ISOLATION OF BETA-

LACTAM
ANTIBIOTIC RESISTANT
MICRO- ORGANISMS
FROM SOIL

A PROJECT SUBMITTED TO SHIVAJI UNIVERSITY, KOLHAPUR.

FOR THE DEGREE OF
MASTER OF SCIENCE IN MICROBIOLOGY

UNDER THE FACULTY OF SCIENCE OF BY

ANUSE NEHA, BAND MANSI AND PATIL PRATIBHA

UNDER THE GUIDANCE OF

Dr. S. R. Waghmare Dr. K. D. Sonawane
Co-guide Guide
 Department of Microbiology,
Shivaji University, Kolhapur

CERTIFICATE
This is to certify that the thesis entitled ― ‘ISOLATION OF
BETA-LACTAM ANTIBIOTIC RESISTANT MICRO-ORGANISMS FROM
SOIL’ is being submitted here with for the award of the Degree of M.
Sc. in Microbiology of Shivaji University, Kolhapur. The work reported
in this Project is based upon the results of the original work carried
out Ms. NEHA ANUSE, Ms. MANSI BAND, Ms. PRATIBHA PATIL under
my supervision and guidance. To the best of my knowledge and
belief the work embodied in this Project has not formed earlier the
basis for the award of any Degree or Diploma or similar title of this or
any other University or examining body.

Place: Kolhapur
Date:

Dr. S. R. Waghmare Prof. K. D. Sonawane

(Co-Guide) (Guide) HoD
 DECLARATION

We hereby declare that the thesis ― ‘ISOLATION OF

BETA-LACTAM ANTIBIOTIC RESISTANT MICRO-ORGANISMS
FROM SOIL’ completed and written by us has not previously
formed the basis for the award of any Degree or Diploma or
other similar title of this or any other University or examining
body.

Place: Kolhapur
Date:

Ms. Neha Anuse

Ms. Mansi Band
Ms. Pratibha Patil
 ACKNOWLEDGEMENT

We take this opportunity to express our sincere thanks

towards the Department of Microbiology, Shivaji University,
Kolhapur for providing us opportunity to work under this
project. Our special thanks to Prof. K. D. Sonawane, Head of
Department, Department of microbiology for providing us
opportunity to do project work, and also giving us permission
to use materials and media from laboratory. Also our sincere
thanks to Dr. Shailesh R. Waghmare for his valuable guidance
and help throughout our project work and also thankful to
Mr. Vikram More sir for their help and guidance in project
work. Thanks to all my classmates who are directly or
indirectly helped in project.
 INDEX
Sr.no Title
1. Introduction

2. Review of literature

3. Aims and objective

4. Materials and
methods
5. Result and
discussion
6. Summary and
conclusion
7. Refrences
 INTRODUCTION
The term antibiotic refers to any substance produced by any microbial entity that is
detrimental to other microbial species. Antibiotic producing organisms are generally
observed in complex environment like soil, the main objective as to why an organism
would produce such antimicrobial substance is, to control and inhibit the growth of
competing organism. antibiotics prove lethal to micro-organisms even at very low
concentration.
They are found to be a potent antibacterial chemotherapeutic agent against wide
array of bacteria both gram positive and gram negative. Hence, they are used to
treat many bacterial infections and hence have found their way into medical
treatment and prevention arena. some limited antibiotics have been observed to
possess anti-protozoal activity, but they prove ineffective as a treatment method
against viruses.

The era exercising chemotherapy by employing antibiotics was instituted by the

discovery of Penicillin in 1928 by the professor of Bacteriology at St. Mary's hospital,
London, Sir Alexander Fleming. The discovery of antibiotics has been regarded as
greatest breakthrough in using antimicrobial components produced by micro-
organisms as chemotherapeutic agents for treatment of infections and as
prophylactic measure. Before the discovery of antibiotics, the mortality rate due to
certain bacteria borne disease were extremely high and the health care facilities
were flooded with infected individuals.

Brief History of Penicillin-

DISCOVERY OF PENICILLIN-
on September 3, 1928, Prof. Alexander Fleming was returning from a holiday, as he
began to sort through some petri dishes with Staphylococcus aureus culture, he
noticed a unusuality on one of the dishes, the plate was completely covered with
colonies of bacteria except for one spot where a blob of fungus was growing. He
isolated that mould and it was found to be a rare strain of Penicillium notatum, he
concluded that the mould had secreted something that had inhibited the growth of
the bacterium. He carried a series of experiments which showed that the supposed
"mould juice" that the fungus secreted was capable of killing wide array of harmful
microbes, streptococcus, meningococcus, diphtheria bacillus to name a few.
Stuart Craddock and Frederick Ridley, were the two assistants of Professor Fleming
that were tasked with extracting pure form of penicillin. The pure form was extremely
unstable hence, they were only able to prepare solution of cured material to have
something to work with. in June 1929, sir Alexander Fleming published his findings
with a passing reference to the potential therapeutic benefits of penicillin, in the
British Journal of Experimental Pathology. on the basis of his findings the only
application of this discovery seemed to be distinguishing penicillin- sensitive and
insensitive bacteria from a mixed culture.
Harold Raistrick, a Biochemistry professor at the London School of Hygiene and
Tropical Medicine tried to obtain a purified form of penicillin but failed.
Howard Florey, Ernst Chain and their colleagues of Sir William Dunn School of
Pathology at the Oxford University began their research to isolate purified form of
penicillin in 1939, but the ongoing war was causing a lot of unavoidable difficulties in
their project. In order to carryout animal experiments and clinical trials they needed
to prepare up to 500 litres of mould filtrate a week and the culture vessels they could
muster were baths, bedpans, milk churns, food tins etc.
This methodology was proving to be tiresome and difficult hence they later designed
a special fermentation vessel for proper culturing and filtrate extraction, this
customized vessel was designed such that the broth beneath the surface of mould
could be removed with ease. To keep an eye on the fermentation, process a team of
women was appointed who were paid 2 pounds per week, they carried out
inoculation and monitored the fermentation process.

