2025-2026 Molecular Bacteriology Surbhi Malhotra
MOLECULAR BACTERIOLOGY
COURSE INFORMATION
• Exam: written exam with mcq (15%) and 3-4 open questions (50%) (illustrations are needed)
• Paper presentation (20%) and practicals (15%)
CONTENT OF LECTURES
1. Classification of antibiotics
2. Genetics of antibiotic resistance
3. Beta-lactams, macrolides and fluoroquinolones
4. Biofilms
5. Mechanisms of clonal evolution of major hospital and community pathogens
à MRSA
à MDR-GNB and plasmid-based transmission
6. Bacterial typing methods
7. Tolerance and persistence
8.
9.
Hypermutators
Primer on metagenomics
What is an Antib
CLASSIFICATION OF ANTIBIOTICS Just a short review (not actual course material)
WHAT ARE ANTIBIOTICS? • A drug that kills (bactericidal) or pre
(bacteriostatic) of bacteria
• AB = a drug that kills (bactericidal) or prevents multiplication –
(bacteriostatic)
Specificityofofbacteria
an antibiotic resides in its
o Bacteriostatic = decreases the growth of bacteria to such level that the host IS can take over
and not the host
and kill them
• Specificity of an AB = its ability to damage the bacteria and not– the
1928: The first
host (targeting antibiotic,
bacterial penicillin, was
pathways
which are not present in the host) Fleming
• The first AB was found in 1928 by Alexander Fleming: penicillin
o He found it by accident: he had a fungal contamination on his bacterial
culture and saw that the bacteria couldn’t grow near that contamination
• Abs are classified based on their target site:
o Metabolic analogues
o Protein synthesis inhibitors
o Cell membrane inhibitors
o Cell wall inhibitors
o DNA replication inhibitors
à All antimicrobial drugs work against growing bacteria (if a bacterium does not produce NAM-NAG
for its cell wall, you can’t block this!)
1
,2025-2026 Molecular Bacteriology Surbhi Malhotra
METABOLIC ANALOGUES
• Metabolic analogues = molecules that mimic metabolites/molecules required in a certain pathway
o Mimicry = imitating someone/something
o E.g. some butterflies have wings which mimic big eyes for protection against predators
• In this case we want an AB that mimics a metabolite/molecule in a pathway of the bacteria and not
the host (remember specificity) e.g. the folic acid pathway
• Folate is needed to produce DNA/RNA
Co-trimoxazole
o Humans need to get folic acid through food (green vegetables) because we can’t
make it ourselves
o Bacteria produce folic acid from PABA (para-aminobenzoic acid) à we can make
AB that mimic PABA: structural analogues Dapsone
1. Sulphonamides Sulphonamiden
2. Trimethoprim
à These two are usually given together as they have a synergetic effect: they
Protein
target different steps of the same pathway (both structural Synthesis
analogues). The
combination of the two is known as co-trimocazole
•
Trimethoprim
Another example is dapsone, a metabolic analogue targeting Mycobacterium Leprae (the mechanism
Metabole DNA
is not very clear)
analogen Inhibitoren
PROTEIN SYNTHESIS INHIBITORS
• Bacterial ribosomes differ from human ribosomes = good target forANTIBIOTICA
AB
• Ribosomes of bacteria Celwand
Eiwitsynthese Inhibitoren
o Small (30s) and big (50s) subunit inhibitoren
o Aminoacyl (A) site = entry site for tRNA
o Peptidyl (P) site = has the peptidyl tRNA (tRNA that carries the
growing polypeptide chain) Celmembraan
Inhibitoren
o Exit (E) site = exit site for tRNA without AA
o How does it work?
