Lecture 2 Pharmacology of antibiotics 19/10/2021

Arianna Rossi, Elena Marchetti

Antimicrobial Pharmacology – 02
Prof. Federico Pea, 19.10.21

Pharmacology of antibiotics (2)

BETA-LACTAMS
Mechanism of resistance vs beta-lactams
We continue the last lesson, talking about the mechanism of resistance of beta-lactams. This is very
important nowadays, since many bugs have become resistant to beta-lactams.
Several different mechanisms of resistance:
a. Decreased permeability of membrane due to porin loss: impossibility of the drug to penetrate in
the extracellular moiety of the bug (between the outer and the inner membrane). This is related to
the loss of porins, which are proteins that allow the penetration through the hydrophilic
compartment in the inner membrane of the pathogen.
b. For Gram positives, the most important mechanism is the modification of PBPs (Protein Binding
Proteins) expression. PBPs are the target of the antibiotics, meaning that when the Gram positives
become resistant to beta lactams, they will be characterized by methicillin resistance. In this
setting, almost all the different beta-lactams cannot be used.
c. For Gram negatives, the most important mechanism is the production of beta-lactamases, which
are enzymes able to hydrolyse the beta-lactam ring. According to this, now we have more than 900
different types of beta-lactamases, meaning that there are a lot of possibilities to make this drug
ineffective against Gram negatives.
This mechanism can be chromosomally determined (<20%) or plasmid mediated (>80%). This is
important because, especially among Enterobacterales, it is plasmid-mediated, allowing the
transfer from one species to another species (and not only between strains).
➔ By looking at the different beta lactams (penicillins, cephalosporins, carbapenems, monobactams),
the characteristic of the beta-lactamases is to hydrolase the beta lactam ring. For this reason, all
the beta-lactams will lose their activity.
➔ The mechanism of beta-lactamases production is inducible, meaning that the more antibiotic we
use, the more resistant mechanism appear. So, we should use the less antibiotics possible and for
the shorter time possible.
Why beta-lactamases are very important for Gram negative, while they’re NOT so relevant for Gram
positives?
Important difference between gram positives and gram negatives:
- GRAM POSITIVES: beta-lactamases are produced but then, as gram positives are not able to retain
beta-lactamases within their structure, they are released in the environment.
- GRAM NEGATIVES have an outer
membrane. Indeed, after the
production of beta-lactamases,
they are retained in the
periplasmic space. This is very
effective because whenever beta-
lactams penetrate through the
porins, they directly find beta-
lactamases and they are
inactivated.

1
Lecture 2 Pharmacology of antibiotics 19/10/2021
Evolution of resistance to beta-lactams
It is related to the different beta-lactams that have been
introduced in the therapeutic armamentarium.
The first ones were introduced during the 2 nd World
War: penicillin. Unfortunately, in few years the first
mechanism of resistance appears, which were
penicillinases.
In the subsequent years, other mechanism of resistance
appeared, especially for Staphylococcus aureus, but also
for different types of Gram negatives (especially
Enterobacterales).
In 1970’s and 1980’s, the first cephalosporinases (AmpC)
were found.
Fortunately, in the 1980’s the beta-lactamases inhibitors were identified. These agents are able to
counteract the mechanism of resistance through competitive inhibition of beta-lactamases, leading to the
recovery of penicillin activity against strains producing beta-lactamases.
- Beta-lactams devoid of any antibacterial activity
- Extracted from Streptomyces or semi-synthetic
➔ Beta-lactamases inhibitors include clavulanic acid, sulbactam, tazobactam (introduced in the early
1980’s).
Nowadays, we have 3 different pre-constituted combinations (very effective) of penicillin and beta-
lactamases inhibitors:
- Amoxicillin + Clavulanic acid
- Ampicillin + Sulbactam
- Piperacillin + Tazobactam
Unfortunately, in the last 20 years, new types of beta-lactamases appear. They are able to destroy almost
all of the old beta lactams.
In a first instance, there was the introduction of ESBL (= Extended Spectrum Beta-Lactamases). These beta-
lactamases are able to destroy almost all the old beta-lactams.
In 1990’s, MβL (= Metallo-Beta-Lactamases) appeared, which are even worse.
Last but not least, there were the appearance of Carpenemases of which the worse is KPCs (= Klebsiella
pneumoniae carbapenemase). These enzymes are beta-lactamases able to destroy even carbapenems.
➔ Indeed, we had no weapons against Gram negatives, so we started to recover very old drugs in
order to counteract these pathogens.
➔ Fortunately, more recently, we have started to introduce also new types of beta-lactamases (as we
will see in the course of the lecture)
Ambler classification and beta-lactams
We will not go too much in depth.

2
Lecture 2 Pharmacology of antibiotics 19/10/2021

It is a very important classification from a clinical and microbiological standpoint because it identifies which
kind of beta-lactamases are produced by which kind of pathogens (thus which beta-lactams are inactivated
and cannot be used with that pathogen).
- First group is related to ESBL.
ESBL is able to inactivate:
• Almost all first to 4th generation cephalosporins
• Older beta-lactamases inhibitors (like piperacillin + tazobactam, or amoxicillin +
clavulanic acid)
- Second group is related to Cephalosporinases (AmpC).
AmpC is able to inactivate most of the beta-lactams
- Third group is related to Carbapenemases (among which KPC is the most important).
The most worrisome group, also found in our hospitals. Italy is one of the two worst European
country.
There are different types of carbapenemases:
• KPC (Klebsiella Pneumonia Carbapenemase) is the most important
• NDM (New Delhi Metallo-beta-lactamase)
• OXA-48 group (appeared more recently)
KPC producers, which are very easily transmitted through plasmids, are able to inactivate almost all
beta lactams. The same is true for NDM and OXA-48 group, actually it is even worse since this kind
of carbapenemases are more effective in destroying beta lactam rings even in new antibiotics.
According to this classification, we need new antibiotics.
Note from sbobinatore: Ambler classification divides beta-lactamases into 4 classes (A,B,C,D), which are
reported in the table, based on the amino acid sequence.
- Class A: ESBL, KPC
- Class B: NDM
- Class C: AmpC
- Class D: OXA-48 group