Another biochemist Norman Heatley extracted penicillin from huge volume of filtrate
coming from the production line that was set up by the oxford professors. Using a
counter current system, he extracted it into amyl acetate and then back into water.
Biochemist Edward Abraham assisted by setting up production, he used a then
newly discovered technique, alumina column chromatography that removes
impurities form penicillin prior to clinical trials.
One of the initial and vital experiments were carried out by Australian scientist
Howard Florey and Ernst Chain who were interested in the findings of Sir Alexander
Fleming, they looked after the microbiological aspects, they started studying the
mould in 1939.
Chain first purified and concentrated the “mould juice” by a tedious and drudging
process of repeatedly freeze drying the product. They worked with mice and showed
that the extracted penicillin could protect the mice against infection from
staphylococcus aureus, this experiment was carried out on May 25, 1940. After this
successful trial Heatley noted “It really looks as if penicillin may be of practical
importance” in his journal.
The first trial on human was carried out on February 12, 1941, a 43-year-old
policeman named Albert Alexander, he became the first person to receive oxford
penicillin. He had gotten infected by accidently scratching his mouth while pruning
roses, he had developed a serious infection, he had huge abscesses affecting his
nose, eyes and lungs. He was desperate and hence agreed to the first clinical trial,
he discovered within days from when he was injected. He made a remarkable
recovery, but soon the supplies of drug ran out and as a result he died few days
later. further trials went well with other patients. The Mouse experiment of Edward
Florey and Ernst Chain sparked interest in both scientific, industrial communities as
well as the military as the war was going on. There were soldiers falling left and right
not particularly because of warfare but by diseases and infections, and the health of
soldiers was of extreme importance as it could define the victory or defeat.
During the war, it was evidently difficult to manage and keep up the production of
penicillin by the biochemists at Oxford University, hence the industrial production of
penicillin was taken up by huge British companies like Glaxo and Kemball Bishop.
This London firm was later bought by Pfizer.

Mass production of penicillin in united states during WWII -

Florey and Chain determined that large scale production of penicillin in Britain was
not feasible because all the pharmaceutical industries were directed towards war.
Sweeping clinical trials and experiments were to be performed to confirm the
effective dose and side effects of the drug. Hence, Florey and Heatley travelled to
the United States during the summer of 1941. There they convinced four major drug
companies, Charles Pfizer & Co., Lederle Laboratories, Merck, E. R. Squibb & Sons
for industrial production of penicillin and carrying out experiments.
Florey and Heatley carried out their work in Peoria, Illinois, where they perfected the
fermentation process for penicillin production. They used corn instead of glucose as
nutrient source and observed approximately 500 times more growth of penicillin than
any previous trial.
They established a team to find more productive strains of penicillin notatum and
found the best specimen growing on an over-ripe cantaloupe in a grocery store of
Peoria.
After the Pearl Harbour attach on December 7, 1941 the scientists and military
strategists decided to join forces to prepare huge amounts of penicillin that could
help them win the war. Total 21 U.S. companies got together and they produced 2.3
million doses of penicillin in preparation of invasion of Normandy. This success of
penicillin during the wartime gave penicillin the name “miracle drug”.

Sir Alexander Fleming, Ernst Chain, Sir Howard Florey was awarded the Noble Prize
in Physiology or Medicine “for discovery of penicillin and its curative effect in various
infectious diseases” in 1945.
INTRODUDTION TO PENICILLIN-
Penicillins are narrow spectrum bactericidal antibiotics belonging to the group of
betas- lactam antibiotics. They are derived originally from Penicillium moulds.
Penicillins are quintessentially antibacterial and are used as a medication against
bacterial infections. There are various derivatives of penicillin but they have one
thing in common that is the beta lactam ring in their structure. Based on the
variations in this beta lactam ring the penicillins are classified as Penicillin G (natural,
benzyl penicillin), Penicillin V, procaine penicillin and benzathine penicillin.