§ The right AA binds theDomains
right tRNA
of 23S rRNA
§ The first tRNA binds the start codon in the P-site
§ The next tRNA will bind the ribosome at the right codon (in the A-site)
§ The 2 AAs are coupled
§ The first tRNA goes away and the second moves to the P-site so a new tRNA can bind
§ When a stop codon is positioned in the A-site, no tRNA can fit the A-site: this codon
is recognized by the release factor which catalyzes the hydrolysis of the peptidyl-
tRNA bond
§ The polypeptide is released through a tunnel and the ribosome disassociated into
subunits
o Special: while mRNA is transcribed, its already translated because the half-life of mRNA is
very short in bacteria
o The ribosomal subunits exist of different rRNA domains
§ 23S rRNA = major binding site for AB
§ 16S rRNA = highly conserved and therefore used for identification of bacteria (up to
species level)
2
,2025-2026 Molecular Bacteriology Surbhi Malhotra
• A few ABs are bacterial protein synthesis inhibitors
• Targeting the tRNA synthesis:
o Mupirocin
§ Selectively binds the bacterial isoleucyl-tRNA synthetase
§ = halts the incorporation of isoleucine into the peptide chain
§ Mostly for gram positives, active against MRSA!
• Targeting the bacterial ribosome
o Fusidic acid
§ Binds and inhibits the peptide chain elongation factors
§ = prevents elongation of the polypeptide
o Lincosamides
§ Binds to 50S: inhibits transpeptidase (binds the new AA to the polypeptide)
§ = prevents peptide chain elongation
§ E.g. lincomycin, clindamycin
o Fenicols
§ Binds to the 50S: inhibits peptide bond formation and prevents ribosomal
translocation
o MASK (Macrolides, Azalides, Streptogramins and Ketolides)
§ This will come back later
§ Macrolides includes azalides and ketolides (but they differ in structure)
o Aminoglycosides
§ Binds to the 30S at the A site: inhibits the peptide chain transfer and interferes with
formation of the initation complex
§ E.g. streptomycin, tobramycin
o Tetracyclines
§ Binds to the 30S: prevents attachment of amino-acyl-tRNA to the A-site
§ E.g. doxycycline, tetracycline, tigecycline
• Lincosamides, macrolides and streptogramines all have a common binding site on 50S meaning that
macrolide resistance will also give resistance to the other two groups because their binding sites are
close to each other
CELL MEMBRANE INHIBITORS
• What is the cell membrane? Phospholipid bilayer
(hydrophilic heads to the outside and hydrophobic tails to
the inside) = amphiphilic molecules! Many protein channels
allow minimal transport over the membrane.
• Daptomycin: works like a detergent, forms micelles and
breaks down the bacterial cell membrane
o For gram positives (too big to pass the outer
membrane of gram negatives)
• Polymyxins: bind phospholipids and lipopolysaccharides
(LPS) in the outer membrane of gram negatives and alters the cell membrane permeability
o This is a toxic AB
o E.g. polymyxin B, colistin (polymyxin E)
3
, 2025-2026 Molecular Bacteriology Surbhi Malhotra
CELL WALL INHIBITORS
The Bacterial Cell Wall
REMEMBER:
• This is different for gram negatives vs gram positives
o Gram negative bacteria: consits of an outer (with LPS) and
inner cell membrane containing a periplasmic space with a
small peptidoglycan layer
o Gram positive bacteria: only an inner cell membrane
surrounded by a thick peptidoglycan layer (cell wall)
o The porins in the outer membrane of the gram negative bacteria
don’t let through all ABs, in gram positives ABs can come in
through passive diffusion (much easier than gram negs)
• Gram staining: differentiate between gram negs and positives
1. Crystal violet (+) stains G- and G+ both purple by binding (-) groups in the bacteria The Bacterial Cell
