Antibacterial drugs:

Classification, mechanism of
action and mechanisms of
resistance
Adapted and translated from Di Bonaventura (2020)
Antibacterial drugs: What we need to know
• What?
• A chemical structure, whether natural or synthetic product
• How do they act?
• By means of binding sites and mechanisms of action
• When should they be taken?
• Depending on their antimicrobial spectrum activity
• Clinical indications
• What are their limitations?
• Side effects on the patient
• Increase or appearance of bacterial resistance
• How can it be inactivated by the microorganisms?
• Mechanisms of bacterial resistance
• Clinical impact of antibiotic resistance
Chemotherapy: Historical highlights
• “Empirical” chemotherapy
• Cortex of cinchona vs. malaria (Indian Peruvians)
• Molds vs. wounds
• Mercury vs. syphilis (16th century)
• “Modern” (scientific) chemotherapy
• Paul Ehrlich (1854-1915)
• Concept of “selective toxicity”
• Arsphenamine (Salvarsan) vs. syphilis (only in vivo)
• Domagk (1935)
• Red colorant (Prontosil) vs. bacterial infections
• Fleming (1929)
• Penicillium notatum vs. staphylococci (penicillin)
Sir Alexander Fleming (1881-1955): “The
intuition” of the researcher
• Scientific creativity assumes different forms.
One of these is connected to serendipity,
which means unexpectedly being lucky (doing
an accidental discovery)
• The greatest discoveries can be taken from
intuition, or the capacity of being “prepared”
to grasp the exceptional meaning of an
apparently insignificant event.
• The scientific success of Alexander Fleming
resides in his openness, in his strong technical
intuition and his propensity towards
naturalistic observation.
• “I found myself in a situation wherein I could
leave my previous job in order to follow the
path that my destiny has shown me” (A.
Fleming)
1924: The discovery of lysozyme…
…the prelude to…
• In autumn of 1921, Fleming, who was
down with a bad cold and moved by
curiosity, cultured his own nasal
secretions on an agarized medium. After
some weeks, he noticed the presence of
signs of microbial inhibition
corresponding to the mucus culture.
• Fleming has de facto discovered
lysozyme, an enzyme present in many
secretions (mucus, tears, saliva) and has
antibacterial activity. Although lysozyme
does not result to being active against the
primary pathogenic bacteria, this
“observation” alerted Fleming to the
potential of natural antibiotics.
 …discovery of penicillin (1928)
• In 1928, upon observing some Petri dishes that he
distractingly forgot on the lab bench during the vacation,
Fleming observed that the bacterial growth was inhibited
near a contaminant mold (Penicillium notatum).
• Fleming hypothesized that the mold produced a
substance, which he called “penicillin,” which was able to
stop the bacterial growth.
• He experimentally verified the efficacy of penicillin with
respect to numerous pathogenic bacteria in humans.
• It is only in 1939 that penicillin was produced in significant
quantities and in a stable form by Howard Florey and Ernst
Chain.
In 1945, Sir Alexander Fleming, Sir Howard Florey
and Sir Ernst Chain received the Nobel Prize for
Physiology and Medicine
Antibacterial drugs
• Antibacterial agents: A compound able to interfere with bacterial
growth and multiplication; unlike disinfectants, it is only active against
bacteria that are metabolically active (exception: polymyxins)
• Antibiotic – a substance of low molecular weight that is produced from the
metabolism of a microorganism: Actinomycetes (Streptomyces), mycetes
(Penicillium, Cephalosporium):
• Mechanism of “self-conservation:” In the presence of other species or in increased cell
density, many microorganisms produce substances (bactericides, microbicides) in order
to compete for space and nutritive substrates
• Characteristic of many telluric microorganisms
• “Semi-synthetic” antibiotics are derived from the structural modification of natural
antibiotics, which are improved for their antibacterial and/or pharmacological
characteristics
• Chemotherapeutic – synthetic product (structural analog of an antibiotic)
Antibacterial drugs: “Ideal” characteristics for
an antimicrobial [agent]
• Adequate antimicrobial activity to prevent the development of “resistant”
variants:
• Biocide activity
• Narrow spectrum of action
• Active at low concentrations (low selective pressure)
• Adequate pharmacological properties:
• Long half-life in the blood plasma
• Reaches adequate tissue concentrations
• Low bonds with plasma proteins
• Possibility of oral or parenteral formulation
• No interactions with other drugs
• Should not be destroyed, neutralized or excreted until it is able to carry out its proper action
• Stable, economical and easy to administer
• Selective toxicity:
• Activity that is aimed against the bacterium and not against the host cell, due
to the structural/functional differences that exist between the pathogen and
host:
• The “target site” of the drug is only present in the microbial cell (ex. Cell wall)
• High capacity of drug penetration in the prokaryotic cells (ex. Tetracycline)
• Different affinity of the drug by means of “targets” that are functionally, but not
structurally similar (ex. Antibiotics that inhibit the protein synthesis on account of the
structural difference of the prokaryotic ribosome)
• The drug does not interact with the host defense mechanisms and with the commensal
flora
• Not toxic for the host:
• Favorable therapeutic index
Antibacterial drugs: Classification
• Nature of inhibition:
• Bactericide (β-lactams, aminoglycosides, antifolates, quinolones). Brings about bacterial
death; indicated in the treatment of severe infections and of the immunocompromised host.
• Bacteriostatic (tetracyclines, macrolides, chloramphenicol). Inhibit the growth of the
bacterium. In the “quiescent” phase, the bacteria is easily attacked by cell-mediated
immunity; for this, it is indicated in an immunocompetent host that by default does not need
a bactericidal action.
• Typology of microorganism (number of bacterial species sensitive to the
antibiotic spectrum):
• Narrow spectrum (It. Spettro ristretto – restricted/limited spectrum) – active vs. a limited
number of species (ex. vs. Gram+, like penicillin); preferrable because they have a small
impact on the commensal flora and cause mild toxic effects
• Broad spectrum – active vs. numerous species, generally whether Gram+ or Gram-
(tetracyclines, cephalosporins); generally associated with an important toxicity profile
Mechanism of action:
• Inhibits cell wall synthesis
• Inhibits cell membrane functions
• Inhibits protein synthesis (30S,
50S, tRNA)
• Inhibits nucleic acid synthesis
• Inhibits metabolic enzymes
Inhibitors of cell wall synthesis:
Glycopeptides, β-lactams
Cytoplasm:
synthesis of the cell
wall precursors