General structure and chemistry of penicillin-

Penicillins contains 6-aminopheiciallanic acid with a side chain of acyl group
attached to it. The chief structural component of the antibiotic is the beta-lactam ring
present that the core, it defines the biological activity of penicillin.
β-Lactams are classified according to their core ring structures:
β-Lactams fused to saturated five-membered rings;
β-Lactams containing thiazolidine rings are named penams;
β-Lactams containing pyrrolidine rings are named carbapenams;
β-Lactams fused to oxazolidine rings are named oxapenams or clavams;
β-Lactams fused to unsaturated five-membered rings;
β-Lactams containing 2,3-dihydrothiazole rings are named penems;
β-Lactams containing 2,3-dihydro-1H-pyrrole rings are named carbapenems;
β-Lactams fused to unsaturated, six-membered rings;
β-Lactams containing 3,6-dihydro-2H-1,3-thiazine rings are named cephems;
β-Lactams containing 1,2,3,4-tetrahydropyridine rings are named carbacephems;
β-Lactams containing 3,6-dihydro-2H-1,3-oxazine rings are named oxacephems; and
β-Lactams not fused to any other ring are named monobactams.
The side Chains R1 & R2 determine the antibacterial and phamocokinetic
characteristics of the antibiotic. As depicted in fig it is evident that penicillin is
structurally composed of acyl side chain, beta-lactam ring in the core and thiazolidine
ring. The N-Acyl group is a side chain that is attached to 6-amino penicillanic acid.
 M

Variations in the R & M chains changes the type and nature of penicillins.

This image was taken from BRTL10.pdf (nitsri.ac.in)

M ring determines the solubility and ingestion rate:
For direct and rapid injection - Na or K
For oral medication - K, Ca or Al
For delayed oil-based injection – procaine or other derivatives
Natural penicillins can be obtained directly from fermentation liquors of Penicillium
chrysogenum, they are available as salt form of sodium, potassium.
Based on the mode of production Penicillins can be classified as-

1. Natural Penicillin-
The natural for of penicillin is Penicillin G (Benzyl penicillin) these are
fermentatively synthesized biosynthetically from Penicillium chrysogenum. For
synthesis of Penicillin G, phenyl acetic acid is added to the culture medium. It
was the first antibiotic that was used for medical trial and for clinical practice.

2. Biosynthetic penicillin
The synthesis of Penicillin V (phenoxy methyl penicillin) is fermentative and it
is derived from Penicillin G. It is prepared using the organism Penicillium
chrysogenum. The precursor added to the medium for production of Penicillin
V is phenoxy acetic acid.

3. Semisynthetic Penicillins-
These antibiotics cannot be produced by biosynthetic pathway and
fermentation, primarily because, the mycelia cannot take up the side chain or
simply because the side chain cannot be activated when inside mycelia.
These antibiotics are manufactured by carrying out a series of chemical
reactions. The process is known as semi synthesis, The enzyme acylase
causes diacylation of Penicillin G, it splits off the side chain leaving only the 6-
aminopeniciallanic acid (6-APA).
New side chains could then be grafted onto the core forming various
derivatives and providing different pharmacological and antibacterial
properties. Some organisms produce beta-lactamase enzymes that
inactivates the penicillin by degrading the beta-lactam ring. Hence the use of
derivatives of penicillin has become obsolete.
They provide improved coverage, effectiveness among wide array of
organisms including gram-negative cocci and bacilli and certain anaerobic
organisms. These semisynthetic penicillins can be administered orally or
parenterally. Some examples are amoxicillin, ampicillin.
Pseudomonads are considerably resistant to these antibiotics hence they are
combined with aminoglycosides.
They are used as surgical prophylaxis, by obstetrics, gynaecologists and for
intra-amniotic infection, endometritis and other pelvic infections.
DISCOVERY OF BETA-LACTAM ANTIBIOTIC ACTION
The chain of investigation that followed after the discovery of beta lactam antibiotics
led to the discovery of few seemingly independent routes for their action, as:
1. Physiological analysis -
Chain, Florey and their colleagues were studying the mode of action of
penicillin antibiotic on the organism Clostridium welchii, they noticed that the
appearance of the organism was changed after exposure to the antibiotic.
Upon further examination, particularly the microscopic observation, elongation
of most of the cells was observed, instead of their normal appearance they
observed as unsegmented filaments ten or more times longer than those seen
in normal cells. On the basis of these new findings, they hypothesized that
penicillin must act on the structure that maintains the cell’s integrity and the
shape. The concept of a cell wall that surrounds the cell or its physical
characters and composition were not known then.