Antimicrobial spectrum of new antibiotics in development

Nowadays, we have two new drugs that have been introduced in the therapeutic armamentarium:
- Ceftolozane/tazobactam
- Ceftazidime/avibactam

In addition, other drugs are in phase 3 clinical trials (aztreonam/avibactam, imipenem/relebactam,

meropenem/RPX7009). Indeed, we hope to have them available in our therapeutical armamentarium in the
next few months.
As for the two new drugs already approved:
- They are effective against ESBL-producers, AmpC-producers and partially against Pseudomonas
(including multi-drug resistance strains).
- By looking at KPC-producers, we see that Ceftazidime/avibactam is partially effective, while
Ceftolozane/tazobactam is not effective.
- Neither MBL-producers nor Acinetobacter baumannii are covered by these antibiotics.
Indeed, these two new drugs are not the final solution to the problem. Whenever new antibiotics are
introduced, the buds defend themselves by producing new mechanisms of resistance. It is very important
that antibiotics are used very wisely and for the shortest time possible.

3
Lecture 2 Pharmacology of antibiotics 19/10/2021
Global distribution of various carbapenemases in carbapenemase producing Enterobacteriaceae
This is the worldwide situation nowadays regarding carbapenemases.

In Italy, the red colour is related to the fact that we have endemic KPC-producers in our hospitals. This
means that very frequently, unfortunately, patients that are admitted to the hospital may be complicated
by hospital-acquired bacterial infections.
That’s why nowadays we have new beta-lactamase inhibitors (including Avibactam that we mentioned
before).
The road to avibactam: the first clinically useful non-beta-lactam working somewhat like a beta-
lactam
Avibactam: new non-beta-lactam beta-lactamase
inhibitor, which is coupled with Ceftazidime.
Image on the right: Comparison of the in-vitro
activity of beta-lactamase inhibitors in terms of
inhibitory concentration of 50% of the enzymes
activity (IC50).
By looking at the KPC-producers, we see that the
concentration needed for Avibactam to counteract
KPC is lower (by far) compared to the one of
Clavulanic acid and Tazobactam (that are old beta
lactamases)
➔ Avibactam: lower concentration needed
to counteract KPC → indeed, it is very
effective in counteracting KPC-producers.

Nowadays, we have two more new beta-lactamase inhibitors: Relebactam and Vaborbactam.
- Relebactam is coupled with Imipenem.
- Vaborbactam is coupled with Meropenem.
These two combinations are in phase 3 clinical trials, so probably in the next few months we will have them
in our therapeutic armamentarium.
Activities of novel beta-lactamase inhibitors
Essentially, there are no major differences between Avibactam, Vaborbactam and Relebactam.
The only relevant difference is in their action against OXA carbapenemases (Ambler class D):
- Avibactam is acting against OXA
- Vaborbactam and Relebactam are not acting against OXA

4
Lecture 2 Pharmacology of antibiotics 19/10/2021

New siderophore beta-lactam

In addition to new beta lactamase inhibitors, there is also this new siderophore beta lactam which is a beta-
lactam with a new type of mechanism of penetration in bacterial cell wall.
The new drug is called Cefiderocol, which has been present in our therapeutic armamentarium for the last
few months. It is derived from Ceftazidime: comparing the two drugs, the scaffold is very similar, but there
is a different side chains, which have important peculiarities:
• Pyrrolidinium group confers very high stability against beta-lactamases and KPC-producers
carbapenemases in general. It is very well protected by the structure of the drug.
• Presence of chlorocatechol side chain, which confers the possibility to chelate iron which is very
important for bacterial replication.

Mechanism of action of cefiderocol

The presence of chlorocatechol also allows another
important mechanism of accumulation. In fact, the
other beta-lactams are able to reach the periplasmic
space only through porins.
Differently, with Cefiderocol, iron is actively
transported → by chelating to iron, Cefiderocol can
penetrate with active transport within the cells, being
able to reach much higher concentration in the
periplasmic space (compared to the other beta-
lactams).

Different spectrum of activity of novel beta-lactams (BL) and/or beta-lactams/beta-lactamase inhibitors

(BL/BLIS)

5
Lecture 2 Pharmacology of antibiotics 19/10/2021
The in vitro activity is the widest and the
most complete, at least theoretically.
Unfortunately, in vivo it is more difficult
to reach a high cure rate even with
Cefiderocol. Anyway, this is still a very
important starting point.
Comparison with other new drugs which
are in phase 1/2 clinical trials: we see
that some of these mechanisms of
resistance are not counteract by these
new molecules’ combination, differently
from what occurs with Cefiderocol.

PK of beta-lactams
The professor suggests studying PK in a productive way, meaning trying to find the right considerations that
are useful in the clinical practice.
Common features of beta-lactams:
• Hydrophilicity → drugs are not able to penetrate the cells, so they are located only in plasma and
extracellular fluid, meaning they have a low volume of distribution (< 20 L). Clinically, this is not a
problem since all pathogens (shown before) are extracellular.
• Linear first-order PK → meaning that if you double the dosage, you double the concentration
(linear progression of dosage and concentration). So, it is very easy to adjust the dosage according
to the renal function.
• Renal function is relevant → all drugs are excreted by the kidneys as unmodified moiety. Indeed,
whenever we have a change in renal function, we have to modify the dosage in order to have the
right exposure to this drug.
PK of penicillins
➢ Only few penicillins (ampicillin, amoxicillin) have oral bioavailability. The others can be
administered only by IV route.
➢ Short elimination half-life (< 2 hours). It is important to determine the frequency of administration
of the drug. In fact, we know that in order to eliminate completely the drug we need four-fold the
elimination half-life. Indeed, we have to administer the antibiotic at least every 6 or 8 hours,
according to the different elimination half-lives.
➢ Very low volume of distribution.
➢ Protein binding is not relevant as it is < 80%.
➢ Renal elimination route for almost all penicillins (exception: oxacillin, divided between biliary and
renal elimination).