Cycloserine:
Inhibits the
incorporation of alanine
in the formation of the
cell wall precursor

Cytoplasmic membrane:
Synthesis of new cell wall Bacitracin:
subunits bound to a lipid Inhibits the
carrier dephosphorylation of
the lipid carrier, so the
Glycopeptides: necessary carrier will
They bind to the D-ala- not be regenerated until
D-ala residues so that the synthesis of cell wall
the subunits will not be continues
incorporated to the
growing peptidoglycan

Cell wall:
Insertion of the new cell
wall units to the growing
peptidoglycan chain

Beta-lactams:
They bind and stop the
enzymes that catalyze
the formation of this
bond
Inhibitors of cell wall synthesis: Glycopeptides
• Has a glycopeptidic structure
• Natural:
• Vancomycin: produced from Streptomyces orientalis
• Teicoplanine: produced from Actinoplanes teichomyceticus
• Mechanism of action:
• Inhibits the elongation (polymerization) of peptidoglycan
• The antibiotic binds to the terminal group D-ala-D-ala of each pentapeptide; in this way, the action of the
enzyme transglycosylase is inhibited. Transglycosylase is responsible for binding the different units of
disaccharide-pentapeptide.
• Narrow spectrum:
• Gram+ cocci (streptococci, staphylococci met-R), bacilli (Listeria, Corynebacterium), anaerobes (Clostridium)
• Gram- are naturally resistant because the glycopeptides are unable to cross the external membrane
• Toxicity:
• Local vein intolerance (phlebitis and thrombophlebitis), allergic reactions (itchiness, orticaries)
• Ototoxicity, nephrotoxicity
Inhibitors of cell wall synthesis: β-lactams
• They represent the most numerous antibiotic class and are most
commonly used.
• These are formed from different bactericide molecules that have a β-
lactam ring:
• Penicillins
• Cephalosporins
• Other β-lactams:
• Clavulanic acid, thienamycin, sulbactam
• Moxalactam
• Monobactams (aztreonam)
• Carbapenems
 Beta-lactam ring

The beta-lactam class of antibiotics

Penicillins: Cephamycine
ex. benzylpenicillin, cloxacillin, ex. cefoxitin
flucloxacillin, ampicillin,
amoxicillin, carbenicillin,
ticarcillin, azlocillin, mezlocillin,
piperacillin

Carbapenems
ex. imipenem

Cephalosporins:
ex. cephalexin, cefaclor,
cefadroxyl, cefuroxime,
cefamandole, celotaxime,
ceftazidime, cefepime, cefpirome
Monobactams
ex. aztreonam
Inhibitors of cell wall synthesis: β-lactams -
penicillin
• It is produced from Penicillium chrysogenum (antibiotic)
• Base nucleus: 6-amino-penicillinanic acid
• Natural (extractives):
• Benzyl-penicillin (penicillin G), phenoxymethyl-penicillin (Penicillin V)
• Active vs. cocci (Gram+ and Gram-)
• Sensitive to β-lactams
• Semi-synthetic (addition of lateral chains to the base nucleus;
spectrum and action intensity greater as against penicillin G):
• Methicillin + isoxazolyl-penicillins (oxa-, cloxa-, dicloxa-, flucoxa-)
• Resistant to β-lactamase
• Aminopenicillins (ampi-, amoxi-)
• Carboxypenicillins (carbeni-, ticar-)
• Ureidopenicillins (pipera-, azlo-, mezlo-)
• Sulfoxyl penicillins (sulbeni-, sun-)
Inhibitors of cell wall synthesis: β-lactams -
cephalosporins
• Produced from Cephalosporium achremonium (antibiotics)
• Base nucleus: 7-amino-cephalosporanic acid
• Natural: Activity