2. Biochemical analysis –
Till the 1950s, the concept of cell wall was relatively new. The first clue for
finding the site of action of penicillin was found after the experimental work of
Park and Johnson, in their experiment, they demonstrated that numerous
uridine peptides accumulated in the cytoplasm of penicillin-treated
Staphylococcus aureus. Later, Park and Strominger carried out another series
of experiments and found that these uridine peptides that were seen
accumulated were similar to those in the cell wall of the bacteria, this
suggested that those uridine peptides were in-fact cell wall precursors that
accumulated because penicillin had halted the cell wall biosynthesis pathway.
These molecules that constituted approximately similar proportion of protein
and sugar moiety were further named as peptidoglycans and those
peptidoglycans that formed the bacterial cell membrane or wall were called as
murein, these terms were used interchangeably. Murein sacculus is the name
given to the rigid murein polymer that is present in the cell wall.
In 1951, Salton and Horne worked on Salmonella pullorum and discovered
the technique to isolate cell wall, it was an extensive procedure that involved
monitoring each step purification by electron microscope. By this method cell
wall components of large number of gram-positive eubacteria were done and
the presence of the N-acetylglucosamine and N-acetylmuramic acid extracts
were observed by qualitative analysis. In addition to these sugar molecules,
several amino acids particularly, D-glutamic acid, D- and L-alanine and L-
lysine were noted, a tetrapeptide chain of L-alanyl-D-glutamyl-L-lysyl-D-
alanine was observed with Glycine as cross-bridge. As the newly discovered
compound showed similarities to the discoveries that Park made while
working on Staphylococcus aureus hence these two investigations were
converged. Hence, it was concluded that since these two organisms
contained the same structural components, the mechanism through which the
penicillin acts must be same too.
 3. Biosynthesis of Murein –
Murein also known as peptidoglycan is the structure that envelopes the
bacterial cell and helps in maintaining the rigidity and shape. To understand
the action of beta-lactam antibiotics on the cell wall synthesis, it is essential to
be familiar with the process by which murein is formed. For understanding this
biosynthetic pathway and for to shed light on the enzymatic reactions
involved, ten years were dedicated and the organisms of study were E.coli
and P. aeruginosa.
The cell wall biosynthesis occurs in following stages:
a) Formation of precursors-
The site where this step occurs is, the cytoplasm. First, the attachment of
UDP-N-acetylglucosamine with three-carbon compound, phosphoenolpyruvic
acid (PEP) is carried out, which leads to the formation of UDP-N-
acetylmuramic acid. Further amino acids are added, this step is catalysed by
a series of enzymes. Stepwise addition amino acids is carried out to form
UDP-MurNAc pentapeptide, which was the compound that Park and Johnson
isolated.
b) Formation of glycoprotein backbone-
This stage occurs in the cytoplasmic membrane. Here the intracellular
precursors that were formed earlier namely the UDP-GlcNAc and UDP-
MurNAc pentapeptide are joined at the glycolic ends and they are joined at
NAM.
c) Formation of cross bridge-
This is the final step that involves transpeptidation and does not require
energy. The transpeptidase enzyme forms cross-linking between two NAM
strands, and the D-alanine carboxypeptidase removes the last D-ala
residue of the pentapeptide strand.
The inhibition of transpeptidase hampers the cell wall synthesis considerably but
inhibition of the action of carboxypeptidase shows no lethal effects.
Penicillin is nearly identical to the D-alanyl-D-ala unit. The most striking similarity is,
that the highly reactive CO-N bond in the beta-lactam ring of the lies in the exact
same position as the CO-N bond present in the D-alanyl-D-ala which is the target for
the transpeptidase enzyme.

4. Biophysical analysis –
In 1949, Cooper and Rowley conducted experiments to study the target of
penicillin, for this they used radioactive penicillin. It was found that penicillin
binds to a specific site on the cytoplasmic membrane of the cell. The places
where the penicillin was found to be bound was termed, penicillin binding
component. It was observed that the entire molecule of the radioactive
penicillin interacts with the PBC. After observing the binding, attempts were
made to remove the penicillin, it was found that neither, acids, neutral buffers
or detergents could remove the bound penicillin. But upon alkali treatment the
penicillin was released as penicilloic acid. Also, physical stress conditions like
 high heat showed destruction of PBC. From all the observations it was
concluded that the penicillin binds covalently to PBC. When this
penicillin-PBC was subjected to sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), the weight was found to be around 40-90 kDa.
Later these components were termed as penicillin binding proteins and they
were numbered according to their molecular weight in descending order. The
PBPs are broadly classified as low molecular weight and high molecular
weight penicillin binding proteins, it was found that the low molecular penicillin
binding proteins are observed abundantly in the bacilli but not in the cocci.

The genetic analysis was done to see the action of these penicillin binding proteins
and to study their role.
This was done by carrying out series of experiments including various mutants and
radioactive penicillin, the mutants were those which did not have certain penicillin
binding proteins.
PBP1 in E. coli was found to be involved in cell elongation process. It also showed
that the same penicillin binding protein had action in polymerization of the glycan
subunit and crosslinking of muropeptides.
The mutants that lacked PBP2 showed morphological alterations, in which the rod-
shaped organisms showed cocci confirmation. The PBP2 therefore is said to
maintain the cell shape. Absence of PBP2 caused inhibition of cell division and the
peptidoglycan synthesis was delayed.
The PBPs 4, 5 and 6 showed DD-carboxypeptidase activity.
The genetic analysis showed that, the beta lactam antibiotics act particularly on the
transpeptidase enzyme and the carboxypeptidase, the high molecular weight PBPs
are responsible for transpeptidation, and inhibition of this step affects the cell division
and multiplication considerably but inhibition on carboxypeptidase activity which
were the low molecular penicillin binding proteins, showed no lethal effect other than
accumulation of pentapeptide units.
MODE OF ACTION –
 Penicillin is a cell wall synthesis inhibiting antibiotic
 It disrupts final cross-linking during cell wall synthesis stage, it mimics
D-Alanyl-D-Alanine residue, which is a substrate for transpeptidase
activity.
 Penicillin binds to a penicillin binding protein, transpeptidase enzyme
and forms an ester linkage with a serine residue.
 The open-ring structure of penicillin forms a sort of steric shield
 It prevents the substrate and water from reaching the ester linkage, this
inhibition is irreversible.
 Cell swells due to water retention and it finally is lysed due to high
osmotic pressure