6
Lecture 2 Pharmacology of antibiotics 19/10/2021
PK of cephalosporins
➢ Only three cephalosporins (cefalexin, cefuroxime, ceftibuten) are orally bioavailable but,
unfortunately, they are not so powerful in terms of spectrum of activity → so they are not used to
counteract very severe Gram-negative bacterial infections.
All the others are administered by IV route.
➢ Elimination half-life is similar to the one of penicillins.
Exception: ceftriaxone, which has a much longer elimination half-life → that’s why it is the only
cephalosporin administered once or twice daily depending on the infection (e.g. in case of
meningitis, we double the dosage to have more antibiotic penetration into CSF).
➢ Volume of distribution is similar to penicillins.
➢ Protein binding is not relevant. Only for ceftriaxone we have a very high protein binding, which
explains why the elimination half-life is longer compared to the other agents.
➢ Renal elimination route (exception once again: ceftriaxone, which undergoes almost 50% biliary
and 50% renal excretion).

PK of carbapenems
➢ Administered by IV route. No orally bioavailable agents.
➢ Short elimination half-life → administered every 8 hours (exception: Ertapenem, which has a
longer elimination half-life→ for this reason, it is administered once daily).
➢ The difference in half-life is related to the difference in protein binding: 90-95% protein binding for
Ertapenem, while < 30% for the other carbapenems.
➢ Renal elimination

PK of monobactams (Aztreonam)
➢ Not orally bioavailable.
➢ PK parameters are very similar to the other drugs.

PK exception: Imipenem
We said that generally beta-lactams are hydrophilic molecules. The only exception among beta-lactams in
terms of PK behaviour is Imipenem. This drug is not so stable in our organism because kidney tubular cells
7
Lecture 2 Pharmacology of antibiotics 19/10/2021
have an enzyme, called dehydropeptidase, able to hydrolyse Imipenem.
In our therapeutic armamentarium, Imipenem is combined with Cilastatin, which is an inhibitor of renal
deydropeptidase, allowing imipenem action against the different pathogens. Of course, the PK
characteristics or Cilastatin should be very similar to those of Imipenem in order to have a similar
concentration in the different compartments of our organism.
PK of novel beta-lactams
PK characteristics of novel beta-lactams (mentioned before) are very similar to the ones of beta-lactams.
The volume of distribution is related to a standard human adult of 70 kg. So, by diving the values in the
table by 70, you find a volume of distribution of 0,2-0,3 L/kg.
➢ Volume of distribution of 0,2-0,3 L/kg.
➢ Elimination half-life is around 1-2 hours → that’s why they have an administration schedule every 8
hours.
➢ Renal route elimination.
➢ Protein binding is not clinically relevant.

Drug-drug interactions of beta-lactams

Beta-lactams have a low potential for pharmacokinetic drug-drug interactions because:
Hydrophilic agents may not penetrate within liver cells (this is true for all hydrophilic drugs in general). As a
consequence:
- NOT metabolised by the CYP450 microsomal system
- They are not able to influence or modulate activity of liver enzymes

They’re eliminated mainly by the kidney in unchanged form by glomerular filtration.

In addition, the ones with a shorter elimination half-life have another additional mechanism of elimination,
which is active tubular secretion (→ shortening the time of drug elimination).
Historical perspective: remember that during the 2nd world war, the role of penicillin was to counteract
infections in soldiers. During the early 1950’s, it was identified an inhibitor of the active tubular secretion of
the penicillin, called Probenecid. Having Probenecid in the therapeutic treatment with penicillin means to
promote the persistence time of penicillins in our blood and body in general (acting as a “penicillin-sparing
agent”) → less need for drug re-administration.
Allergic reactions:
Penicillins in general, and cephalosporins very frquently may generate some allergic reactions. This is
related to the fact that these drugs undergo some degrees of spontaneous degradation, generating some
particular very low molecular weight moieties that can promote the appearance of different types of
allergic reactions.
With the exception of skin reactions, these are rare reactions.
More frequent with parenteral administrations.
- First-type reactions (MOST FREQUENT): caused by the release of histamine and other substances
similar histamine related to IgE production due to hypersensitivity
• Early hives
8
Lecture 2 Pharmacology of antibiotics 19/10/2021
• Quincke oedema
• Anaphylactic shock: rare, but it is the most worrisome situation as it can lead to
exitus very rapidly as it promotes a very severe hypotension with the risk of cardiac
arrest.
- Second-type reactions (called cytolytic)
• Leukopenia
• Thrombocytopenia
• Haemolytic anemia
- Third-type reactions: due to the complement (immunocomplex)
• Vasculitis
• Glomerulonephritis (most frequent)
• Arthralgias (most frequent)
- Fourth-type reactions: (delayed hypersensitivity)
• Atopic dermatitis (most frequent).
For this reason, beta-lactams are never used in topical applications (for skin administrations or eye
drops, etc.), otherwise the probability of inducing this kind of hypersensitivity reactions would be
very high.
The anamnesis is very important: we have to ask to the patients if they had any kind of beta-lactams
allergic reactions as these are related to the previous sensitization to allergens (so they may manifest in the
subsequent administration).
Keep in mind that the first type reactions are called immediate reactions, just because they appear no
longer than 30-60 minutes after the drug administration. This could also lead to shock, and by using the IV
route these reactions can appear even after 2 minutes.
ARTICLE: “Amoxicillin rash in infectious mononucleosis”
Not all allergic reactions are related to the drug itself. This (below) is a picture published 1 month ago in
the New England Journal of Medicine, in which we see a typical skin reaction related to amoxicillin
administration in a child affected by mononucleosis (caused by EBV infection).
EBV virus may predispose to this kind of rush if you use antibiotics like beta-lactams, especially
aminopenicillins. It has been shown for 30-40 years ago. It is NOT an allergic skin reaction, but it is surely
promoted by the viral infection.
➔ Indeed, viral infection (especially EBV virus) can promote some kind of allergic dermatitis when
using beta-lactams.
Beta-lactams are not needed to treat mononucleosis as it is a viral infection. However, since mononucleosis
is frequently characterised by the presence of lymph nodes enlargement in head and neck and ulcers in the
mouth (which can suggest the presence of tonsillitis), physicians could use antibiotics.
The professor underlines that this is not correct since it is a viral infection which does not benefit from
antibiotic treatment.

