Lecture 1 Pharmacology of Antibiotics 05/10/2021

Arianna Gobbato – Alice Dalsass

Antimicrobial Pharmacology – 01
05/10/2021 – professor Federico Pea

PHARMACOLOGY OF ANTIBIOTICS
I copied in Italics what was written in slides but not mentioned by the professor.
The slides present in the pdf supplied by the professor have a terrible quality, so I have not put some images
for this reason.

Introduction to antibiotics
Historical aspects
Before the discovery of antibiotics we were in the “dark age”  there was an high risk of death due to
sepsis in particular in the obstetrics field: both mothers and newborns died because of bacterial infections.

Ignaz Semmelweiss when was in Vienna started to work both at the obstetric clinic and as an anatomo-
pathologist and he figured out that by washing your hands you could prevent bacterial infections.
So he is responsible for the introduction of “washing the hands” as a form of prevention.
After the introduction of washing hands with clorexidin indeed there was a drop in incidence of bacterial
infections.

Chemotherapy
In the early XX century scientists started to study chemical substances and developed chemotherapy.
Definition of chemotherapy  “Chemotherapy is the doctrinal and methodological aspects that are aimed
at the research of artificial and natural chemical substances with selective toxicity against prokaryotic or
eukaryotic cells, that are responsible for infections, infestations, neoplasms and immunological disorders”.

The founder of chemotherapy:

Paul Erlich  defined as “magister mundi” because he was the first one to identify agents able to
counteract the activity of several pathogens. For this reason and for his research on the immunological
aspects he was awarded the Nobel prize in 1908:
- “Corpora non agunt nisi fixata”  this sentence means that the drug, in order to be active, needs
to bind to a particular receptor on cell surface (drug-receptor binding theory).
- In 1910 he performed the first anti-treponema drug (anti-syphilis): Salvarsan.
From that moment on, the antimicrobial chemotherapy was developed.

Definition of antimicrobial chemotherapy  pharmacological discipline for the research and study of
“etiotropic” molecules, with selective toxicity toward microbes.
We have drugs against peculiar targets that differ in prokaryotic and eukaryotic cells.
Antimicrobial pharmacology is responsible for:
- Infections it is how we define the action of viruses, bacteria or fungi on our body.
- Infestions  if the pathogens are elmints and schistosomes.

Chemotherapy  it is a “therapy with chemical substances” and it is splitted into different arms:
- Antibiotics  if we are dealing with bacteria (in our coruse we will focus on antibiotics).

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
- Anti-tubercolar drugs  if we are dealing with mycobacteria
- Anti-fungals  if we are dealing with fungi
- Anti-virals  if we are dealing with viruses
- Anti-neoplastic drugs  if we are dealing with neoplastic cells

The common feature of all these different drugs is “ selective toxicity”  since the target of these drugs is
characteristic only of prokaryotic cells and not of eukaryotic cells, you can kill bacteria without harming the
cells of the human organism.

Antibiotics
Definition of antibiotics drugs for the treatment of bacterial infections.
What happened after the introduction of antibiotics?

First step forward in the setting of community-acquired meningitis, which is a diseases caused mainly by
three different pathogens: Neisseria Meningitidis, Streptococcus Pneumoniae and Hemophilus Influenzae.
Before the introduction of antibiotics the probability of death in patients with meningitis was close to
100%.
At a certain point Simon Flexner introduced the use of the serum taken from patients who survived
meningitis, managing to heal some patients. The mortality in this way decreased.

However the real change happened with introduction of antibiotics.

The first antibiotics were introduced in the early ’40s and many different antibiotics continued to be
introduced till the end of the last century.
In this way mortality dropped significantly, that associated to meningitis too (mortality associated to
pneumococcal meningitis is still of 25% unfortunately).

Antibiotics classification
Antibiotics can be classified according to different types of classifications. We will go through only some of
these. The others are explained very well in books and I’m not going to talk about them.
Different types of classification in relation to:
- Mechanism of action
- Type of action
- Intrinsic characteristics
- Chemical-physical properties
- Mode of antibacterial activity

Targets of antibiotics
The target consists of molecules typical of the prokaryotic cell.
The different antibiotics can act by targeting:
- Bacterial cell wall (not present in eukaryotic cells)
- Cell membrane
- protein synthesis inhibition  by targeting the two different ribosome subunits
- DNA synthesis inhibition by targeting DNA-erase or DNA-polymerase (an example of the latter is
Rifampicin).