• Cephalosporin C Gram(-)
Resistance to β-lactamases
Staphylococci

• Semi-synthetic:
• 1st generation* (cephalothin, cephazolin)
• 2nd generation (cefoxitin, cefuroxime)
• 3rd generation (cefotaxime, cefoperazone)
• 4th generation (cefepime, cefodizime)
• Cephamycin (cefuroxine, cefamandole)
• Natural (Streptomyces)
• Structure similar to cephalosporins (methoxyl group in C7)
• Extended spectrum of action (Gram- and in particular, anaerobic bacteria)
Inhibitors of cell wall synthesis: β-lactams –
Mechanism of action
• Main target: bacterial cell wall
• Characteristic target of the microbial cell (selective toxicity)
• Inhibits the final stage of polymerization in peptidoglycan
synthesis (transpeptidization reaction), “selectively”
binding itself to the involved enzymes (Penicillin Binding
Proteins – PBPs: carboxylpeptidase, transglycosylase,
transpeptidase)
• Bactericide activity on cells dividing actively:
• Accumulation of wall precursors cause the activation of
autolytic mechanisms (murein-hydrolase)
• Production of “lasso” peptidoglycan
• Arrest of bacterial growth
• Spheroplast formation
• Cell lysis
Inhibitors of cell wall synthesis: β-lactams –
Action spectrum
• Penicillins:
• Active vs Gram+ (aerobes and anaerobes):
• S. aureus, S. pyogenes (streptococcal angina, bactremia, septicemia, pharyngitis, otitis media)
• S. pneumoniae (meningitis, pneumonia)
• Active vs some Gram-:
• Proteus, E. coli (urinary infections)
• Salmonella, Shigella (gastroenteritis)
• Cephalosporins:
• Also have an extended spectrum against Gram- due to the resistance to β-lactamases
(active vs penicillin) and a greater penetration through the external membrane:
• Haemophilius influenzae type B, Streptococcus pneumoniae, Neisseria meningitidis
(meningitis)
Inhibitors of cell wall synthesis: other β-
lactams
• Inhibitors of β-lactamases
• Structural derivatives of 6-amino-penicillanic acid (by way of modification of
the thiazolidine ring)
• Clavulanic acid (Streptomyces clavuligerus), sulbactam, tazobactam
• Have poor antibacterial activity, carry out protective action against the β-
lactamases by functioning as “suicidal” inhibitors: used in combination of a
penicillin (amoxicillin/clavulanic acid, ampicillin/sulbactam,
piperacillin/tazobactam) and bind themselves covalently to the β-lactamases
by irreversibly blocking their activity in order to protect the penicillin.
• Moxalactam
• Structural derivative of 7-amino-cephalosporanic acid (modification of the
dihydrothiazolidine ring)
• Extended spectrum of action, peaked resistance against β-lactamases
• Monobactams (aztreonam)
• Chromobacterium violaceum
• Base nucleus is a monocyclic β-lactam (absence of heterocyclic ring)
• Relevant activity vs. Gram-negatives (excluding anaerobes), peaked resistance
against β-lactamases
Inhibitors of cell wall synthesis: β-lactam -
toxicity
• Penicillins:
• Immediate hypersensibility reaction; penicillin allergy is frequently “crossed”
with cephalosporins.
• Anaphylactic shock
• Idiopathic reaction (ampicillin): cutaneous rash, 25% in ongoing treatment
• Neurotoxicity (benzylpenicillin in doses > 40-50 million U, in patients with
renal failure/insufficiency)
• Cephalosporins:
• Local intolerance (cephalosporins – parenteral 1st generation)
• Allergic reactions
• Hematic reaction (thrombocytosis, eosinophilia)
• Gastroenteric disturbances (modest diarrhea)
Inhibitors of normal cell wall function –
polymyxins
• Antibiotics (Bacillus polymyxa)
• Polypeptides with cyclic structure
• Polymyxins A and B (colistin) are the most commonly used.
• Mechanisms of bactericide action:
• Acts as a cationic detergent that destroys the phospholipid structure of the
membrane Polymyxin B
• Narrow spectrum of action:
• Gram- (exception: Proteus spp)
• Cutaneous and mucous infection (wound, ear, eye); bactremia, UTI,
meningitis
• Urinary bladder disinfectant
• Elevated neurotoxicity and nephrotoxicity are associated with
the intravenous (IV) administration (“second-choice”
antibiotics)
• Mainly used topically (pomade/cream, ointment); used also
through os (mouth) (colistin is used for intestinal
decontamination in neutropenic patients)
Colistin
Antibacterial drugs: Classification –
Mechanism of action
DNA synthesis inhibitors: Quinolones
• Antibacterial agents of synthetic origin (chemotherapeutics)
• Classified in generations on the basis of their action spectrum:
• 1st generation: nalidixic acid
• 2nd generation: ciprofloxacin, ofloxacin, norfloxacin
• 3rd generation: levofloxacin, moxifloxacin, gatifloxacin, sparfloxacin
• 4th generation: trovafloxacin
• Mechanism of bactericidal action
• Bind the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV so that the DNA will not
unravel itself so that its replication will not proceed
• Broad spectrum
• Enterobacteria
• Gram+ and Gram- cocci
• Toxicity
• Gastroenteric disturbances
• Neuropsychic disturbances (cephalea, vertigo, convulsions in elderly patients)
• Growing cartilage (not advised during gestation and breastfeeding)
 Nalidixic acid Ciprofloxacin
Topoisomerase IV DNA gyrase

Replication direction

Moxifloxacin Trovafloxacin
 Characteristics of highly represented quinolones (Di Bonaventura, 2020)

Drug Category* General activity spectrum

Nalidixic acid First generation Gram-negative bacteria (except for Pseudomonas)