9
Lecture 2 Pharmacology of antibiotics 19/10/2021
Toxicity of beta-lactams
They have a very low toxicity:
- Neurotoxicity (→ convulsions)
• Especially with high doses (IV perfusion) of Penicillin G, Carboxypenicillins and
Imipenem or in case of renal insufficiency (since it causes a drug overexposure for the
patient leading to this kind of adverse events).
• Keep in mind that in babies (< 6 months) BBB is not matured → beta-lactams may cross
the BBB.
• Sensitivity in elderly, and in patients with a neurological history (like epilepsy)
- Hydro-electrolytic imbalances (→ hypernatremia, hyperkalemia)
Penicillin G has a very high load of sodium or potassium. Indeed, by using this drug at very high
dosages, you may experience hypernatremia or hyperkalemia.
Toxicity of cephalosporin
The same is true for cephalosporins in terms of tolerability (extremely reduced toxicity).
- Neurotoxicity: especially in case of renal dysfunction.

➔ The use of cephalosporins has been consistently reduced in our hospitals because they are not
effective against anaerobes in general, differently from penicillins and carbapenems.
An important bug present in our gut is Clostridium difficile (not aggressive if counterbalanced by
the other bugs present in our gut). However, with cephalosporins, we make a selection of C.
difficile → in this way, it becomes pathogenic and produces some toxins, causing acute
pseudomembranous colitis.
Toxicity of carbapenems
- Neurotoxicity (→ convulsions)
This occurs with Imipenem.
- Risk factors favouring the onset of adverse events: elderly, high doses, kidney failure,
neuropsychiatric disorders, alcoholism

Indications of beta-lactams
Regarding dosages, the professor states that we can “read them later” (not sure of what he meant).
I underlined the indications that the professor said to be the most important ones.
Indications - penicillins
Penicillin G:
- Streptococcal endocarditis (in Italy it is not possible to use it)
Actually, nowadays for the following indications we have more powerful antibiotics to be used.
- Streptococcal tonsillitis (beta hemolytic group A)
- Pneumococcal pneumonia (pen S)
- Pneumo/meningococcal meningitis (pen S)
- Actinomycosis
- Primary/secondary syphilis
- Gonorrhoea
- Erysipelas, necrotizing fasciitis s. beta hemolyticus
- Gas gangrene, diphtheria, tetanus
Benzatinpenicillin: long-acting form of penicillin as the drug is released very slowly after IM administration.
It was applied also in Italy to counteract rheumatic fever during the 1950’s-70’s.
- Rheumatic fever prophylaxis (very powerful – still used in some parts of the world where rheumatic
fever is present)

10
Lecture 2 Pharmacology of antibiotics 19/10/2021
Oxacillin
It is a typical anti-staphylococci penicillin, and it is acting only against Methicillin-Susceptible
Staphylococcus Aureus (MSSA).
- MSSA-related endocarditis, bacteremia, pneumonia
Aminopenicillins:
The most frequently used combination is amoxicillin plus clavulanic acid (AMOXI/CLAV). Amoxi/Clav is also
frequently used in the community, not only in the hospital. In fact, there is also an oral formulation.
- CAP pneumonia which could be related to S. pneumonia, H. influenzae, Moraxella catarrhalis.
However, keep in mind that CAP pneumonia can also be caused by intracellular pathogens, which
are not counteracted by beta-lactams (as they are unable to penetrate within cells).
➔ Whenever there are risk factors for intracellular pathogens, beta-lactams are not indicated.
- Ampicillin has a very important role in meningitis due to Listeria monocytogenes (could be present
in elderly, frequently related to not-well-pasteurized cheese) and in Streptococcal endocarditis
- Upper respiratory tract infections (URTIs)
- Skin and soft tissue infections
- Complicated urinary tract infections (c-UTIs)
- Biliary infections (amoxicillin)
- Abdominal infections (E. Coli, E. Faecalis)
Ureidopenicillins:
Piperacillin/Tazobactam is the most important agent we have in the hospital. Used in:
- Severe infections due to Enterobacterales and P. Aeruginosa
Keep in mind that it is NOT active against ESBL-PRODUCERS pathogens → in this case,
unfortunately, we should move to carbapenems.
- Infections due to Bacteroides fragilis
It is active also against anaerobes like Bacteroides fragilis, which is present in our gut, but can be
responsible for intra-abdominal infections after surgery.
Indications - cephalosporins
There are 1st and 2nd generation agents, even though both are used for clean surgery prophylaxis. This
means that during an elective surgery (for example: orthopedic or cardiac), we administer a single shot of
drug before skin incision in order to avoid the contamination of the wound (which could be complicated by
the infection).
- 1st generation: clean surgery prophylaxis (Cefazolin 2g single shot)
- 2nd generation: clean surgery prophylaxis (Cefamandole 2g single shot)
- 3rd-4th generation:
o Community acquired meningitis
o Community acquired pneumonia (CAP)
Ceftriaxone (2g q 12-24H) and Cefotaxime (2g q 8H) are mainly used to treat
community acquired meningitis and CAP. The difference between the two is that
ceftriaxone has a different formulation according to the disease.
→ Ceftriaxone is used once daily for CAP (every 24 hours), while you have to double
the dosage for community acquired meningitis (every 12 hours)
The reason is that in general all beta-lactams are hydrophilic drugs, so they can
penetrate through the BBB (blood brain barrier), when it is damaged according to the
infection. But when the BBB recovers, the penetration rate will be shorter so we will
need very high blood concentration to achieve therapeutic concentration in CSF.
o Enterococcal endocarditis
→ Double beta-lactam combination: Ampicillin + Ceftriaxone (introduced 10-15
years ago). This is related to the fact that, on one hand, aminoglycosides are
related to very severe adverse events. Plus, on the other hand, frequently there are
very high levels of Enterococci resistance against aminoglycosides.

