, and proteins.
Outer surface may have an overall negative or
positive charge depending on its constituent
triglycerides.
An excess of phosphatidylglycerol would result in an overall anionic charge
whereas an excess of lysylphosphatidylglycerol would result in an overall
cationic charge.
Penicillin has a free carboxylic acid which if ionized would be repelled from the
negative type of membrane.
The fatty portion of the coating may act as a barrier to the polar hydrophilic
penicillin molecule.

Protein channels can transport penicillin type of molecules but most of them are
usually closed.
 2- High levels of transpeptidase enzyme are produced by some Gram-negative bacteria.

3- Modification of the transpeptidase enzyme is more frequent in Gram-negative bacteria.

Due to this modification penicillins can not bind with transpeptidase. This is done by a mutation in bacteria.

4- Presence of beta-lactamase.
Beta-lactamases are present between the cell wall and its outer coating in Gram-negative bacteria.

5- Transfer of the beta-lactamase enzyme

Bacteria can transfer small portions of DNA from one cell to another through structures called plasmids.
These are small pieces of circular bacterial DNA. If the transferred DNA contains the code for the beta-
lactamase enzyme, then the recipient cell acquires immunity.
This is the same process that we use to produce drugs like insulin.

Solution to above problems

The search for broad-spectrum antibiotics has been one of trial and error which involved making a huge
variety of analogues.
UP NEXT
Side effects
Antibiotics are screened for any negative effects before their approval for clinical use, and are usually considered safe
and well tolerated. However, some antibiotics have been associated with a wide extent of adverse side effects ranging
from mild to very severe depending on the type of antibiotic used, the microbes targeted, and the individual patient.
[22][23] Side effects may reflect the pharmacological or toxicological properties of the antibiotic or may involve
hypersensitivity or allergic reactions.[4] Adverse effects range from fever and nausea to major allergic reactions,
including photodermatitis and anaphylaxis.[24] Safety profiles of newer drugs are often not as well established as for
those that have a long history of use.[22]

Common side-effects include diarrhea, resulting from disruption of the species composition in the intestinal flora,
resulting, for example, in overgrowth of pathogenic bacteria, such as Clostridium difficile.[25] Taking probiotics during
the course of antibiotic treatment can help prevent antibiotic-associated diarrhea.[26] Antibacterials can also affect
the vaginal flora, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area.[27]
Additional side effects can result from interaction with other drugs, such as the possibility of tendon damage from the
administration of a quinolone antibiotic with a systemic corticosteroid.[28]

Some antibiotics may also damage the mitochondrion, a bacteria-derived organelle found in eukaryotic, including
human, cells. Mitochondrial damage cause oxidative stress in cells and has been suggested as a mechanism for side
effects from fluoroquinolones.[29] They are also known to affect chloroplasts.[30]