Ciprofloxacin Second generation Same coverage as the first generation, including Pseudomonas
Enoxacin spp. and some Gram-positives (Staphylococcus aureus but not
Lomefloxacin Streptococcus pneumoniae)
Norfloxacin
Ofloxacin
Levofloxacin Third generation Same coverage as the second generation, but with an increased
Gatifloxacin activity with respect to Gram-positives (Penicillin-sensitive and
Moxifloxacin resistant Streptococcus pneumoniae); moreover, some are active
Sparfloxacin against anaerobes
Trovafloxacin Fourth generation** Same coverage as the third generation, but with activity with
respect to anaerobes
• All except the first generation compounds are fluoroquinolones (*)
• Associated with cases of grave hepatic compensation; its use is reserved to terminal clinical situations (**)
DNA synthesis inhibitors: Novobiocin
• Antibiotics (Streptomyces spheroides, S.
niveus)
• Mechanisms of bactericidal action
• Bind the GyrB subunit of DNA gyrase
(topoisomerase II), preventing the de-
spiraling of DNA, and hence, its synthesis
• Action spectrum – synergic action with
that of the quinolones (combination
therapy)
• Selective toxicity – different enzymatic
structures between prokaryotes and
eukaryotes
Antibacterial drugs: Classification –
Mechanism of action
RNA synthesis inhibitors: Rifamycin
• Antibiotics (Nocardia mediterranea)
• Rifamycin (mostly used in clinical practice), rifabutin, rifapentine
• Mechanism of bactericide action
• Blocks the initial stage of transcription by means of the β subunit of the RNA-
polymerase
• Action spectrum
• Tubercular mycobacteria and atypicals
• Gram+ cocci (prosthetic endocarditis caused by streptococci, S. aureus)
• N. meningitidis (prophylaxis of meningitis) and H. influenzae
• Selective toxicity
• Gastroenteric disturbances
• Hepatotoxicity (elevated doses)
Antibacterial drugs: Classification –
Mechanism of action
Inhibitors of folic acid synthesis: Sulfonamides
• Synthetic antibacterial agents (chemotherapeutics). Structural
analogs of para-aminobenzoic acid (PABA).
• Sulfamethoxazole, dapsone, para-aminosalicilic acid
• Mechanism of bacteriostatic action: competes with PABA for the
active site of dihydropteroate synthase, the enzyme involved in the
biosynthesis of tetrahydrofolic acid (THFA), which is necessary for the
synthesis of purines and pyrimidines, and therefore, the synthesis of
nucleic acids
• Selective toxicity: many bacteria synthesize THFA, while the human
cells are unable to synthesize it – they utilize exogenous folic acid
(from the diet)
• Action spectrum – extensive, Gram+ but above all Gram- (except
Pseudomonas spp), cause of urinary infection. Dapsone
(Mycobacterium leprae), para-aminosalicylic acid (M. tuberculosis).
• The drug is administered orally, often combined with trimethoprim
(as co-trimoxazole, so that resistant strains will not appear), it is
metabolized in the liver and excreted through the kidneys
• Toxicity: evidence of side effects, may cause erythema and repression
of the bone marrow (Stevens-Johnson syndrome)
 Para-aminobenzoic acid (PABA)
+ pteridine
Sulfonamide ring Para-aminobenzoic
structure acid (PABA)
Dihydropteroate
Sulfonamides
synthase

Dihydropteroic
acid

Dihydrofolate
Sulfadiazine Sulfamethoxazole synthase

L-glutamate

Dihydrofolic
acid

Dihydrofolate
Trimethoprim
reductase

2 NADPH

Dapsone Para-aminosalicylic
acid (PAS)
2 NADP

Tetrahydrofolic acid
(THFA)

Purine Pyrimidine
Inhibitors of folic acid synthesis: Trimethoprim
• Chemotherapeutic (synthetic), belongs to the pyrimidine-simile
groups. It is the structural analog of the aminohydroxypyrimidinic
group of folic acid.
• Mechanism of bacteriostatic action: Competes with folic acid for the
active site of the dihydrofolate reductase, the enzyme involved in the
biosynthesis of tetrahydrofolic acid (THFA), indispensable for the
synthesis of purines and pyrimidines, hence for the synthesis of
nucleic acid.
• Selective toxicity: Even though dihydrofolate reductase is present in
both host and bacterial cells, trimethoprim has a greater affinity for
the bacterial enzyme.
• Action spectrum: Gram- (except Pseudomonas spp). In combination with
sulfamethoxazole, it is active vs urinary pathogens and Salmonella typhi,
pneumonia (Pneumocystis jiroveci), nocardiosis, soft ulcers.
• It is administered orally or intravenously, often in combination with
sulfamethoxazole. It is metabolized in the liver and excreted by means of the
kidneys. In patients with kidney failure, it is excreted more rapidly than the
sulfonamides, which may lose the synergic effect in some way.
• Toxicity: When it is administered alone or in combination with
sulfamethoxazole, it can trigger neutropenia, nausea and vomiting especially
among AIDS patients.
Folic acid
Inhibitors of folic acid synthesis:
Sulfamethoxazole + Trimethoprim
• Sulfamethoxazole and trimethoprim are often administered in
association with each other, as co-trimosaxole.
• The advantage of a combined therapy with respect to a single drug is
due to the following:
• Simultaneous resistance to both molecules is improbable
• The two drugs act in a synergic way, that is, the resulting action of the
combination drugs is greater with respect to the sums of the effects that are
derived from the use of a single molecule. In this way, it is possible to use
smaller concentrations of each molecule to obtain the same therapeutic
effect, limiting/preventing in this way the rise of resistant strains.
Antibacterial drugs: Classification –
Mechanism of action
Inhibitors of protein synthesis: Selective
toxicity
• Even though protein synthesis occurs in a similar way in both
prokaryotes and eukaryotes, the selective toxicity of these drugs are
based on the existence of some significant structural differences that
exist in the prokaryotic and eukaryotic ribosomes.
Inhibitors of protein synthesis:
Mechanism of action