11
Lecture 2 Pharmacology of antibiotics 19/10/2021
o Severe infections due to Enterobacterales (NOT ESBL-PRODUCERS)
→ For the toxicity profile (regarding C. difficile selection), cephalosporins can be
used but are not recommended for Enterobacterales.
o Pseudomonas aeruginosa
Only Ceftazidime and Cefepime (which are 4th generation cephalosporins) are able
to fight against Pseudomonas aeruginosa, which is a non-fermentative Gram
negative differently from Enterobacterales (→ ceftazidime 2g q 8H; cefepime 2g q
8H).
- 5th generation
o MRSA-related pneumonia and /or SSTIs (Ceftobiprole 500 mg q 8H; Ceftaroline 600
mg q 8H)
MRSA = Methicillin Resistant Staphylococcus Aureus.
SSTI = Skin and Soft Tissue Infection.
Ceftobiprole and Ceftaroline are the first cephalosporins, and beta-lactams in
general, that are active against MRSA-related pneumonia. They are able to
counteract MRSA as they can bind very effectively to PBP2A (which is the most
important PBP present in MRSA).
Indications - carbapenems
They are at the peak of our therapeutic armamentarium, so we must avoid using them when they are not
necessary.
- Most frequent scenario: hospital infections with ESBL-producing Enterobacterales that are resistant
to ALL other beta-lactams in at risk subjects:
• Neutropenic
• Immunosuppressed
• Critically ill patients
- Infections due to Acinetobacter baumannii, which usually appears in clusters in hospitals. When
appearing in clusters, frequently it has a high rate of resistance against almost all antibiotics.
Among beta-lactams, only carbapenems are effective against A. baumannii, but unfortunately more
than 30% of the strains are resistant also to carbapenems.
Indeed, carbapenems are not a definite solution against A. baumannii.
Indications - new BL/BLI
Ceftazidime/Avibactam, Meropenem/Vaborbactam, Imipenem/Relebactam are indicated in case of
infections due to carbapenemase producers (KPC).
Ceftolozane/Tazobactam indicated in case of infections due to XDR Pseudomonas.
XDR = extensively resistant Pseudomonas.
Indications - monobactams
Aztreonam is a very old drug, which was recovered because it is the only active against MBL producer
Enterobacterales.
In fact, MBL (metallo-beta-lactamase) resistance is very worrisome regarding Enterobacterales. All drugs
shown before are not active against MBL producers.
- Infections due to MBL producer Enterobacterales
Recommended doses for patients with varying degrees of renal impairment
To conclude beta-lactams, we will talk about the right way to adjust the dosage in case of renal impairment

12
Lecture 2 Pharmacology of antibiotics 19/10/2021
In the table, we see the dosage used in case of creatinine clearance > 50 ml/min. Moreover, it is written
how to reduce the dosage in case of renal dysfunction.
However, this is not effective in clinical practice because beta-lactams are time-dependent agents
(meaning you have to maintain a constant concentration of antibiotic above the MIC).
When you have to reduce the dose, you may face two situations:
- Wrong situation: extending the dosing interval between one dose and the other one may allow the
concentration to decrease more in depth (compared to the correct situation of administering the
dosage at the same dosing intervals).
- Correct situation: unchanged dosing interval (8 hours) while reducing the dosage.
Here we see a table with the correct way of adjusting dosages (regarding
Ceftolozane/Tazobactam), which is recommended by the professor. The dosing interval is
unchanged even in patients with end-stage renal disease (drug given every 8 hours). In this way, we
can maintain more easily the antibiotic concentration above the MIC. This is the best way to
maintain the effectiveness of the drug.

The opposite happens for concentration-dependent agents.

AMINOGLYCOSIDES
The discovery of streptomycin: Selman Waksman
Selman Waksman was an important researcher who discovered Streptomycin, which was the first drug
acting against Mycobacterium tuberculosis. Indeed, it was very important to identify such kind of agent. For
this reason, in 1952 a Nobel Prize was awarded to Selman Waksman.
Nowadays, Streptomycin is not used anymore since it is very toxic, especially ototoxicity and
nephrotoxicity.
In the early 1960’s and 1970’s, new aminoglycosides have been identified, which are polycationic polar
molecular produced by Streptomycetes and/or semisynthetic. They are:
• Amikacin
• Gentamicin
• Netilmicin
• Tobramycin
Spectrum of activity
The characteristic of aminoglycosides is that they are very active against Gram negatives.
In the early 1970’s, it was made a selection where Amikacin was considered more powerful for P.
aeruginosa, but this is not true nowadays.
➔ Nowadays, we continue using Amikacin and Gentamicin for all Gram negatives including P.
aeruginosa.

13
Lecture 2 Pharmacology of antibiotics 19/10/2021
Mechanism of action
Inhibition of protein synthesis by binding to the 30S ribosomal subunit.
Aminoglycosides and penetration in the bacterial cell
Different behaviour between Gram positives and Gram negatives:
- Gram negatives have an outer membrane, meaning that you have to penetrate within the outer
membrane through the porins.
However, unfortunately, the target of aminoglycosides is intracellular, so they need also to pass the
cell (inner) membrane, which may be passed only through active transport (as they are hydrophilic
agents), which is oxygen dependent. Indeed, this kind of transport is inactivated in presence of 2
peculiar condition:
• Absence of oxygen, and
• Reduction of pH
➔ So, in presence of these conditions, aminoglycosides do not work.
This is why anaerobes, Streptococci and Enterococci are inherently resistant to aminoglycosides.
- Gram positives: cell membrane is present, but no outer membrane.
Regarding cell membrane, the previous concept (active transport) is valid also for gram positives.

Nowadays, aminoglycosides are less used than in the past. We do not use aminoglycosides alone, but
rather in combination with beta-lactams (synergistic effect), in order to allow aminoglycosides to
penetrate within the cells because the activity of beta-lactams is to destroy the bacterial cell wall → in this
way, aminoglycosides can escape this resistance mechanism of Streptococci and Enterococci.
Type of antibacterial activity
- Bactericidal
- Concentration-dependent agents: Cmax/MIC ratio should be > 10. To obtain this goal and make the
drugs effective, they should be administered in 1 daily dose. This is allowed by the fact that,
irrespective of PK behaviours with a short half-life (2-3 hours) that would suggest us to administer
the drug every 8-12 hours, we have post-antibiotic effect. This means that even when the
antibiotic concentration is very low, the bacteria remain blocked in their growth → in this way, the
drug can be administered once daily (instead of twice daily).