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Classification based on mechanism of action:
- Inhibition of the synthesis of the bacterial cell wall:
 Beta lactams
 Glycopeptides, Lipoglycopepetides.
- Alteration of the bacterial cell wall
 Lipopetides
Others act during different steps of metabolic reactions (you can read it in every type of book).
- Inhibition of metabolic reactions:
 Biosynthesis of nucleic acids
 Fluoroquinolones, Rifamycin
 Protein biosynthesis
 Aminoglycosides, macrolides, tetracyclines, glyclycycline, oxazolidinones,
chloramphenicol.
 Biosynthesis of folic and folinic acid
 Sulfonamides, trimethoprim

Classification based on the type of action = bactericidal Vs bacteriostatic action:

To define the type of action of a substance you start with a predefined inoculum of bacteria in presence of
culture medium, you put them inside an oven in standardized conditions and you should normally see
bacteria proliferating very rapidly, with exponential growth.
On these basis, you define as:
- Bactericidal  an agent that in these conditions is able to cause irreversible cell damage to
>99.99% of cells over 24 h.
- Bacteriostatic  if you cannot cause this percentage of damage over 24h.
These agents can stop or slow bacterial metabolism and/or replication, but are not able to
completely defeat bacterial growth.
It has been suggested that for bacteriostatic agents, to be active in clinics, it is very important the
role of the host immune system. This means that the majority of these agents cannot be used in
immune compromised patients.

Principles of MIC test:

It is the principle that establishes the susceptibility of pathogens to a peculiar antibiotic.
MIC  “minimum inhibitory concentration able to prevent bacterial growth”.

The test is done in vitro. We start by putting some culture medium within different tubes.
In every tube we have a predefined inoculum of bacteria (10-6 cfu for mL) and we put different antibiotic
dilutions in different tubes. We also keep one tube as control, where we don’t put any antibiotic.
Obviously in the control, bacteria are able to proliferate very rapidly because no antibiotic is present (the
color of the dye introduced in the tube changes from yellow to red when there is bacterial growth).
In presence of antibiotics whereas we can have large or small bacteria growth, depending on the type of
antibiotic dilution (depending on how much antibiotic you put in the tube).
The first tube where there is absence of bacteria growth defines the MIC (minimum inhibitory
concentration).
MIC test is the basis to understand whether a certain antibiotic can be used in clinics or not.

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Based on type of action we can distinguish among  Bactericidal and bacteriostatic antibiotics.
(professor did not read all the types of antibiotics present in this slide).

Points of weakness of Bactericida Vs Bacteriostatic classification:

At a certain point in the past some physicians started to claim that this classification is not so relevant from
a clinical point of view.
One of the points of weakness of this classification is: what if an antibiotic is not able to destroy exactly
99.99% of bacteria in 24h but a percentage close to it? Can we still talk about bactericidal antibiotic or is it
bacteriostatic?

Points of weakness taken from slides  referred to a study on gram+ bacterial infections:
- Those agents that are called “bactericidal” usually fail to kill every organism (if, for instance, the
inoculums is large )within 18–24h after the test.
- most so-called “bacteriostatic” agents kill some bacteria within the 18–24h after the test, often
more than 90%-99% of the inoculum, but not enough (>99.9%) to be called “bactericidal.”
- The in vitro microbiological determination of whether an antibacterial agent is bactericidal or
bacteriostatic may be influenced by growth conditions, bacterial density, test duration, and extent
of reduction in bacterial numbers.
- The clinical definition is even more arbitrary.
- Most antibacterials are better described as potentially being both bactericidal and bacteriostatic.
- Conclusions Although bacteriostatic/bactericidal data may provide valuable information on the
potential action of antibacterial agents in vitro, it is necessary to combine this information with
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Lecture 1 Pharmacology of Antibiotics 05/10/2021
pharmacokinetic and pharmacodynamic data to provide more meaningful prediction of efficacy in
vivo.