• Tetracycline (Tet)
• Streptomycin (Stp)
• Glycylcycline (tigecycline (Tig))
• Chloramphenicol (Cam)
• Lincosamide (clindamycin (Cln))
• Oxazolidinones (linezolid (Lnz)
• Streptogramin A (SA)
• Streptogramin B (SB)
• Aminoglycosides hygromycin B
(HygB), neomycin (Neo),
paromomycin (Par)
• Macrolides (erythromycin (Ery))
• Ketolides (telithromycin (Tel))
Inhibitors of protein synthesis:
Aminoglycosides
• Amino sugars bound by way of glycosidic link to an aminocyclitol nucleus
• Natural
• Streptomycin
• Neomycin
• Kanamycin
• Gentamycin
• Tobramycin
• Sisomycin
• Semi-synthetic
• Amikacin
• Netilmycin
Inhibitors of protein synthesis:
Aminoglycosides
• Mechanism of “multi-step” bactericide action: The aminoglycosides
cross the membrane by means of an oxygen-dependent process,
hence binding the 30S ribosome subunit (S12 protein):
• It inhibits the formation of the initiation complex (inhibits the action of the
fmet-tRNA, which modifies the attachment site of the ribosome), which is
necessary for the protein synthesis in prokaryotes
• Inhibits the translocation from site A to site P, blocking the formation of the
peptide chain in neo-synthesis
• Induces reading errors in the mRNA codons: “anomalous” protein formation,
which, inserted in the membrane, form pores that can increase antibiotic
entrance to the cell interior. Paradox effect: streptomycin dependence
Inhibitors of protein synthesis:
Aminoglycosides
• Extended action spectrum:
• Gram- (grave infections, sepsis, pulmonitis, including P. aeruginosa and Acinetobacter)
• Gram+ (when combined with β-lactams): streptococci, staphylococci
• Inactive vs. anaerobes
• Mycobacteria (streptomycin, amikacin, kanamycin)
• Prophylaxis/therapy for abdominal surgical infections
• Generally administered by means of endovenous therapy:
• Not absorbed in the intestinal level, does not penetrate the soft tissues and does not go
beyond the blood-brain barrier; excreted renally
• Toxicity:
• Therapeutic index (Ctox/Cter) is quite “reduced”
• Potentially nephrotoxic and ototoxic (loss of the ciliated cells and permanent damage to the
organ of Corti)
• Necessity of monitoring the hematic levels, especially in cases of renal failure
Inhibitors of protein synthesis: Tetracycline
• Tetracycline: antibiotic (Streptomyces spp)
• Possesses a large tetracyclic structure, with many sites that can be
substituted chemically:
• Semi-synthetic tetracyclines
• Oxytetracycline
• Chlortetracycline
• Doxycycline
• Demeclocycline
• Mechanism of bacteriostatic action:
• Penetrates the cells with Mg2+ complex, bind the ribosomal subunit 30S,
thereby impeding the access of the aminoacyl-tRNA to the receptor site A of
the ribosome. It then blocks the protein synthesis.
Inhibitors of protein synthesis: Tetracycline
• Extended action spectrum (the term “broad spectrum” was first coined with
regards to the tetracyclines):
• Intracellular pathogens (genito-urinal infections caused by mycoplasms, Chlamydia, rickettisiae)
• Gram+ cocci (acne) and Gram- (Neisseria)
• Gram- (airway infection)
• Spirochetes (syphilis)
• Some protozoans
• Selective toxicity:
• High penetration in the bacterial cell (vs. eukaryotic cell)
• Can inhibit the eukaryotic protein synthesis, but it rarely reaches toxic concentrations
• Toxicity:
• Superinfections (S. aureus, Candida), following the death of the commensal flora (particularly intestinal)
• Dental alterations (coloration of teeth during development) and bones (delay in bone development)
• Hepatotoxicity
• Cutaneous alterations: photosensitivity
Inhibitors of protein
synthesis: Macrolides New macrolides In vitro activity vs.
erythromycin
Human
pharmacokinetics
Antibiotics (Streptomyces spp)
Large cyclic molecules (14, 15 or 16 atoms) Roxithromycin (14-atom Comparable High blood
that contain a macrocyclic lactonic nucleus ring) concentration peaks
with ketone function bound to different
sugars: (half life = 12 h)
Erythromycin Azithromycin (15-atom Enhanced action High concentration
Roxithromycin ring) against Gram- in tissue,
Azithromycin bacteria administered once a
Clarithromycin day
Clanthromycin (14-atom Enhanced action The blood
ring) against Gram+ and concentration peak
erythromycin Legionella spp is higher than
erythromycin

