PENICILLIN MECHANISM

Penicillin is a widely used antibiotic prescribed to treat staphylococci and streptococci bacterial infections.
Penicillin belongs to the beta-lactam family of antibiotics, the members of which use a similar mechanism
of action to inhibit bacterial cell growth that eventually kills the bacteria.

Bacteria cells are surrounded by a protective envelope called the cell wall. One of the primary
components of the bacterial cell wall is peptidoglycan, a structural macromolecule with a net-like
composition that provides rigidity and support to the outer cell wall. In order to form the cell wall, a single
peptidoglycan chain is cross-linked to other peptidoglycan chains through the action of the enzyme DD-
transpeptidase (also called a penicillin binding proteinPBP). Throughout a bacterial lifecycle, the cell
wall (and thus the peptidoglycan crosslinks) is continuously remodeled in order to accommodate for
repeated cycles of cell growth and replication.

Penicillins and other antibiotics in the beta-lactam family contain a characteristic four-membered beta-
lactam ring. Penicillin kills bacteria through binding of the beta-lactam ring to DD-transpeptidase,
inhibiting its cross-linking activity and preventing new cell wall formation. Without a cell wall, a bacterial
cell is vulnerable to outside water and molecular pressures, and quickly dies. Since human cells do not
contain a cell wall, penicillin treatment results in bacterial cell death without affecting human cells.

Gram-positive bacteria have thick cell walls containing high levels of peptidoglycan, while gram-negative
bacteria are characterized by thinner cell walls with low levels of peptidoglycan. The cell walls of gram-
negative bacteria are surrounded by a lipopolysaccharide (LPS) layer than prevents antibiotic entry into
the cell. Therefore, penicillin is most effective against gram-positive bacteria where DD-transpeptidase
activity is highest.

Resistance
Bacteria reproduce quickly and are prone to genetic mutations when growing in the presence of
environmental pressures, such as an antibiotic. Over time, genetic mutations that provide a survival
advantage may arise in the bacterial population, allowing bacteria to continue to grow and multiply in the
presence of antibiotic. This leads to the creation of a resistant strain, which can only be killed through the
use of alternative, stronger antibiotics. The potential for antibiotic resistance increases through repeated
or improper use of an antibiotic. Bacterial strains can become resistant to more than one antibiotic,
leading to the creation of superbugs that are extremely difficult to treat medically.

Bacteria generate antibiotic resistance through a number of mechanisms. Some bacteria can become
resistant to penicillin by producing beta-lactamase, a bacterial enzyme that destroys the beta-lactam ring
of penicillin and makes it ineffective. One common example is Staphylococcus aureus, which produces
high levels of beta-lactamase and causes infections in the blood, skin, or lungs. Most Staphylococcus
aureus strains are now resistant to penicillin. In response, scientists have developed a synthetic form of
penicillin that is resistant to beta-lactamase, termed penicillinase-resistant penicillins or second
generation penicillins. These include dicloxacillin, oxacillin, nafcillin, and methicillin. Soon after
development, researchers quickly identified Staphylococcus aureus strains resistant to methicillin, termed
methicillin-resistant Staphylococcus aureus(MRSA). MRSA use a second resistance method to overcome
methicillinnamely by upregulating a low-affinity form of penicillin binding protein that does not bind
the antibiotic and whose activity is not inhibited. To date, MRSA has demonstrated blanket resistance to
all beta-lactam antibiotics and is an extremely serious health risk.

Threat to Public Health

In 2014, the World Health Organization reported that antibiotic resistance is a worldwide threat to public
health. In areas around the world, disease-causing bacteria are already resistant to all initial forms of
antibiotic and are quickly developing resistance mechanisms to treatments of last resort. Some antibiotic-
resistant bacteria are highly contagious and can quickly spread throughout a family or community,
creating a serious public health risk. As bacteria continue to gain resistance to some of the strongest
antibiotics available, pharmaceutical development of new antibiotic agents is in decline. This is due to
several reasons, including low profitability due to short treatment cycles, lack of new therapeutic targets
or strategies for killing bacterial cells, and low tolerability in the medical community for side effects.

http://www.news-medical.net/health/Penicillin-Mechanism.aspx

THE MECHANISM OF ACTION OF PENICILLIN. PENICILLIN ACYLATES THE ACTIVE SITE OF BACILLUS
STEAROTHERMOPHILUS D-ALANINE CARBOXYPEPTIDASE

Abstract
Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step
in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and
Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of
the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that
2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of
the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related
penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of
Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of
14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently
labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was
shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore,
the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several
new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2)
use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in
concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent
for peptides.

https://www.ncbi.nlm.nih.gov/pubmed/7372662
 PENICILLIN ANTIBIOTICS

In 1929 Alexander Fleming, a British biologist, inadvertently discovered penicillin. He had observed
bacterial Staphylococci colonies disappearing in cultures that were contaminated with mold.

Fleming eventually extracted the compound from the mold that had been responsible for destruction of
the bacterial colonies. The product was named penicillin, after the Penicillium mold from which it was
derived. Some examples of penicillins include: amoxicillin, ampicillin, bacampicillin, oxacillin, penicillin

Antimicrobial Spectrum of Penicillins

Penicillins have a bacteriocidal effect on Gram-positive bacteria. In Gram-positive cells, peptidoglycan
makes up as much as 90% of the thick, compact cell wall, and is the outermost layer.

Penicillins are not effective against Gram-negative bacteria, which have cell walls in which peptidoglycan
is not the outermost layer, but that lies between the plasma membrane and a lipopolysaccharide (LPS)
outer membrane. Penicillin cannot access the peptidoglycan of Gram-negative cells.

Infections Penicillins Are Used Against

Penicillins are used to treat Gram+ bacterial infections which, depending on the specific microbe involved,
cause a wide range of illnesses including skin infections, dental infections, ear infections, respiratory tract
infections, urinary tract infections, and gonorrhea.

http://www.scienceprofonline.com/microbiology/mode-of-action-of-penicillin-
antibiotic.html

MORDANT

A mordant is classically defined as an ion that binds a chemical dye and holds it down, such that
the dye remains stuck on the organism. However, any chemical that keeps a dye in place can also
be considered as a mordant. Classically defined, mordants are usually ions such as metal ions or
halide ions, but can be any molecule that serves the purpose of holding down a dye. However, a
molecule called phenol is a non-ionic mordant that is discussed below. Some mordants bind both
the dye and proteins on the microorganism. Most mordants are ions because the electrical
charge on the ion attracts the electrical charge on a chemical dye. Thus, when the ion binds the
dye, they form a large complex that precipitates -- meaning they become a solid and are no longer
dissolved in the solution. Mordants hold down, or weigh down, the dye so it does not wash away
during the remainder of the staining procedure. Washing is done so that only the true staining
regions are visualized.