Classification based on modality of antibacterial activity:

Experimental animal model simulated what happens if you have a severe infection in blood stream and you
are using different types of antibiotics.
- In some cases you have a rapid response
- In other cases you have a slow response (slow decrease in bacterial growth)
Notice that bactericidal and bacteriostatic antibiotics are both represented in the two panels related to
rapid response and slow response respectively.
Thus we understand the classification bactericidal Vs bacteriostatic is not important from a clinical point
of view because there is no correlation with the rapidity of response.

Consequently we have another classification based on the mode of antibacterial activity:

- Time-dependent antibiotics  slow response: you have to wait 48-72h before a clinically
meaningful response can be visible.
- Concentration-dependent antibiotics  very rapid response, usually in 24-48h

Literature suggests us that nowadays what is very important in the assessment of antibiotic effectiveness
in vivo is  the relationship between SC (serum concentration) profile of the antibiotics and the MIC
(in vitro data collected testing the susceptibility to antibiotic).
We have 3 different possibilities to demonstrate this relationship, because you can correlate the MIC with:
- the peak plasma concentration
- the minimum plasma concentration
- or with the overall exposure you have in serum (AUC = concentration Vs time)

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Consequently nowadays we have a new classification of antibiotics that is based on modality of anti-
bacterial activity:

- time-dependant antibiotics  the most important ones are: beta-lactams, glycopeptides,

oxazolidinones and macrolides.
To understand how they work we look at this experiment with a beta-lactam antibiotic.
We start with inoculums of 6-10 cfu/mL of antibiotics in the different tubes containing bacteria.
What happens when you have a time-dependent agent is that:
 if you have a concentration equal to MIC  you are able to counteract bacterial proliferation.
 If you have a concentration that is x4times the MIC  you have increased effectiveness,
 but if you increase the concentration up to x10 or x100 times you don’t have further increase
in effectiveness.
 When we are using time-dependent agents as beta-lactams, what is very important is to have
in the serum and in tissue of our patients, a concentration of antibiotics >MIC.
It is not relevant if you have a concentration of antibiotic x10 x100 times greater than MIC
because you don’t have any further benefit. (Cmax >MIC)

Nowadays in clinical practice we look at the Antibiogram list of different antibiotics that can be
used to treat the pathogen responsible for patient’s infection, containing the specific MICs for
every type of antibiotic.

For time-dependent antibiotics, you must maintain a constant concentration of antibiotic above
the MIC, thus the dosing regimen is based on multiple daily doses (3 or more doses/day usually)
Indeed if you look at pharmacokinetics, you see that most time-dependent antibiotics have a very
short half-life.

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- Concentration-dependant antibioitcs  the most important are: aminoglycosides,

fluoroquinolones, azalides, tetracyclines, glicylciclines, lipopeptides.
By increasing the dosage of the antibiotic we assist to a linear increase in its effectiveness.
By increasing the dosage we will have both:
- Increased effectiveness
- More rapid effect

Dosing regimen suggested with concentration-dependant antibiotics  we should optimize the

exposure in terms of peak-serum concentration (Cmax) to MIC, greater of at least 10 folds.
(optimal exposure: Cmax/MIC >10 )
From slides  the objective is to have high peak levels and/or AUC.
 Cmax/MIC >10
 AUC/MIC >125
In this way can obtain a rapid antibacterial activity.
With concentration-dependent antibiotics we use the 1-daily-dose regimen to optimize the dose.

- For severe infections we can use an administration that is more effective in achieving the target,
that is continuous infusion  in this way you have a stable concentration in serum and tissues of
our patient

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Susceptibility to antibiotics
It is also important to understand how the definition of “susceptibility to an antibiotic” has changed in
clinics over time. It has been defined by the two regulatory agencies EUCAST (in Europe) and CLSI (in USA).
This new definition is a revolution because:

- before this revolution  the antimicrobial susceptibility test was based on the distribution of MIC,
obtained through tests on wild-type strains.
Considering the Gaussian distribution, before the revolution the regulatory agency had put the
break-point at the end of the curve. But this was NOT a guarantee that the antibiotic could be
effective in humans, because we didn’t know if that dosing regimens could guarantee sufficient
drug levels in the serum and in the tissue of different patient.
(I have not completely understood the meaning of this paragraph but these were the words of the
professor).