14-atom ring
• Mechanism of bacteriostatic action: It binds to the 23S rRNA
found within the 50S ribosomal subunit. In this way:
• The peptide bond formation between the amino acids at the A and P
sites is inhibited
• Translocation is blocked, by way of stopping the release of
“unloaded” tRNA, following the formation of the peptide link,
thereby blocking protein synthesis
• Extended action spectrum:
• Gram+ and Gram- cocci
• Erythromycin: alternative to penicillin vs streptococci (in allergic
subjects)
• Gram- - Legionella pneumophila, Campylobacter jejuni, Yersinia,
Bordetella pertussis
• Intracellular pathogens: mycoplasms, Chlamydia (atypical
pneumonia, genital infections), rickettsia
• Toxicity:
• Hepatotoxicity (high doses)
• Gastroenteric disturbances (vomiting, nausea, hepatitis):
erythromycin – oral route
 Inhibitors of protein synthesis:
Chloramphenicol
• Antibiotic (Streptomyces venezuelae), although is
actually obtained through synthesis
• Simple structure: one nitrobenzene nucleus
• Other derivatives are synthesized, although they are
seldom used in therapy
• Mechanism of bacteriostatic action:
• Binds the 50S subunit, thereby blocking the peptidyl
transferase enzyme, thereby impeding the formation of the
carbo-amide bond
• It can also recognize eukaryotic ribosomes (70S
mitochondrial subunit), causing dose-dependent medullar
toxicity
• Extended action spectrum:
• Gram+ and Gram- cocci (Neisseria); Gram-, enteritogens in
particular (Salmonella, Shigella, Brucella, Vibrio)
• Bacterial meningitis, cerebral abscess (H. influenzae, S.
pneumoniae), ocular infections
• Gram+ and Gram- anaerobes
• Rickettsia, Chlamydia, Mycoplasma
• Oral administration, IV and also topical, is well-absorbed
(especially in the blood-brain barrier level). Metabolized in
the liver, excreted through urinary tract
• Relevant toxicity:
• Dose-dependent suppression of the bone marrow (structural
analog of nitrobenzene), in cases of long-term treatment; reversible
• Aplastic anemia; irreversible, but very rare (1 case/30,000 patients)
• Toxic in newborns, above all among those premature (lack of
hepatic enzymes, “gray syndrome”)
 Inhibitors of protein synthesis: Lincosamides
• Lincomycin (Streptomyces lincolnensis)
• Clindamycin (chlorinated derivative of lincomycin) is the most
important drug of this class.
• Mechanism of bacteriostatic action:
• It binds with the 50S subunit, thereby impeding the formation of the
peptide bond in sites A and P
• Intermediate action spectrum (Clindamycin):
• Anaerobes, Gram+ (Clostridium spp) and Gram- (Bacteroides spp) Lincomycin
• Gram+ (osteomyelitis caused by S. aureus, in particular)
• Inactive vs Gram- aerobes (low penetration of the external membrane)
and Clostridium difficile (first evidence of pseudomembranous colitis
following therapy)
• Administration by means of oral route, intramuscular, intravenous –
clindamycin penetrates the bone tissue, polymorphonucleated
leukocytes and macrophages. It is metabolized by the liver, and its
derivatives are excreted with the feces.

Clindamycin
Inhibitors of protein synthesis:
Streptogramins
• Antibiotics (Streptomyces)
• Classic formulation: Streptogramin A (dalfopristin) +
streptogramin B (quinopristin)
• Mechanism of action: Quinopristin
• They bind to the 50S subunit, inhibiting the elongation of the (streptogramin B)

peptide chain at different levels (synergic interaction):

streptogramin A (inhibition of peptide bond), streptogramin B
(chain elongation)
• Bacteriostatic (as single unit), streptogramin A and B are
bactericides that work together
• Narrow action spectrum:
• Gram+ cocci
• Enterococcus faecium, but not E. faecalis
Dalfopristin
• Active vs. multi-antibiotic resistant strains (VRSA, vancomycin- (streptogramin A)
resistant S. aureus; VRE, vancomycin-resistant Enterococcus)
Inhibitors of protein synthesis: Oxazolidinones
• Chemotherapeutics (synthetic) – Linezolid
• Mechanism of bacteriostatic action:
• Binds to 50S subunit to inhibit the formation of the initiation
complex
• Narrow action spectrum:
• Gram+ (Staphylococcus, Enterococcus, Listeria
monocytogenes, Clostridium difficile)
• Active vs. multi-antibiotic resistant strains (VRSA,
vancomycin-resistant S. aureus; MRSA, methicillin-resistant
S. aureus)
• Mycobacterium tuberculosis
• Toxicity:
• Gastroenteric disturbances (anorexia, nausea, vomiting)
Anti-tuberculosis drugs: Isoniazid,
ethambutol, pyrazinamide
• The therapy for infections caused by M. tuberculosis is made complicated
by a series of structural, functional and pathogenic characteristics:
• Structural and chemical complexity of the cell wall that opposes the entry of the
antibiotics
• Bacteria are localized intracellularly (difficult for antibiotics to enter)
• Slow growth (necessity for a long-term therapy up to 9 months, low compliance and
high probability of developing drug resistance)
• TB is a secondary infection to the AIDS epidemic (difficulty of rolling out the therapy
in developing countries)
• The anti-tubercular drugs that are in actual use are: isoniazid, ethambutol,
rifampicin, pyrazinamide and streptomycin.
• They are generally used in combination [with other drugs] to prevent drug
resistance.
• Isoniazid
• Hydrazide of nicotinic acid
• Bactericide: inhibits mycolic acid synthesis
• Neurological complications
• Ethambutol
• Synthetic
• Bacteriostatic: inhibits the polymerization of
arabinoglycan in the cell wall
• Optical neuritis
• Pyrazinamide
• Nicotinamide analog
• Mechanism is not yet clear: it is converted to
pyrazinoic acid that acidifies the intracellular
environment with lethal effects; inhibits the
synthesis of mycolic acids
• Favors the development of resistance: combined use
with other molecules
• Hepatotoxic
Antimicrobial drug resistance: Definition
• Antibiotic-resistant microorganism –
microorganism that cannot be inhibited in
its growth or cannot be killed by attainable
pharmacological concentrations of the
molecule on the site of infection following
the administration of a “usual” therapeutic
dose.
• (Acquired) antibiotic resistance indicates a
partial or a total loss of activity on the
part of the antibiotic vs. the formerly
sensitive microorganism.
• It predicts the probable failure of the
antibiotic therapy.
Antimicrobial drug resistance: Types
• Types of resistance:
• Natural (intrinsic): the refraction on the action of the molecule depends on the lack
of the biological target or the presence of peculiar cell structures that interfere with
the mechanism of action of the molecule
• A mechanism is evolved on the species level: intrinsic (innate), which is immutable over time
and it is manifested in ALL strains of the SAME species
• Gram- (E.coli) vs. glycopeptides and penicillin (incapacity to cross the external membrane)
• Chlamydia and mycoplasms vs. β-lactams (absence of cell wall)
• As an expected, notable characteristic, natural resistance does not place particular
problems on the clinician in choosing the antibiotic therapy.
• Acquired: it is made of resistant strains that are normally sensitive to a particular
antibacterial drug:
• Chromosomal – spontaneous mutation
• Extra-chromosomal – acquisition of genetic determinants by of plasmid or transposon
location
• Acquired resistance can be the cause of failed therapies whenever an antibiotic
therapy is begun without doing an antibiotic sensitivity test (in vitro) of the pathogen
against the drugs (Kirby-Bauer test)
“Acquired” resistance to antimicrobial drugs:
Basis of genetic resistance
• Chromosomal resistance
• Caused by spontaneous mutations: very rare (1 event for every
10^6-10^8 cells), causes 10-15% of the resistance Chromosomal mutation:
• Resistance can be caused by a mechanism: Mutant selection
Antibiotic therapy
• “Single-step” – one mutation can cause the altered synthesis of a
protein, or the substitution of an amino acid (dihydropteroate
synthase)
• “Multi-step” – necessity of more mutations to modify the protein
(PBP – penicillin binding proteins)
• The presence of the antibiotic creates a selective action (selects
the resistant mutants, thereby inhibiting the sensitive cells)
• Resistance concerns only the antibiotic against which the
resistant mutants are isolated
• The same mutants can also be resistant to other antibiotics with
similar characteristics (crossed resistance or cross-resistance)
• It is of vertical transmission (from mother to daughter cell)
• Extra-chromosomal resistance
• Elevated frequency of propagation, responsible for most resistances (around
90%)
• It begins from acquiring new genetic information coming from other
microorganisms, by means of the mechanisms of genetic transfer
(conjugation, transformation, transduction)
• It is caused by localized genes on mobile extra-chromosomal elements
(plasmids, transposable elements)
• The resistance concerns more antibiotics (multiple resistance)
• The mobile elements can contain different resistance determinants (or different genes
that code for resistance to antibiotics from different classes) that are organized to form
an integron (or “cassette”)
• Of a horizontal transmission – diffusion among cells that belong to the same
species or among different species (“contagious” resistance)
Plasmid resistance:
Diffusion of resistant plasmid Resistant “cassette A” “Cassettes” of
genetic
resistance
donor donor