2. Decolorization with alcohol and aceton: only G- loses its color (aceton dissolves the outer
membrane of G- whereas the color can escape, the peptidoglycan layer for G+ is to thick for
• Structure of peptidoglycan
the color to go away)
3. Safranin will now color G- pink (counter staining) cytoplasm
• Structure of peptidoglycan
o Sugarbackbone: NAM-NAG sugars (N-acetylmuramic acid and N- NAG-NAM-NAG-NAM
|
L-Ala NAG-NAM-NAG-NAM
acetylglucosamine) | |
L-Ala
D-Glu
o NAM is bound to a tetrapeptide: the third AA of the tetrapeptide (gram | |
D-Glu
L-diA-(AA) -NH
dependent) will form a crosslink with the last AA of the tetrapeptide from
n 2
| |
D-Ala L-diA -(AA) -NH n 2
another chain = 3,4-oligopeptide bridge |
D-Ala
|
D-Ala
o G-: lysine Transpeptidation D-Ala
|
o G+: diaminopimelic acid and
o The NAG-NAM sugars are coupled with glycosidic bonds: transglycosylation Transglycosylation
reaction
o The AAs are coupled with a peptide brige: transpeptidation
• Cell wall synthesis
o Cytoplasm: NAM + NAG + pentapeptide
o Flip: the structure is flipped to extracellular through binding to bactoprenol
o Extracellular: transglycosylation and transpeptidation
à PBP (penicillin binding protein) = bifunctional enzym that carries out both the
transpeptidase and transglycosilase function
CELL WALL INHIBITORS
Mechanism of Action of Vancomycin
• Glycopeptides
o Vancomycin and teicoplanin G M
G M G M G M
o Vancomycin: binds to D-ala-D-ala and prevents the transpeptidation =
physical blockage
§ This molecule is very large and can’t enter gram negative
bacteria Vancomycin binds to
D-Ala-D-Ala; prevents
§ Only works if the peptide crosslinks are not formed yet Vancomycin transpeptidation
• Cycloserine
• B-lactams
o Binds to PBP and thus inhibits transpeptidation
o 4 groups: penicillins, cephalosporins (4 generations), monobactams (aztreonam) and
carbapenems (imipenem and meropenem)
o Different kind of penicillins
§ Small spectrum (e.g. penicillin)
4
MOLECULAR BACTERIOLOGY
COURSE INFORMATION
• Exam: written exam with mcq (15%) and 3-4 open questions (50%) (illustrations are needed)
• Paper presentation (20%) and practicals (15%)
CONTENT OF LECTURES
1. Classification of antibiotics
2. Genetics of antibiotic resistance
3. Beta-lactams, macrolides and fluoroquinolones
4. Biofilms
5. Mechanisms of clonal evolution of major hospital and community pathogens
à MRSA
à MDR-GNB and plasmid-based transmission
6. Bacterial typing methods
7. Tolerance and persistence
8.
9.
Hypermutators
Primer on metagenomics
What is an Antib
CLASSIFICATION OF ANTIBIOTICS Just a short review (not actual course material)
WHAT ARE ANTIBIOTICS? • A drug that kills (bactericidal) or pre
(bacteriostatic) of bacteria
• AB = a drug that kills (bactericidal) or prevents multiplication –
(bacteriostatic)
Specificityofofbacteria
an antibiotic resides in its
o Bacteriostatic = decreases the growth of bacteria to such level that the host IS can take over
and not the host
and kill them
• Specificity of an AB = its ability to damage the bacteria and not– the
1928: The first
host (targeting antibiotic,
bacterial penicillin, was
pathways
which are not present in the host) Fleming
• The first AB was found in 1928 by Alexander Fleming: penicillin
o He found it by accident: he had a fungal contamination on his bacterial
culture and saw that the bacteria couldn’t grow near that contamination
• Abs are classified based on their target site:
o Metabolic analogues
o Protein synthesis inhibitors
o Cell membrane inhibitors
o Cell wall inhibitors
o DNA replication inhibitors
à All antimicrobial drugs work against growing bacteria (if a bacterium does not produce NAM-NAG
for its cell wall, you can’t block this!)