- After the revolution (2012)  the EUCAST proposed an algorhythm based on preclinical and clinical
PD and PK studies that is used to identify the PD target.
Thus these studies are the basis to identify our PD target that we could achieve in the serum and in
the tissues of our patients, according with the dosing-regimens studied during clinical trials.
The methodological aspect called population pharmacokinetics, gives us the opportunity to
understand which is the clinical breakpoint we are able to achieve according to the dosing regimen
we are using (I think that with the following example the concept will be clearer).

Example:
Our example is related to meropenem (beta-lactam).
The pharmacodynamic (PD) target of this antibiotic is a concentration for the 50% of time above
the MIC, according to animal models.
According to this PD target and according to the licensed dosing regimen (1g every 8h), through the
Montecarlo simulation, EUCAST calculates the probability of achieving this target (50% of time
above MIC) according the different distribution of MIC of pathogen.
 If MIC = or < 2  you have 60%-70% of time above MIC
 If MIC >2  you have less than 50% of time above MIC
According to this simulation EUCAST decided that the clinical breakpoint of meropenem against this
pathogen is 2mg/mL.

Nowadays we base our antibiotic choice mainly on: PK-PD relationship.

Classification based on physical and chemical properties:

The antibiotics can be lipophilic or hydrophilic.
 Hydrophilic antibiotics are:
o beta-lactams,
o glycopeptides
o aminoglycosides
- They have a PK common feature  limited Volume of Distribution. This means they are unable to
penetrate the cells of the body and this affects also their PD  hydrophilic agents are not able to
act against intracellular pathogens (like some pathogens of pneumonia: Legionella Pneumoniae,
Clamidya, Mycoplasma).
- Another important characteristic of hydrophilic drugs is that they are eliminated though kidneys,
thus it is very important to know the renal function of the patient, in order to modify the dosage
accordingly.
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Lecture 1 Pharmacology of Antibiotics 05/10/2021

 Lipophilic antibiotics are:

o Macrolides
o Fluoroquinolones
o Tetracyclines
o Chloramphenicol
o Rifampicin
o Oxazolidinones
- They have a large volume of distribution  they can diffuse through plasma membrane according
to concentration gradient  They are active against intracellular pathogens.
- They are eliminated though liver metabolism, so the renal function is not important in determining
the dosage of these antibiotics. You have to modify the dosage only in patients with liver disease.

Choice of antibiotic therapy

It is important to keep in mind:
- PD of the drug:
 spectrum of activity (that we have in vitro)
 antibacterial activity:
o time-dependant or
o concentration dependant.
- PK of the drug  we have to consider that the drug concentration in the serum and in the tissues
of our patient is not stable, but instead changes over time (according to administration,
distribution, metabolism and excretion).

 Thus to have an effective antibiotic therapy we have to merge PK and PD and to look at PK-PD
relationship.
This principle may allow us to study the best effectiveness over time of antibiotics and so we
have obtained clinical efficacy.

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The antimicrobial puzzle therapy:

15 years ago we introduced the idea of anti-microbial puzzle therapy.
Before that time, the antibiotic choice was based on the in vitro activity (establishment of MIC in vitro), but
these studies did not take into account some important variables like:
- site of infection
- the type of pathogen we have
- the pathophysiology of the patient

Infection site:
Infections can be variably difficult to treat, according to the site of infection.
Two examples:
- pneumonia  most of pathogens are located in the extracellular space called “epithelial lining
fluid” (ELF). In this case it is very important that the antibiotic is able to achieve the PD target that is
in the ELF site, in order to be effective.
- Endocarditis  The penetration of antibiotics inside vegetations may be very heterogeneous,
which explains why some antibiotics cannot eradicate the microorganism in large vegetations.
For vegetations the antibiotic treatment should be bactericidal, and the bactericidal activity should
ideally be obtained rapidly and maintained until cure is established;
But:
o PK  the penetration of some antibiotics inside the vegetation may be very heterogenous
which may explain why some antibiotics cannot eradicate the microorganism in large
vegetations;
o PD  the concentration of antibiotics within the vegetation must be sufficiently high and
sustained.