Action of Penicillin on Gram positive VS Gram negative bacteria-

Gram positive bacteria have more peptidoglycan content in their cell
wall hence the penicillin can show more activity. The gram-negative
cell wall comprises of outer lipopolysaccharide layer that is not
permeable to penicillin also they have relatively low peptidoglycan
content, hence the drug cannot show much effect.
 AIMS AND OBJECTIVES

Aim:

Objective:

REVIEW OF PROJECT
 ANTIBIOTIC-
Antibiotic is a secondary metabolite produced by micro-organisms predominantly
moulds, that inhibits the growth of other micro-organisms at low concentrations.
The antibiotic of interest here was the one acting on the cell walls synthesis of
bacteria, particularly Benzyl penicillin (Penicillin G)

PENICILLIN G –
o It belongs to Beta- lactam antibiotics group
o It inhibits cell wall synthesis in Gram positive bacteria by binding and
inactivating penicillin binding proteins present in the bacterial cell wall.
o They form ester linkages and inhibit transpeptidation reaction, thereby
causing hinderance on cross linking mechanism.
o The cell bursts due to osmotic pressure.
o It is effective only on the growing cells in their lag phase and once the
organisms attain stationary phase, they serve to be ineffective as cell wall
synthesis has already occurred.
o They show little activity against gram negative bacteria, particularly gram-
negative cocci, that lack outer membrane and non-beta lactamase
producing anaerobes.

CHARACTERISTICS OF PENICILLIN G –
1. Absorption-
It gets inactivated by gastric juices hence needs to be given intramuscularly
via syringe.

2. Metabolism –
They are metabolized to a minor degree; this may affect serum half-life in
renal failure since these drugs are not absorbed completely and hence tend to
accumulate

3. Excretion –
Excretion of drug occurs mostly via tubular secretion. Their accumulation can
prove lethal and reduces renal function markedly.

4. Distribution –
Penicillins are found in lung, liver, kidney, muscle bone and placenta.
They do not penetrate the white blood cells.
More concentration is observed in bile than in serum

5. Untoward reactions to penicillins –

 Penciliins have shown to act as hapetns they combine with human proteins,
penicilloyl is a major antigenic determinant that is produced by the cleavage of
the beta-lactam ring, which allows an amide linkage to the body proteins.

6. Determination of allergy -
Wheal and flare reaction to intradermal injection has been observed.
The drug should not be prescribed to the patients that show immediate
reaction.
Desensitization by repeated administration of increasing doses could be done
but it has a high risk hence, alternative therapy agents’ treatment should be
sought.

7. Administration-
Penicillin G is commonly given parenterally: in the form of salts of sodium and
potassium.
It shows delayed absorption hence the levels persist for 12 hours for Procaine
Penicillin G, for benzathine penicillin it is 12-28 days.

MECHANISM OF ACTION OF PENICILLIN G –

As mentioned earlier the penicillin antibiotics inhibit cell wall synthesis by binding to
certain penicillin binding proteins. The full mechanism is not elucidated.
 The cell wall of bacteria is made up of peptidoglycan containing alternating N-
acetyl muramic acid and N-acetyl glucosamine residues. These are linked
together by cross linkage of amino- acid residues.
 Formation of peptidoglycan can be divided in following stages: precursor
formation in cytoplasm, linkage of precursor products together to form long
polymer of NAG & NAM, and finally the formation of crossbridge.
 Penicillin G binds to the penicillin binding proteins that are essentially
enzymes, transpeptidases, carboxypeptidases and endopeptidases that play
a role in formation and maintenance of cell wall.
 Penicillin G acts as a structural analogue of acyl-D-alanyl-D-alanine which is
the substrate for transpeptidase enzyme.
 As a consequence, the transpeptidation reaction is stopped and the cross
bridge is not formed.
 The cell wall structure weakens and lysis of cell occurs.
  Significance of Penicillin binding proteins –
Penicillin- binding proteins are essentially the enzymes involved information or
assembly of bacteria cell wall. They are found embedded in the cytoplasmic
membrane. These are readily detectable by sodiumdodecylsulfate (SDS) gel
electrophoresis and fluorography. Most of the bacteria contain 1000 to 10,000 PBPs
per cell. In E. coli almost 12 penicillin binding proteins have been classified. These
PBPs are broadly classified based on their molecular mass as High-molecular-
weight PBPs and the other class is Low-molecular- weight PBPs, these typically
include carboxypeptidases. It has been observed that the high molecular weight
penicillin binding proteins are more sensitive to beta-lactam antibiotics like Penicillin
G and cephalosporin than the low molecular weight penicillin binding proteins.
To explain the action of High-molecular-weight and Low-molecular-weight pencilline
binding proteins:
The high-molecular-weight (HMW) are also called the class A PBPs, they are
bifunctional and catalyse trans glycosylation (polymerization of sugar chains) and
transpeptidation invitro. The HMW class B molecules carry out transpeptidation
(cross-linking). The LMW PBPs act as carboxypeptidase, which helps in cross-
linking and maturation of cell wall. The HMW are found among almost all of the
gram-negative and gram-positive bacteria, however, the functions that these carryout
among each bacterial group varies considerably, since the composition and
peptidoglycan content of each is vividly different.
The high-molecular weight penicillin binding proteins are further classified on the
basis of their mode of action as, PBP: 1a, 2a and 3. Out of these, PBPs, 1a and 2a
possess the dual action of transpeptidation and transgycosylation. The enzymes are
transpeptidase-transglucosylase, these two enzymes aid in elongation of the glycan
or sugar chain and the transpeptidase help in the formation of cross-link that
connects the N-acetylmuramic acid and N-acetylglucosamine.
Formation of septum in the cell wall which is an essential step for cell division is
carried out by PBP3. The pbpA gene encodes for PBP2 encourage elongation of
lateral cell wall and aid in maintenance of the rod shape of any given organism,
hence if there is any mutation in the pbpA gene or there is any obstruction in the
activity of PBP2 the cell confers a spherical shape and due to this considerable
change in the morphology of the organism, cell lysis occurs after some generations.
PBP2 is considered a major factor required for overall process of cell wall synthesis
as it performs almost 70% of the process of cell wall synthesis elongation process.
The class of Low-molecular weight penicillin binding proteins comprises of PBP: 4, 5,
6a, 6b and 7. The LMW PBPs are rather superfluous, any mutation in gene coding
for them or their expression does not affect vitality of cells, most of the important
function like chain elongation, septum formation is carried out by HMW PBPs. These
function as DD-Carboxypeptidases. All the low molecular weight penicillin binding
proteins account for almost 50% of the penicillin binding capacity of bacterial cells.
Carboxypeptidase essentially involves degradation of the pentapeptide side chain to
tetrapeptide side chain as observed in Staphylococcus aureus. The N-acetyl
muramic acid residues carry these pentapeptide side chains and the
carboxypeptidases act on these for formation of the tetrapeptide side chains that are
to be used for formation of cross links. Hence, the most adverse effect that could
occur in the absence of these carboxypeptidases is the accumulation of
pentapeptide side chains in the murein sacculus.
The experimental work described in Note: Role of Penicillin-Binding Proteins in the Initiation
of the AmpC β-Lactamase Expression in Enterobacter cloacae - PMC (nih.gov) shows that during
accumulation of these pentapeptidases a signalling molecule, 1,6-
anhydromuramylpentapeptide sends the signal for induction of beta-lactamase
induction, it sends the signal by converting AmpR from a repressor into an activator.
Imipenem and cefoxitin are some of the strong beta- lactamase inducers. These two
binds to the essential penicillin binding proteins which leads to conservation of
pentapeptide chains in the murein.