1
,2025-2026 Molecular Bacteriology Surbhi Malhotra
METABOLIC ANALOGUES
• Metabolic analogues = molecules that mimic metabolites/molecules required in a certain pathway
o Mimicry = imitating someone/something
o E.g. some butterflies have wings which mimic big eyes for protection against predators
• In this case we want an AB that mimics a metabolite/molecule in a pathway of the bacteria and not
the host (remember specificity) e.g. the folic acid pathway
• Folate is needed to produce DNA/RNA
Co-trimoxazole
o Humans need to get folic acid through food (green vegetables) because we can’t
make it ourselves
o Bacteria produce folic acid from PABA (para-aminobenzoic acid) à we can make
AB that mimic PABA: structural analogues Dapsone
1. Sulphonamides Sulphonamiden
2. Trimethoprim
à These two are usually given together as they have a synergetic effect: they
Protein
target different steps of the same pathway (both structural Synthesis
analogues). The
combination of the two is known as co-trimocazole
•
Trimethoprim
Another example is dapsone, a metabolic analogue targeting Mycobacterium Leprae (the mechanism
Metabole DNA
is not very clear)
analogen Inhibitoren
PROTEIN SYNTHESIS INHIBITORS
• Bacterial ribosomes differ from human ribosomes = good target forANTIBIOTICA
AB
• Ribosomes of bacteria Celwand
Eiwitsynthese Inhibitoren
o Small (30s) and big (50s) subunit inhibitoren
o Aminoacyl (A) site = entry site for tRNA
o Peptidyl (P) site = has the peptidyl tRNA (tRNA that carries the
growing polypeptide chain) Celmembraan
Inhibitoren
o Exit (E) site = exit site for tRNA without AA
o How does it work?
§ The right AA binds theDomains
right tRNA
of 23S rRNA
§ The first tRNA binds the start codon in the P-site
§ The next tRNA will bind the ribosome at the right codon (in the A-site)
§ The 2 AAs are coupled
§ The first tRNA goes away and the second moves to the P-site so a new tRNA can bind
§ When a stop codon is positioned in the A-site, no tRNA can fit the A-site: this codon
is recognized by the release factor which catalyzes the hydrolysis of the peptidyl-
tRNA bond
§ The polypeptide is released through a tunnel and the ribosome disassociated into
subunits
o Special: while mRNA is transcribed, its already translated because the half-life of mRNA is
very short in bacteria
o The ribosomal subunits exist of different rRNA domains
§ 23S rRNA = major binding site for AB
§ 16S rRNA = highly conserved and therefore used for identification of bacteria (up to
species level)
2
,2025-2026 Molecular Bacteriology Surbhi Malhotra
• A few ABs are bacterial protein synthesis inhibitors
• Targeting the tRNA synthesis:
o Mupirocin
§ Selectively binds the bacterial isoleucyl-tRNA synthetase
§ = halts the incorporation of isoleucine into the peptide chain
§ Mostly for gram positives, active against MRSA!
• Targeting the bacterial ribosome
o Fusidic acid
§ Binds and inhibits the peptide chain elongation factors
§ = prevents elongation of the polypeptide
o Lincosamides
§ Binds to 50S: inhibits transpeptidase (binds the new AA to the polypeptide)
§ = prevents peptide chain elongation
§ E.g. lincomycin, clindamycin
o Fenicols
§ Binds to the 50S: inhibits peptide bond formation and prevents ribosomal
translocation
o MASK (Macrolides, Azalides, Streptogramins and Ketolides)
§ This will come back later
§ Macrolides includes azalides and ketolides (but they differ in structure)
o Aminoglycosides
§ Binds to the 30S at the A site: inhibits the peptide chain transfer and interferes with
formation of the initation complex
§ E.g. streptomycin, tobramycin
o Tetracyclines
§ Binds to the 30S: prevents attachment of amino-acyl-tRNA to the A-site
§ E.g. doxycycline, tetracycline, tigecycline
• Lincosamides, macrolides and streptogramines all have a common binding site on 50S meaning that
macrolide resistance will also give resistance to the other two groups because their binding sites are
close to each other
CELL MEMBRANE INHIBITORS
• What is the cell membrane? Phospholipid bilayer
(hydrophilic heads to the outside and hydrophobic tails to
the inside) = amphiphilic molecules! Many protein channels
allow minimal transport over the membrane.