Over time researchers figured out that some deep tissue infections should be treated with different dosing
regimens compared to infections restricted to blood flow (bacteremia).
 If you put the antibiotic directly in blood stream through IV injection, you reach very high
concentrations in blood stream, so it is not difficult to deal with bacteria either if you are
dealing with time-dependent or concentration dependent agents.
But if you are dealing with pneumonia, endocarditis or osteomielitis, you have to think that the
antibiotic should migrate from blood stream to infection site.
According to the classification of hydrophilic Vs lipophilic antibiotics  hydrophilic antibiotics
are not capable to distribute to different tissues, and only a fraction around 30% the
concentration we have in the serum is able to reach the extracellular space of heart, lungs,
bone and joint site.

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Wide use of antibiotics:

-issue of resistance
Important principle  whenever you use antibiotics, you have a selective pressure upon microbes and
microbes will become resistant to that antibiotic.
Thus the more antibiotic you use, the more the microbe becomes resistant.

We don’t have to destroy bacteria because they are very important for human life.
On this table (from university of Los Angeles) we compare microbes to humans for what concern:
- Number of individuals on earth  there are much more bacteria than humans
- Mass
- Generation time  much more rapid for bacteria
- Time on earth bacteria are present on earth from before

This is to say that we will deal only with pathogenic bacteria, and not with all bacteria present on earth.
Keep in mind that antibiotics are not only used in clinics, but they are also widely used in other fields like
agriculture and animal farming and this is a real problem.

In the last 20 years resistance rate has been constantly increasing.

The more antibiotics you use, the more the pathogen will become resistant.
The problem anyway is not present when we use antibiotics appropriately, but it ensues when we make an
inappropriate use of antibiotics.

Study  tested which was the probability of selective antibiotic resistance when antibiotics were
administered to children.
They studies 119 children with acute otitis media or respiratory tract infections.
They divided the population under study in two groups: one was treated with antibiotics and the other with
no antibiotics.
(acute otitis media is not always treated immediately with antibiotics. Usually we prefer to wait and see.
Only if the child’s health is worsening we administer antibiotics).
Children were tested with a pharyngeal swab to individuate the MIC of pathogens after 2 and 12 weeks.
They figured out there is a change in bacterial susceptibility in children treated with antibiotics.
- In children treated with antibiotics  no change in MIC of course
- In children treated with antibiotics  MIC increases after 2 weeks, but after 12 weeks MIC turns
back to its pre-treatment value.
After exposure to antibiotics you select pathogens with higher MIC for that antibiotic and the re-
sensitization of pathogen to that antibiotic will take 3-4 months.

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
This is why it is very important to use antibiotics for the shortest period possible.
Even in very severe infections the duration of treatment should not be longer than 7-10 days (in the past
they used to keep it for months).

Resistance to antibiotics over time:

This is the timeline of antibiotics since the early ‘40s of
last century.
Bacteria are able to adopt mechanisms to survive even in
presence of new antibiotics, that’s why we are
continuously searching for a new antibiotic toward which
pathogens continue to mount resistance.

-how antibiotics affect our microbiota

Every time we use antibiotics we cause a profound change also in our microbiota.
This is why we should use antibiotics only when it is really necessary.

Study  they tested the attitude of antibiotics prescribed for non-bacterial upper respiratory tract
infections.
We should consider that 50% of upper respiratory tract infections are actually caused by viruses, thus it is
completely meaningless to prescribe antibiotics for an upper respiratory tract infection.
This study was performed in Canada, which has one of the most evolved public health systems.
Taking into account patients over 65 years, the number of patients who were inappropriately treated with
antibiotics for conditions of non-viral origin (like acute nasopharyngitis or acute bronchitis which are
basically always viral) was as high as 36-45%.

Two populations where antibiotics are inappropriately prescribed are:

- Elderly over 65y (as we have just seen in the study)
- Pediatric population
William Osler quotation that the professor really appreciates: “ one of the first duties of the physician is to
educate the masses, not to take medicine”.

There are 4 main bacteria resistant to basically all antibiotics we have in clinics:
- Staphylococcus aureus  resistant to meticillin
- Enterococci  resistant to vancomycin
- Pseudomonas aeruginosa  resistant to carbapenems
- Acinetobacter  resistant to carbapenems.
These 4 pathogens are responsible for about 50% of in-hospital deaths related to bacterial infection.