14
Lecture 2 Pharmacology of antibiotics 19/10/2021
Mechanism of resistance – aminoglycosides
1. Enzymatic inactivation by bacterial enzymes (most frequent and effective)
• Presence of adenyl-transferase, phosphotransferase, acetyltransferase, which increase
the molecular weight dimension of the drug → in this way, they prevent the drug from
reaching the target (ribosomes).
• Extra-chromosomal resistance
• Cross-resistance between the various aminoglycosides
2. 30S ribosomal subunit modification
• Rare, only for streptomycin
• Chromosomal resistance (mutation)
• Absence of constituent prokaryotic ribosome protein
PK of aminoglycosides
PK behaviour is typically the one of low-molecular weight hydrophilic drugs.
- No oral route since the drug is not absorbed → you have to administer it by IV route
- Very low protein binding (5-30%, clinically irrelevant)
- Poor diffusion into liquor (CSF), aqueous humor, vitreous humor, prostate, bile
- Short elimination half-life (around 2-2,5 hours)
- No biotransformation (similarly to beta-lactams) → low potential for PK drug-drug interactions
- Rapid elimination in active form by renal route (90% GF)
- According to this, there is also the possibility to use it for topical indications:
• To treat some intestinal infections (Neomycin, Paromomycin per os)
• To treat conjunctivitis, by using some eye drops (Gentamicin, Tobramycin in eye drops)
These are the PK parameters:

Toxicity of aminoglycosides
The toxicity profile is very relevant, that’s why the use of aminoglycosides is very limited nowadays.
It is related to two main organs:
- Kidney: due to dose-dependent accumulation into tubular cells of the renal cortex.
There are some transporters in our kidney tubular cells that are able to concentrate the drug within
the cells (concentration may reach values which are 50-100 times higher compared to plasma)
→ perturbation of phospholipid metabolism → degenerative lesions of intracellular organelles.
To counteract these adverse events, we should keep in mind that there are some risk factors:
• Age > 65 yo
• Prolonged treatment / high doses (> high C min)
• Kidney failure
• Method of administration (with single dosage, less probability of adverse events)
• Concomitant administration of other nephrotoxic drugs (Vancomycin, Cyclosporine,
Indomethacin)
To prevent nephrotoxicity risk, we can:
• Monitor serum concentration (TDM = Therapeutic Drug Monitoring) in order to
15
Lecture 2 Pharmacology of antibiotics 19/10/2021
maintain it within a pre-defined window → in this way, we have more probability of
effectiveness, and we can contain the risk of adverse events.
• The other way is to monitor serum creatinine in order to control if we are dealing with
nephrotoxicity. An increase in serum creatinine is a warning for the risk of
nephrotoxicity.
Why once daily dosing is better than multiple daily dosages?
This is a study published in Critical care medicine
(2004), in which they compare these two kinds of
administrations (single vs multiple) in this specific case
of Apramycin.
The baseline renal function was similar in the two
groups. The total daily dosage administered was even
higher in patients receiving once daily dosage
compared to multiple dosage. Irrespective of this, the
frequency of nephrotoxicity occurrence was much
lower in the once daily dose group.
There are two enzymes in the urine (normally located
within tubular cells of kidney) → if these enzymes
appear in the urine, this means you have damage of

the tubular cells. As we can observe in the graphs below, if you administer the drug in multiple daily doses,
you have a higher incidence of these adverse events compared to administration of once daily dosage
(single shot).
Explanation (graphs below): here we see the once daily dose (green), and multiple daily doses (red).
They are different in terms of performance:
- Once daily dosage (green): very high peaks and very low plateau levels
- Multiple daily dosage (red): lower peaks with higher plateau levels
By looking at the concentration within tubular cells (renal cortical concentration):
- With multiple daily doses, there is a much higher accumulation within renal cortex cells compared
to the single daily dosage. This is because the drug is actively transported. In fact, with small doses
administered more frequently, it is more probable that the active transport is driven effectively →
with the result of concentrating the drug within cells.
- Differently, with once daily dosage, the transporter is saturated and not all the drug is uptake by
the renal cortex → meanwhile you are able to eliminate much of the drug because the elimination
half-life is very short → accumulation in the cortex is reduced
➔ For this reason, aminoglycosides are used in once daily dosage.

16
Lecture 2 Pharmacology of antibiotics 19/10/2021
Since his explanation was quite confusing, I’ve added below also the graph description taken from
the article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921289/

- (Irreversible) Ototoxicity
It is a major issue. Ototoxicity has been observed early with Streptomycin, which was widely used
for tuberculosis in the 1950’s and 1960’s, but it is also found with the new aminoglycosides.
➔ So, now we limit the duration of treatment with aminoglycosides (e.g. max 3 days, in combination
with beta-lactams). They are very powerful and effective drugs in lowering the bacteria load, but on
the other hand, if we reduce the duration of treatment, we can prevent the onset of these adverse
events.
• Cochlear:
Degeneration of the external ciliated cochlear canal
Late and irreversible
Tinnitus and hearing loss (initially for high frequencies)
It may progress even after discontinuation
• Vestibular:
Posterior labyrinth damage
Early and reversible
Dizziness and spinning sensations
Risk factors: age (infants, elderly), high doses, long treatments > 7-10 days, kidney failure, C min > 1
microg/ml (gentamicin) or 3-5 microg/ml (amikacin), pre-existing hearing loss.
Prevention of ototoxicity risk: TDM, serum creatinine, audiometry.
- Neuromuscular block
➔ The time of administration of aminoglycosides should be of at least 30 minutes (infusion time). This
is because of the risk of neuromuscular block.
• Rare, but potentially serious
• Due to inhibition of presynaptic Ach release + blockade of the postsynaptic receptor for
Ach
• Powered by Curare, Succinylcholine which are agents that also act at this level
• Risk factors: myasthenia gravis

17
Lecture 2 Pharmacology of antibiotics 19/10/2021
Dosing regimens of aminoglycosides
The dosage is related to the body weight (kg).
In neonates, the dosage could be even higher or similar to the adults’ dosage. This is because during early
phases of life, we have much more extracellular water than intracellular water (then this changes in the
first weeks or months of life). Thus, since these drugs accumulate extracellularly, if you have higher volume
in extracellular moiety, you have to administer higher dosage.