Integrons

recipient Transconjugant Resistant “cassette B”

Transposons

Plasmid resistance transferred through transposon: Chromosome or plasmid

diffusion of resistant gene
Resistant “cassette C”
“Acquired” resistance to antimicrobial drugs:
“Contagious” resistance
Antimicrobial drug resistance: Resistance
mechanisms
• The mechanisms responsible for antibiotic resistance can be traced to the
following types:
• Drug inactivation by enzymes, whose metabolites are biologically inactive:
• β-lactamases (β-lactams)
• Transferases (aminoglycosides, chloramphenicol)
• Modification of drug target and the subsequent lack of recognition on the
part of the drug
• Penicillin-binding proteins (penicillins, cephalosporins)
• DNA-gyrase subunit (quinolones, novobiocines)
• β-subunit of RNA polymerase (macrolides, lincosamides)
• DPS (sulfamides), DHFR (trimethoprim)
• Alteration of the cellular permeability for
the drug and its [intracellular] reduction:
• Reduction of cellular permeability for the
drug (Pseudomonas vs aminoglycosides)
• Increased efflux of the drug (tetracycline)
• Inactivation of the enzyme that activates
the “pro-drug” and insufficient “drug”
activation
• katG mutation (isoniazide)
• Antibiotic resistance can be caused by
one or more mechanisms of
resistance that can coexist and
interact in a synergic way.
• Particularly in the case of antibiotics
that present several resistance
mechanisms, the antibacterial
activity will be the reverse function
of the utilization of the molecule in
the therapy.
• In these cases, the possibility of
developments of resistance will be
greater in the context of a selective
pressure (antibiotic therapy).
Resistance mechanisms: Drug inactivation by
enzymes
• Production of β-lactamase
• β-lactamase – enzyme that hydrolyzes the amide bond of the β-lactam ring that causes its opening with the
formation of biologically inactive metabolites
• Typology: described in hundreds of enzymatic variants, on the basis of their different affinity
towards the various β-lactams
• Penicillinase – direct vs penicillin (6-aminopenicillanic acid – penicillanic acid)
• Cephalosporinase – direct vs cephalosporin (7-aminocephalosporanic acid – aminocephalosporanic acid)
• Broad spectrum – metal-β-lactamase, ESBL (Extended Spectrum Beta-Lactamases)
• Localization:
• Gram+ - inducible (in the presence of the antibiotic), exocellular (liberated in the extracellular environment)
• Gram- - constitutive (independent from the presence of the antibiotic), intracellular (periplasmic space)
• Control:
• Chromosomal (cephalosporinase in Gram-)
• Plasmidic (S. aureus, P. aeruginosa, H. influenzae)
• Transposon (Tn2-Tn3, Enterobacteriaceae/Pseudomonas)
 β-lactamase