It is estimated that in 2050 we will have more deaths related to antibiotic resistance than deaths related to
cancer.

The main ways though which pathogens can cause resistance:

- Blocking the entrance of the drug in the cell (loss of porins)
- Changing the target of the antibiotic (target mutations)
- Producing enzymes able to destroy the antibiotic (beta-lactamase in periplasmic space and
antibiotic-modifying enzymes)
- Overproduction of transmembrane efflux pumps
- Ribosomal mutations ofrmodifications

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Study published in 2018  “Surveillance of antimicrobial resistance in Europe during 2017”:

They figured out that in Italy Klebsiella pneumonia and Acinetobacter are resistant to the majority of
antibiotics that are available on the market.

Antibiotics classes
SULPHAMIDICS

It is not a real class of antibiotics.

They were introduced in clinics before the discovery of penicillin by Alexander Fleming.
They were discovered by Gerhard Domagk at the beginning of XX century.
He was a chemist who worked in Germany and, while testing a lot of different compounds, he figured out
that some of them had anti-bacterial activity.
1935  “red prontosil” was the first sulphamidics to be discovered and it was active against staphylococci
and streptococci (commonly causing skin infections).

Mechanism of action:
Sulphamidics are able to block the metabolism of bacteria.
To understand how they work we should first study the process that leads to purine and pyramidine
synthesis in bacteria:
- starting from two compounds (Paraminobenzoic acid (PABA) and dihydropteridine), the enzyme
dihydropteroate synthase, is able to synthesize dihydrofolic acid.
- Then the enzyme dihydrofolate reductase is able to convert dihydrofolic acid into tetrahydrofolic
acid  which is needed to synthesize purines and pyramidins.

Sulphamidics are able to block this first enzyme (dihydropteroate synthase) with a consequent block of
bacterial metabolism.

Cotrimoxazole:
The most used sulphamidics is cotrimoxazole, which is given by the association of trimethoprim and
sulfamethoxazole.
Cotrimoxazole is more powerful than other sulphamidics because it is able to inhibit not only the first
enzyme (dihydropteroate synthase), but also the second one (dihydrofolate reductase) in the process of
purine/pyramidine synthesis.

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Notice that trimethoprime moiety of cotrimoxazole is very similar to the Paraminobenzoic acid (PABA)
region of dihydrofolic acid.

PK of cotrimoxazole
- Oral or IV administration
- Bioavailability of 70-80%
- Wide Vd  since it is lipophilic it can widely distribute to deep tissues and it is also able to cross
BBB.
- Eliminated by renal route
- Strong inhibitor of CYP-2C9  important in patients under treatment with warfarin because the S-
enantiomere of warfarin is metablized by CYP-2C9.

Adverse reactions to cotrimoxazole

- Cristalluria due to the fact that the drug tends to precipitate at normal urine ph.
In order to avoid cristalluria it is important to hydrate the patient and to administer bicarbonates
that make urines more alkaline.
- Iperkalemia
- Hemolytic anemia  G6PDH is very important to neutralize oxidative stress. In patients with
G6PDH deficiency the patient may develop hemolytic anemia when treated with cotrimoxazole.
- Hypersensitivity :
o Agranulocytosis, aplastic anemia
o Lyell’s syndrome, steven

Indications of cotrimoxazole:
- Pneumonia related to Pneumocystis Jirovencii (fungi)  important opportunistic infection typical
of immune compromised patients.
- MRSA-related infections (resistant to beta-lactams)  meticillin test is always done in vitro, to see
if the staphylococcus is resistant or not.
- UTI if Entrobacterales susceptible (poor use)
- Toxoplasmosis

BETA-LACTAMS

They are the most important antibiotics we have.

They have been introduced by Alexander Fleming, who discovered penicillin.
Story of discovery  During a weekend Fleming left some plates of Staphylococcus Aureus on his desk.
After the weekend he noticed that there was an area of the plate where bacteria did not grow. The reason
was the presence of fungi, able to produce penicillin.
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(halo of inhibition around penicillin)

Howard Florey and Ernst Chain collaborated with Fleming in an important

experimental animal model (of late ‘30s) which demonstrated for the first time
the relevant antibiotic activity of penicillin there were two groups of mice:
both groups were challenged with streptococci, but only one of the two groups
was treated with penicillin. The result was that after 24h the untreated group of
mice was completely dead, while the group treated with penicillin was still alive.