Fig: The image was taken from- https://www.researchgate.net/figure/Fig-1-

Schematic-representation-of-the-two-main-reactions-catalyzed-by-HMM-PBPs-
during_fig1_273469243
 DRUG RESISTANCE

The term drug resistance refers to decrease in effectivity of antimicrobial agents that
are prescribed to a person, simply put it is the condition where the micro-organisms
continue to multiply in the presence of drug.
Soon after the discovery of antibiotics, it was widely believed that humans had finally
won against the battle of infections bacteria. The factors that have contributed to
development of this drug resistance majorly is overuse of drug i.e., prescribing
antibacterial drugs for unrelated infections like viral
1. Drug Modification or Inactivation-
This mechanism involves synthesis of certain enzymes that could chemically modify
an antimicrobial compound, thereby inactivating them and reducing their activity.
Example: Enzymatic hydrolysis of the beta-lactam ring which is the core factor
responsible for activity of beta-lactam antibiotics like penicillin, they break the ring by
synthesizing enzyme like beta-lactamase, inactivation of antibiotics acting on nucleic
acid synthesis like rifamycin occurs through glycosylation, phosphorylation or
ribosylation of adenosine di-phosphate.

2. Prevention of cellular uptake-

This mechanism is commonly observed in the gram-negative bacteria, and the
reason is that they possess lipopolysaccharide layer. They confer changes in the
outer LPS membrane, by changing composition, porin channel selectivity or prion
channel concentration.
Example: Pseudomonas aeruginosa resists the drug carbapenem by decreasing the
amount of its OprD porin, which is the primary portal of entry for carbapenem drug
into the other membrane of the pathogen.

3. Active Efflux-
Many of the gram positive and gram-negative organisms possess this mechanism of
drug resistance. These efflux pumps are present embedded in the cell membrane,
they actively transport the antimicrobial drug out of the cell, this helps preventing the
accumulation of drug within the cell to the level at which it may prove lethal to the
organism. Some organisms have shown to possess more than one of these efflux
pumps rather than single efflux pump, hence they can transport multiple types of
antimicrobials, some of the antibiotics that are thrown out of the cell by this
mechanism include, beta-lactams, tetracyclines and quinolones.
Example: norfloxacin, tetracycline drugs are thrown out of the cell membrane by E.
coli.
4. Target modification-
Most of the antimicrobial drugs and compounds are target specific, and these binds
specifically to a region on that target hence, once the target is modified the binding of
the drug would be poor or ineffective. This could be done either preventing the
binding of drug or reducing the effect of the drug due to inept binding. This
mechanism is usually genetically determined and occurs due to mutations in the
genes encoding for the targets of the antibacterial.
Example: a) Genetically modifying the penicillin binding proteins that are the target
for beta-lactam antibiotics, this change in the PBPs helps in producing resistance
against all of the beta-lactam antibiotics, this mechanism is observed in the strains of
Streptococcus Pneumoniae.
b) Changes in the lipopolysaccharide structure in the gram-negative bacteria helps in
developing resistance against drug like polymyxin.
c) changing the structure of DNA gyrase helps to resist the action of fluroquinolones.
d) changes in the ribosomal subunits helps provides resistance to macrolides,
tetracyclines and many aminoglycosides.