- Once daily dose → higher efficacy, less toxicity (already explained)

Why higher efficacy? In the graph, we see
the pathogen MIC and total daily dosage of
240 mg administered once daily (OD) vs 120
mg administered twice daily (BID).
As these drugs are concentration-
dependent, if you consider the Cmax/MIC
ratio as a measure of effectiveness, we see
that Cmax/MIC ratio is much higher in the
once daily dose profile → indeed, we can
conclude that it is more efficacious and less
toxic.
Indications of aminoglycosides
Most of these indications are still found in the guidelines, but several clinicians prefer to move from
aminoglycosides to other agents (seen in the other lectures) which are less toxic.
The only that are used are always administered in combination therapy with other agents (here below there
are some examples).
There is still indication for aminoglycosides when there is the need to decrease very rapidly the bacterial
burden related to Gram-negative infections in clinically unstable patients with febrile neutropenia (who are
typically haematological patients with malignancies). When these patients are clinically unstable, it is
permitted (not recommended) to add an aminoglycoside to the backbone of therapy (represented by an
anti-pseudomonas beta-lactam) in order to obtain a more rapid decrease of the bacteria burden.
Used almost exclusively in the HOSPITAL setting to treat severe nosocomial infections mainly in association
with a beta-lactam:
- Severe infections (mainly bacteremia and / or endocarditis) due to:
• streptococci, enterococci (gentamicin + ampicillin)
• MSSA (gentamicin + oxacillin); MRSA (gentamicin + daptomycin + rifampicin)
• Enterobacterales (broad spectrum beta-lactams + aminoglycoside)
• Pseudomonas aeruginosa (amikacin + piperacillin or ceftazidime / cefepime or
meropenem / imipenem)
- Empirical treatment in clinically unstable patients with febrile neutropenia (aminoglycoside +
piperacillin or ceftazidime / cefepime or meropenem / imipenem)
2015 European Society of Cardiology: Guidelines for the management of infective endocarditis

18
Lecture 2 Pharmacology of antibiotics 19/10/2021
The professor gives us these schemes to look at the different antibiotic combinations according to the
different pathogens to understand how these combinations may be used.

Other indications of aminoglycosides

- Paromomycin can be used for topical action in intestinal infections.
“Topical” as it is administered by the oral route, and you obtain the activity directly in the gut.
- Kanamycin (a kind of evolution of Streptomycin) is the 2nd or 3rd choice in the treatment of
tuberculosis. The 1st choice is represented by Isoniazid, Rifampin, Pyrazinamide, and
Ethambutol. However, when we have Mycobacteria which are resistant to one or more of these 1st
line drugs, we can use Kanamycin.
- For very severe pneumonia infections in ICU, there is the opportunity of using Gentamycin (GM)
or Tobramycin (TBM) by high-pressure aerosol nebulizer in order to reach very high concentration
directly in alveolar moiety. Used in topical therapy of
• Ventilator-associated pneumonia (VAP)
• Pneumonia in the patient with cystic fibrosis

19
Lecture 2 Pharmacology of antibiotics 19/10/2021
FLUOROQUINOLONES (FQ)
Fluoroquinolones are another important class of antibiotics.
Mechanism of action of fluoroquinolones
They are able to block the DNA duplication system by interacting with DNA gyrase and topoisomerase IV.
• Inhibition of the DNA duplication system
• Substitution of DNA A by DNA gyrase (topoisomerase II) and/or by topoisomerase IV
Spectrum of action
Nowadays, differently from what occurred in the 1960’s and 1970’s when we had the 1st generation
fluoroquinolones (e.g. Nalidixic acid), we have the 2nd, 3rd and 4th generations, which are represented by
Ciprofloxacin, Levofloxacin, Moxifloxacin which can be used both in the community and in the hospitals.
However, nowadays, their use has been consistently reduced due to resistance and due to warnings from
European community in terms of risk of toxicity.

First, distinguish between Levofloxacin and Ciprofloxacin on one hand, and Levofloxacin and Moxifloxacin
on the other hand in terms of spectrum of activity:
- Ciprofloxacin, Levofloxacin: active against
• Gram negatives,
• Enterobacterales,
• P. aeruginosa (at least theoretically → nowadays, in our hospitals almost 40-50% of
Pseudomonas are resistant to FQ, but in other parts of the world Pseudomonas
maintain much higher susceptibility)
By looking at Gram positives, we have only two fluoroquinolones essentially:
- Moxifloxacin, Levofloxacin: active against
• Pneumococci, especially
➔ For this reason, they are also called respiratory fluoroquinolones
While Ciprofloxacin is not active against Gram positives.
• Another important aspect to keep in mind is that fluoroquinolones are active against
atypical pathogens responsible for Community-acquired pneumonia (CAP) (mainly
Mycoplasma, Legionella, and Chlamydia). So, these are powerful weapons as
monotherapy to counteract both intracellular and extracellular pathogens in CAP.
• Another possibility is to use them as anti-tubercular agents (against M. tuberculosis).

20
Lecture 2 Pharmacology of antibiotics 19/10/2021

➔ This is a double agent worth because if we use empirically fluoroquinolones, theoretically not
knowing if the patient has CAP or tuberculosis, we are probably counteracting both of these agents.
In other words, using a FQ in a patient with a pneumonia could represent an issue in the
identification of tuberculosis. So, it is better to make diagnosis before using a FQ.
Delafloxacin: new fluoroquinolone (introduced 2 years ago), found in our therapeutic armamentarium. It is
not so relevant nowadays. It is a peculiar FQ as it is acting against MRSA (methicillin resistant
staphylococcus aureus), differently from the other fluoroquinolones.
Once daily or maximum twice daily. Sometimes twice daily administration is needed because of tolerability
issue in order to avoid adverse effects.
Mechanism of resistance to fluoroquinolones
There are two main mechanisms:
- DNA mutations gyrase or topoisomerase IV
• Especially for Gram-negative
• Gene gyrA, gyrB (- frequent) for DNA gyrase
• ParC gene for topoisomerase IV (Gram positive)
- Efflux pumps (most relevant from the epidemiological point of view)
• Both Gram-positive and Gram-negative
• Genes NorA, PmrA (S. pneumoniae)
PK of fluoroquinolones
- Bactericidal
- Concentration-dependent: we have to
maintain a Cmax/MIC ratio > 10 (same seen with
aminoglycosides) to reach maximal activity. This
is why we use this drug once daily or maximum
twice daily. Twice daily may be needed because
of drug tolerability as with once daily
administration, we may reach too high
concentration leading to severe adverse events
(especially neurotoxicity).