The First clinical trial was in 1941 during the Second World War.
Penicillin was a great advantage for soldiers who participated to the war, but
there were problems related to penicillin production.
To counteract this problem the “Morning Milk” method was introduced: since penicillin is excreted in
urines, urines of soldiers were collected, purified and re-used for other soldiers.

Penicillin G was the first type of penicillin to be produced. It is an important drug even nowadays.
During the 2nd world war in 1943 penicillin was used for the first time in Sicily.

There are four subgroups of beta-lactams:

- Penicillin
- Cephalosporins
- Monobactams
- Carbapenems

Beta-lactams common features:

- Beta-lactam ring  every time a beta-lactam ring is active, we have antimicrobial activity.
- Mechanism of action  it is common through the four different subgroups:
o Time-dependent antibacterial action
o Inactive towards intracellular pathogens (L.penumophila, C. pneumoniae, M. pneumoniae)
- Mechanism of resistance  very complex aspect. It is a mechanism shared by many classes.
- Wide therapeutic index  therapeutic index is the ratio between dosage responsible for toxicity
and the dosage responsible for efficacy.
Wide therapeutic index means that the dosage that causes toxicity is hundred folds higher than
the dosage responsible for efficacy.
- Low incidence of toxicity

Beta-lactam ring:
This is the beta-lactam ring. You see if is located in different
positions in different subgroups.

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Mechanism of action:
It is based on inhibition of bacterial cell wall (bactericidal).
Specifically beta-lactams are able to interact with two pentapeptides, of which NMAS (N-acetylmuramic
acid) is the most important. Physiologically these pentapeptides get linked together thanks to
transpeptidases which indeed are able to link the different peptide units of bacterial cell wall to form
peptydoglycans.
 To prevent the formation of bacterial cell wall, beta-lactams inhibit these transpeptidase called
PBP (penicillin-binding protein)  this is the target of beta-lactams.
 As consequence of inhibition of peptidoglycane synthesis, bacteria are not able to survive and
so they release auto-destruction enzymes called autolysins.

Types of transpeptidases
There are 6 different types of transpeptidase (PBPs). It is not important that you know all of them.
The most important ones are:
- in gram-negatives are n°1 and n°3 and
- in gram positive is the n°2.

We have nowadays for the first time a beta-lactam that is active against an MRSA (almost all beta-lactams
are inactive against MRSA): this is thanks to the development of new moieties able to counteract the PBP-II.
PBP-II is the mechanism though which staphylococci are able to become resistant, because they produce
10-100 folds higher levels of PBP-II with respect to normal  so they are able to avoid the action of most
beta-lactams.

Differences between gram + and gram-

- Gram +  no structure outside bacterial cell wall

- Gram-  peptidoglycans strand is thinner than the one of gram positives.

However they have an outer membrane outside cell wall.
 Thus if you have to inhibit the proliferation of gram- by targeting bacterial cell wall, you need
an hydrophilic molecule (porin). Porins allow beta-lactams to reach bacterial cell wall.

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Beta lactams and antibacterial activity:
Beta-lactams are:
- Bactericidal
- Time-dependant  keep in mind that we must refract the dosage.

PENICILLINS
Classification of penicillins:
- Natural:
 Penicillin G or Benzylpenicillin  historical penicillin of Fleminger experiment
 Penicillin V or Phenovymethilpeniccilin
- Semisynthetics (the ones used nowadays):
 Aminopenicillins (os-iv):
o Ampicillin
o Amoxicillin
 Carboxypenicillins (iv)  not used in Italy, but used in developing countries.
o Carbenicillin
o Ticarcillin
 Isoxazolylpenicillins( os-iv)
o Oxacillin
o cloxacillin
o dicloxacillin
o flucoxacillin
 ureidopenicillins (iv)
o piperacillin
o methicillin