5. Enzyme Bypass-
This includes overproduction of the target enzyme such that there is a sufficient
amount of antimicrobial-free enzyme to carry out the proper enzymatic reaction.
Other mechanism includes devising a bypass that circles around formation of
fictional target enzyme, both of these enzymes have been found in sulphonamide
resistance.
Example: Staphylococcus aureus show vancomycin resistance by decreasing the
cross-linkage of peptide chains in the bacterial cell wall, which increases the target
sites for the antibiotic, this increased binding of vancomycin in the outer cell wall
provides a blockage that prevents free drug molecules from penetrating the site to
where they act and block cell wall synthesis.

6. Target mimicry-
This is rather new mechanism, it involves production of protein that bind and
sequester drugs, this prevents the drug from binding to the target site.
Example: Mycobacterium tuberculosis produces a protein with regular pentapeptide
repeats that mimics the structure of DNA.
Even before being administered to humans as chemotherapeutic agent, some
organisms showed resistance toward penicillin.
 BETA LACTAM ANTIBIOTIC RESISTANCE

Most common methods employed by organisms to resist the action of beta lactam
antibiotics include:
1. Preventing the entry inside the cell:
This mechanism is observed mostly in the gram-negative organisms. Almost
all of the gram-negative bacteria posses an asymmetric phospholipid bilayer
and a lipopolysaccharide (LPS) layer as its outer membrane which is the outer
membrane, and the inner membrane consists of peptidoglycan. The space
that separates the inner membrane with the cytoplasmic membrane is called
the periplasmic space. The penicillin binding proteins are located in the
cytoplasmic membrane. The outer membrane shows special proteins termed
as porins, these act as conduits and provide the characteristic of semi
permeability to the membrane. The lipopolysaccharide layer is the outermost
layer that comprises of:
a) Lipid A –
It consists of β-glucosamine-(1→6)-glucosamine-1-phosphate base
with fatty acid esters attached to both carbohydrates. The number of
acyl groups and the length of acyl chain varies according to the
organism. This section is hydrophobic and it imparts toxic property to
the gram-negative bacteria.
b) Hydrophilic core –
This is the hydrophilic core comprising of saccharides. This laver
mostly consists of common hexoses, including glucose, galactose and
N-acetylglucosamine, it is structurally diverse
c) The O-antigen-
It is essentially a repeating oligosaccharide unit that is comprised of
two to six different sugars. This is highly antigenic and is the primary
structural component of lipopolysaccharide it is the determining factor
that helps differentiate between bacteria. It has been assigned and
used to differentiate the serogroups of E. coli, Salmonella enteric and
Vibrio cholerae.
Large number of different types of proteins are embedded in the outer
membrane, these proteins include:
a. Murein lipoprotein (Lpp) –
It carries fatty acid moieties which anchors it into the outer
membrane, one-third of these lipoprotein remains embedded in
the peptidoglycan layer. It is therefore concluded that it
maintains the integrity of outer membrane and it is responsible
for the interactions between outer membrane and peptidoglycan
layer.
b. Outer membrane proteins –
These are a class of unique integral membrane proteins that
remain anchored in the outer membrane. They form a beta-
 barrel structure made of 8 to 26 strands. Large extended loops
are observed between the strands on the extracellular side of
the membrane and on the periplasmic side short loops are
observed. These outer membrane proteins show high stability in
extreme environments and perform various functions but have
similar structural and biological properties. OmpA is most deeply
studied outer membrane protein. Its role is in regulating
adhesion and biofilm formation as observed in
A. baumannii. The main porins that are responsible for uptake
of these antibiotics are OMP F and OMP C, the uptake
depends on molecular size of the antibiotic, electric charge
and hydrophilicity of the molecule.
As observed, most of them enter through the porin channels, hence mutation
that will decrease the number of these porin channels across the membrane would
reduce the amount of drug intake as observed in the members of the
Enterobacteriaceae family. Mutation could also be done so as to change the
selectivity of these channels.
2. Efflux Pumps –
Some gram-negative bacteria for example, E. coli and Pseudomonas
aeruginosa possess tripartite multidrug efflux system that not only expels out
the antibiotics but also other cytotoxic substances out from the cell membrane
directly into the medium by bypassing the periplasm and outer membrane.
This tripartite efflux pump system that is observed in Escherichia coli is
AcrA/AcrB/TolC, this pump expels multiple antibiotics, dyes, bile salts and
detergents. TolC is a trimetric outer membrane factor (OMF) which forms a
beta barrel pore in the outer membrane of the organism, it exhibits a long
periplasmic beta-helical channel. AcrA is a perplasmic membrane fusion
protein (MFP) which serves as a linker between the pump AcrB which is a
trimeric resistance nodulation cell division pump and the TolC. The Acriflavine
Resistance Channel Protein B or the AcrB performs the vital task. This pump
is the primary pathway through which E. coli expels all of the cytotoxic
substances. The AcrB performs functions like, recognition of substrate and a
proton-drug anti-porter.
The over-expression of this AcrB efflux system has posed the threat of
generating potent multidrug resistant phenotypes of E. coli, it has high
effencicy as compared to other efflux transporter, because of this serious
medical concerns have risen regarding antimicrobial therapy.
Structure of AcrB protein:
It is composed of three layers, each of them is parallel to the inner membrane
these include-
a) Transmembrane domain- It is composed of 12 transmembrane alpha-
helices, the bundle is composed of 12 transmembrane alpha-helices. This
section anchors the protein in the inner membrane and plays a major role in
proton translocation.
 b) Porter domain - It contributes the most in terms of function in the AcrB
protein. . The porter domain consists of four subdomains (for each of the three
promoters) these domains are: PN1, PN2, PC1 & PC2. each of these
subdomains is made up of two geta-alpha-beta sandwiches.
The subdomain closest to the core is PN1 and other subdomains encompass
the core.
c) TolC docking domain - This structure is comparable to funnel. Through this
the AcrB pumps substrates int to he TolC and later they are thrown out of the
cell.
The AcrB protein is a trimer with three chains having identical primary
structures. All of these chains are asymmetric; but have different conformation
in the porter chain. These different confirmations of the promoters show the
three different stages of the efflux cycle, which is the cycle involved in removal
of cytotoxic substances.
These three stages are:
1. Access- at any given time any of the potential substrate may it be antibiotic,
zwitter ion, dye etc may enter into the vestibule from the outer leaflet of the
membrane through a small channel that forms a gap between the PC1 and
PC2 subunits.
2. Binding- in this phase the previously formed cleft shrinks slightly, because
of which the binding pocket expands slightly so that the substrate could pass
through the vestibule, the PN2 subdomain gets altered and this creates a
hydrophobic pocket at the interface of PN1 and PC1 subdomains. The drug is
not released during this phase because the central helix of the extrusion
promoter that blocks the exit.
3. Extrusion - in this phase the drug is expelled out of the shrinking pocket
and once the drug is outside, the entrance is closed and the exit remains
open.
After completion of these three confirmational changes the promoter returns
to its original position and the cycle is repeated.
The study carried out in reference shows, Beta-lactamase -negative,
ampicillin resistant Haemophilus influenzae that they collected from Japan,
France and North America indicated that mutations in the fts encoding for the
Penicillin binding protein 3 gave the organism ability to resist against
ampicillin. This resistant was due to the efflux pump observed in the
haemophilus influenzae AcrB.