Common PK features of fluoroquinolones (completely different from the ones of hydrophilic agents):
- Very liposoluble with high pKa → very high oral bioavailability, so we can administer orally the
same dosage given IV reaching the same level of exposure in the tissues.
- Good distribution in different tissues because they have low levels of protein binding (< 30-50%)
(Delafloxacin 84%).
- At the same time, they may penetrate in the plasmatic membrane → indeed, very high volume of
distribution (> 1 L/kg) → as it is > 1L/kg, the drug is able to accumulate very well within cells
(intracellular penetration), which act as reservoir from which the drug is released.
➔ Very good tissue penetration → this is why Levofloxacin and Moxifloxacin are used to treat
infections in deep tissues (like infections of bone and joint, prostate, meningitis due to
Pneumococci, bronchial secretions, ELF, CSF, aqueous humor).
- Linear PK
This is the summary of the different PK parameters (table below). This is only to show you that delafloxacin
has a different PK behaviour → LOWER volume of distribution compared to the other FQ.

21
Lecture 2 Pharmacology of antibiotics 19/10/2021

Warning on FQ oral bioavailability

From a clinical standpoint, we should consider that the
FQ excellent bioavailability can be importantly reduced
by the coadministration of agents containing positive
cations like aluminium/magnesium or calcium.
➔ When using FQ, the recommendation is to
AVOID the concomitant administration of
antacids and/or MILK (or dairies) as this may
affect the drug effectiveness.
Time out: 3-6 hours before or 2-3 hours after.
In case of renal insufficiency
Another difference between the different FQs is
related to the route of elimination. In fact, in case of renal insufficiency:
- Dosing adjustment for Levofloxacin
- NO dosing adjustments for Ciprofloxacin because it is also eliminated by the liver and by trans-
intestinal elimination. So, renal elimination is compensated by an increase in intestinal secretion
and liver biotransformation.
- NO dosing adjustments for Moxifloxacin because it is eliminated through the phase 2 processes in
the liver.
Adverse reactions of fluoroquinolones
Generally, well tolerated. However, it is important to keep in mind that very high level of FQ may be related
to neuropsychic disorders (sometimes also convulsions).
- Dose-dependent GI disorders (1.7-5%): nausea/vomiting, dyspepsia, gastralgia, abdominal pain,
diarrhea
- Hypertransaminasemia (2-4.5%)
- Neuropsychiatric disorders (0.5-1.5%): headache, excitement, drowsiness, visual disturbances,
dizziness, hallucinations, convulsions. Ciprofloxacin > levofloxacin.
Risk factors:
• Neurological disorders
• Renal failure
• Older age
• High doses
• Co-treatment with NSAIDs
These adverse events can be favoured in certain situations: for example, if you use Levofloxacin in
an old patient with renal failure, who is taking also NSAIDs, the probability of neurological events is
increasing even more than 10-fold.
- Photosensitization or phototoxicity (0.6-1.5%)
These drugs may photosensitize the patient. This means that when using FQs, the patient should
not expose himself to the sun because this may favour these adverse events.
Risk factors: sun exposure
- Myalgia and arthralgia, tendinitis (0.9%) - the most severe of those is the rupture of the Achilles
tendon.
We have to consider 2 important risk factors:
22
Lecture 2 Pharmacology of antibiotics 19/10/2021
▪ Coadministration of systemic corticosteroids may favour these events (increasing
their incidence of more than 40-fold).
▪ In elderly, these events can appear even after few (3-4) days of treatment.
- Cardiovascular (0.2-0.4): FQ may favour QT prolongation
→ Arrhythmias can be promoted when QT is longer than 500 ms.
Indeed, some adverse events may be prevented by:
1. Not exposing to the sun
2. Avoiding systemic corticosteroid
Image on the right: Quinolone-associated rupture
of the Achilles’ tendon (published some years ago
in New England journal of Medicine).
Evidence of tendinitis provoked by
fluoroquinolones treatment – a case control study
It was published 15 years age. According to this
study, the combination of FQ and corticosteroids
may increase the probability of developing these
side effects.
The combination of a recent use of FQ and
systemic corticosteroids may produce a 45-fold
higher probability of Achilles’ tendon rupture, compared to using the two drugs separated.

Indications of fluoroquinolones
Theoretically, the indications are very wide, but nowadays there are some restrictions in their use due to
the appearance of adverse events.
- Urinary tract infections (2nd-3rd generation)
→ FQs use has been consistently decreased. In the early 2000’s, FQ were widely used in community
to treat cystitis (very simple infections). Now this indication has been reduced because they may
favour the appearance of resistance and of side effects.
- They maintain their role in the treatment of acute and chronic prostatitis (3rd generation) thanks to
the excellent deep tissue penetration
- They have a role in bone and joint infections (MSSA and Gram-negative bone and joint infections)
(3rd generation) → especially, Levofloxacin is very powerful for these infections.
• For MSSA: Levofloxacin + Rifampicin
- Lower respiratory tract infections (3rd generation)
- Tuberculosis (3rd generation)
- Acute bacterial skin and soft tissue infections (3rd generation)
- Sexually transmitted infections (2nd-3rd generation): gonococcus R, soft venereal ulcer, chlamydia
trachomatis

23
Lecture 2 Pharmacology of antibiotics 19/10/2021
Dosing regimens of fluoroquinolones
According to the recommendations, these are the dosages
(table on the right).
You can read them after.
EMA recommendations on fluoroquinolones
According to EMA recommendations, 3 years ago (2018) there was a warning about the incidence of
adverse events (especially, neurotoxicity and the risk of tendon rupture).
➔ For this reason, EMA recommended to contain the use of fluoroquinolones only in very severe
situations.
The professor reads the following slide:

Contraindications of fluoroquinolones
Contraindications are related to the risks linked to FQ administration.
- Paediatric age, as this drug may affect the growth period of children (cystic fibrosis).
- Pregnancy and breastfeeding
- Neurological diseases

In the next lecture we will see the last class of antibiotics (macrolides) and we will look at antifungals.

24