Spectrum of activity:
 penicillin G and penicillin V active against some simple gram+ and gram- like,
 streptococcus spp,
 neisseria meningitides,
 neisseria gonorroeae,
 some anaerobes of our mouth, peptococci
 spirochetes, treponema pallidum (syphilis) penicillin is still the first
 peptrospetococci
 isoxacillin (oxacillin, cloxacillin, dicloxacillin)
 staphylococcus aureus - methicillin susceptible (MSSA)
 aminopenicillins (ampicillin, amoxicillin)  wide spectrum of activity in particular against gram-
(for example against enterobacteria that are responsible for urinary tract infections).
 MSSA (streptococcus penumoniae, H.influenzae, M. catharrhalis, Proteus mirabilis,
Salmonella spp, Shigella spp, Escherichia coli, Listeria monocytogenes, Enterococcus
faecalis, Helicobacter pilori)
 Anaerobes, peptococci, peptrostreptococci
 Ureidopenicillins (piperacillin)  the most important penicillin that we have in our hospitals.
It is used against several gram- infections, and for anaerobes.
It has a wider spectrum of activity compare to ampicillin against gram- bacteria.
 Pseudomonas spp, Enterobacter spp, Klebsiella spp (gram-)
 Anaerobes (Bacterioides spp)
 Carboxypenicillins (carbenicillin, ticarcillin)

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
 Wider spectrum of activity compared to ampicillin  for Streptococcus spp, E. Faecalis,
Klebsiella spp, L. Monocytogenes.
 Wider spectrum of activtity compared to penicillin  for P. Aeruginosa.

CEPHALOSPORINS
Classification:
They are classified into 5 generations according with the period they have been introduced:
- 1st generation  active mainly against gram+ (in particular against MSSA and streptococci)
o Cefazolin
nd
- 2 generation  some activity against gram- (E- Coli, Klebsiella spp, Proteus spp), but many
gram- bacteria are able to mount resistance against Cefuroxime, so it is not commonly used.
o Cefuroxime (the only one important)
o Cefaclor
o Cefoxitine
o Cefotetan
- 3rd generations  important mainly to be active against Enterobacterales and Streptococcus.
o Cefotaxime  active against most Enterobacterales, Pseudomonas Aeruginosa
o Ceftriaxone  against Serratia, Neisseria gonorrhoeae.
NB: both Ceftriaxone and Cefotaxime are active against Streptococcus Pneumoniae.
o Ceftazidime  active against Streptococcus Pneumoniae and S. Aureus
th
- 4 generations  mainly active against Pseudomonas Aeruginosa.
o Cefepime
th
- 5 generations  the first drugs active against MRSA!!! (thanks to their capacity to target PBP-IIa).
o Ceftobiprole
o Ceftaroline

NB = The majority of cephalosporins are inactive against Anaerobes and Enterococci.

Anaerobes an Enterococci are located in our gut, thus whenever we deal with an intra-abdominal infection
due to translocation of bacteria from the gut to peritoneum or other districts, cephalosporins are not a
possibility of choice.

Ceftaroline fosamil  anti-MRSA activity thanks to the high affinity for PBP-IIa.

CARBAPENEMS
They are the most precious and fragile antibiotics that we have in the cathegory of beta-lactams.
They are:
- Ertapenem
- Imipenem
- Meropenem

They are very important because they are able to counteract many gram- (like Enterobacterales) that are
resistant to basically all other beta-lactams.

However they are very fagile and we have several Enterobacteral which have become resistant to
carbapenems.
This is why we have to keep in mind that even though they have a wide spectrum of activity, we must use
them ONLY when we have an infection resistant to all other beta-lactams!

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Lecture 1 Pharmacology of Antibiotics 05/10/2021
Carbapenems are resistant to extended spectrum beta-lactamase (ESBL). Prof did not add anything to this
sentence.

Exception  Ertapenem = is the only carbapenem inactive against E. Faecalis and P. Aeruginosa.

MONOBACTAMS:
- Aztreonam:
o (Monocyclic structure)
o Spectrum of activity limited to gram- bacilli (Enterobacterales and P. Aeruginosa)
o it is a very old drugs (not frequently used in the past) becoming more important nowadays
because we have some resistant strains of Enterobacterales that are susceptible only to
Aztrenam.
These Enterobacterales strans indeed produce MBL (metallo-beta-lactamases) that are able
to inactivate all beta-lactams antibiotics except aztreonam.

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