3. Destruction of antibiotic. –
It is the main mechanism through which bacteria resist the beta-lactam
antibiotics. This mechanism entails destruction of the main component of
beta-lactam antibiotics which is the beta-lactam ring, this is achieved by
synthesizing enzyme named beta-lactamase that essentially hydrolyses the
beta-lactam ring.
These beta-lactamase’s act on the amide bond of the beta-lactam ring, which
leaves the antibiotic ineffective. These beta-lactamases were first described in
 1940s, only one year since the chemotherapeutic activity of penicillin was
discovered. This posed the treat to the patients that were being treated
particularly for the Staphylococcus aureus. In the 1960s, another beta-
lactamase enzyme coded by plasmid that was capable of hydrolysing
ampicillin was found, this compound was named TEM and it was named after
the patient in which it was found, Temoneira.
The genes that are responsible for coding beta-lactamases are termed as bla,
they are found to be present in the chromosome or localized in MEGs as a
part of accessory genome.
To date more than 1000 different beta-lactamase enzymes have been found
and described. Most of them are found to be a part of normal bacterial evolution.
They are classified in two schemes,
1. Ambler classification-
This classification is done based on the amino acid sequence identity and it
separates the beta lactamases in four groups:
Class A- these enzymes show wide range of biochemical activates form the
narrow spectrum beta-lactamases to those capable of destroying almost all of
the narrow spectrum beta-lactam antibiotics including, carbapenems. Most of
the extended beta lactamases (EMBLs) belong to this class. They are
generally inhibited by clavulanic acid or tazobactam. These have serine
residue in their catalytic site, this property is similar to the class C and D
enzymes.

Class B- these are also known as metallo-beta lactamases. They utilize the
zinc metal ion instead of the serine residue for the nucleophilic attack on the
beta-lactam ring. These are inhibited by the presence of chelating agents like
EDTA, they too are active against wide array of beta lactam antibiotics. But
unlike the class A beta-lactamases, these metallo-beta lactamases are not
inhibited by clavulanic acid or tazobactam. They were found to be encoded by
genes that are usually located in the chromosome of non-pathogenic bacteria.
But in the 1990s these were observed in the clinical strains of
Enterobacteriaceae, Pseudomonas and Acinetobacter species.

Class C- these are active against cephalosporins and hence are sometimes
referred to as cephalosporinases. These are genetically encoded and are
found to bye synthesized by the gram-negative bacteria. They differ from
class A penicillinase as they posses the ability to cleave oxacillins.

Class D- this class of beta-lactamases have been reported in E. coli,

Enterobacter spp., K. pneumoniae and P. aeruginosa. They are genetically
encoded and can be transferred intra and in inter species. These are capable
of degrading isoxazolyl beta lactams like methicillin and oxacillin.