• Daptomycin: works like a detergent, forms micelles and
breaks down the bacterial cell membrane
o For gram positives (too big to pass the outer
membrane of gram negatives)
• Polymyxins: bind phospholipids and lipopolysaccharides
(LPS) in the outer membrane of gram negatives and alters the cell membrane permeability
o This is a toxic AB
o E.g. polymyxin B, colistin (polymyxin E)
3
, 2025-2026 Molecular Bacteriology Surbhi Malhotra
CELL WALL INHIBITORS
The Bacterial Cell Wall
REMEMBER:
• This is different for gram negatives vs gram positives
o Gram negative bacteria: consits of an outer (with LPS) and
inner cell membrane containing a periplasmic space with a
small peptidoglycan layer
o Gram positive bacteria: only an inner cell membrane
surrounded by a thick peptidoglycan layer (cell wall)
o The porins in the outer membrane of the gram negative bacteria
don’t let through all ABs, in gram positives ABs can come in
through passive diffusion (much easier than gram negs)
• Gram staining: differentiate between gram negs and positives
1. Crystal violet (+) stains G- and G+ both purple by binding (-) groups in the bacteria The Bacterial Cell
2. Decolorization with alcohol and aceton: only G- loses its color (aceton dissolves the outer
membrane of G- whereas the color can escape, the peptidoglycan layer for G+ is to thick for
• Structure of peptidoglycan
the color to go away)
3. Safranin will now color G- pink (counter staining) cytoplasm
• Structure of peptidoglycan
o Sugarbackbone: NAM-NAG sugars (N-acetylmuramic acid and N- NAG-NAM-NAG-NAM
|
L-Ala NAG-NAM-NAG-NAM
acetylglucosamine) | |
L-Ala
D-Glu
o NAM is bound to a tetrapeptide: the third AA of the tetrapeptide (gram | |
D-Glu
L-diA-(AA) -NH
dependent) will form a crosslink with the last AA of the tetrapeptide from
n 2
| |
D-Ala L-diA -(AA) -NH n 2
another chain = 3,4-oligopeptide bridge |
D-Ala
|
D-Ala
o G-: lysine Transpeptidation D-Ala
|
o G+: diaminopimelic acid and
o The NAG-NAM sugars are coupled with glycosidic bonds: transglycosylation Transglycosylation
reaction
o The AAs are coupled with a peptide brige: transpeptidation
• Cell wall synthesis
o Cytoplasm: NAM + NAG + pentapeptide
o Flip: the structure is flipped to extracellular through binding to bactoprenol
o Extracellular: transglycosylation and transpeptidation
à PBP (penicillin binding protein) = bifunctional enzym that carries out both the
transpeptidase and transglycosilase function
CELL WALL INHIBITORS
Mechanism of Action of Vancomycin
• Glycopeptides
o Vancomycin and teicoplanin G M
G M G M G M
o Vancomycin: binds to D-ala-D-ala and prevents the transpeptidation =
physical blockage
§ This molecule is very large and can’t enter gram negative
bacteria Vancomycin binds to
D-Ala-D-Ala; prevents
§ Only works if the peptide crosslinks are not formed yet Vancomycin transpeptidation
• Cycloserine
• B-lactams
o Binds to PBP and thus inhibits transpeptidation
o 4 groups: penicillins, cephalosporins (4 generations), monobactams (aztreonam) and
carbapenems (imipenem and meropenem)
o Different kind of penicillins
§ Small spectrum (e.g. penicillin)
4