Hydrolyzed β-lactam
Active β-lactam
Localization of the β-lactamases Classification of the β-lactamases
• Resistance to aminoglycosides
• The most important acquired resistance to aminoglycosides is represented by
the production of enzymes (transferase) that modifies the molecular
structure.
• In this way, the molecule is no longer able to interact with the ribosome (30S
subunit)
• The genes that codify these enzymes are on the plasmid or on transposon
elements.
• The typology of the enzymes possessed by the microorganism determines the
spectrum of resistance (extended or narrow)
• Resistance to chloramphenicol
• Inactivation by means of the addition of an acetyl group by means of the
acetyl transferases (chloramphenicol acetyl transferases, CAT), coded at the
plasmid or transposon.
chloramphenicol

Chloramphenicol
acetyl transferase

Acetylated
chloramphenicol
(inactive)
 Resistance mechanisms: Modification of the
cellular target
• The modification of the target (of the antibiotic) is due to mutation events; this does
not compromise the biological functionality but it prevents the drug interaction.
• Very common examples and clinically relevant:
• Mutation of genes that code for DNA-toposisomerase type II (fluoroquinolones)
• Mutations of genes that code for the β-subunit of the DNA-dependent RNA-polymerase (Rifampicin)
• Production of mutated Penicillin-Binding Proteins, with reduced affinity for the β-lactamases (S.
pneumoniae):
• New sequences that code for the PBPs are acquired by means of transformation, hence they recombine with
“original” genes that code for the PBPs, forming genetic “mosaics.”
• The molecular target can also undergo a biochemical modification that makes it
insensitive to the antibiotic:
• Methylation of the rRNA 23S through the specific methylase (erm gene) that confers macrolide and
lincosamide resistance to many Gram+
• Methylation of the rRNA 16S through the specific methylase (rmt, arm genes) that confers
aminoglycoside resistance to Gram-
• Impeded access to the antibiotic (glycopeptide) to the dimer D-ala+D-ala: transformation to D-
ala+lactate (enterococci) or the synthesis of a protein that creates stearic impediment
(staphylococci)
Resistance mechanisms: Alteration of the
cellular permeability for the drug
• The expression of the resistance phenotype is a consequence of the
reduction of the intracellular concentration of the drug, which can be
derived from a reduced uptake (entrance through external
membrane, porins) or by an increased expulsion of the drug (active
efflux system)
• The reduced uptake of the drug can be due to the:
• External membrane (Gram- vs glycopeptides)
• Diminished/insufficient expression of porins (oprD2 porin of P. aeruginosa vs
carbapenems)
• The increased expulsion of the drug is due to the presence of efflux
systems that can expel the drug: by going against the concentration
gradient, using ATP (or the membrane proton gradient) as a source of
energy. Their increased expression is associated with the appearance
of the resistance phenotype.
• There exist chromosomal “multidrug” systems that are able to expel
molecules belonging to different classes:
• MexA-MexB-OprM in P. aeruginosa vs. β-lactams, chloramphenicol,
macrolides, quinolones, tetracycline
• NorA in S. aureus vs. quinolones and chloramphenicol
• “Targeted” systems, plasmids that are able to expel only a specific
class of antibiotics:
• Gens tet (tetracycline), mef (macrolides), qepA (quinolones)
Structure and function of the efflux pump of the “MexAB-OprM” complex of P. aeruginosa. The antibiotics are
“sequestered” in the periplasmic space, from the cytoplasmic membrane, and/or in the periplasmic space by MexB, D, F,
or Y (RND, resistance-nodulation-division exporter proteins). MexA, C, E, or X (MFP, membrane fusion proteins) functions
as “canals” between the cytoplasmic membrane and the external [membrane]. OprM, J, or N (gated outer membrane
porin proteins) allow the final stage fo the removal of the antibiotic from the cell.
Antimicrobial drug resistance: “Temporal”
evolution
Antimicrobial drug resistance: “Natural”
causes
 Antimicrobial drug resistance: “Artificial”
causes
• The development of antibiotic resistance is
correlated to the level of antibiotic use.
• The abuse (or incorrect use) has increased
the incidence and selection of mutations
associated to resistance:
• Inappropriate use (pediatric for viral infections,
erroneous prescriptions, use without doctor’s
supervision)
• Surgical prophylaxis
• Empirical use (etiologic agent unknown)
• Increased use of “broad-spectrum” antibiotics
• Poor compliance of the patient
• Use of antibiotics as a “probiotic” in animal feeds
Antimicrobial drug resistance: “Artificial”
causes – use of antibiotics in zootechnology
Antimicrobial drug resistance: “Artificial”
causes – use of antibiotics and resistance
Antimicrobial drug resistance: Diffusion
 Antimicrobial drug resistance: Damage to the
host
• Toxicity of the organ (oto-, neuro-,
nephro-, gastro-, hemato-, odonto-,
etc)
• Allergic reactions
Alteration of the teeth
• Alterations of the qualitative and due to tetracycline use
quantitative relationships between the
microbial species (dismicrobism),
causing superinfections
• Enterocolitis by Clostridium difficile
• Vaginal candidiasis (C. albicans)
Dismicrobism and superinfection
Antimicrobial drug resistance: The “size” of
the phenomenon
 Antimicrobial drug resistance: How to reduce
its spread
• Avoid the repetitive use of the same molecule
• Modify the antibiotic to evade the resistance
mechanism
• Use combinations of antibiotics
• Reduce the consumption of antibiotics
• Health education, limit its use in animal feeds
• Increase patient compliance
• Health education
• Develop new molecules
• Natural peptides: magainin (from frogs and sharks),
BMAPs (bovines)
• Optimize techniques to determine antibiotic
sensitivity