Wk 1.3 Bacterial Genetics and Antibiotic Resistance

Learning Outcomes

Lecture 3

  • Understand the make up of bacterial genomes

  • Understand what changes can occur to bacterial genomes (mechanisms of genetic diversity) & how these can effect bacterial pathogenicity

  • Hint: What is meant by & what are homologous recombination, transposition, mutation, transformation,

conjugation, transduction?

  • Understand the role of plasmids in bacterial function

Bacterial DNA

  • Organized as a single double stranded circular chromosome in most prokaryotes.

  • Exceptions: Linear (e.g., Borrelia) or multiple chromosomes (e.g., Vibrio).

  • DNA is supercoiled and associated with basic proteins (histone-like).

  • Contains plasmids: small extrachromosomal double stranded DNA.

DNA Replication in Bacteria

  • Initiation at a single point (origin) with synthesis at the replication fork. (place where at which the helix is unwound)

  • Two replication forks move outward from the origin until the a whole replication is copied

  • Chromosome is a single replicon, using the rolling circle mechanism during conjugation in E. coli.

  • At least 30 proteins are involved in E. coli replication.

Gene Structure

  • One gene-one enzyme hypothesis and one gene-one polypeptide hypothesis (cistron).

  • Some genes encode rRNA and tRNA.

  • Sequence of nucleic acid transcribed to generate an RNA product

  • Genes typically consist of discrete sequences that produce one way and one product

  • Single starting point and one reading frame (no overlapping)

    • Some do overlap

  • Introns present in some bacterial genes.

Gene Expression

  • Bacteria regulate genome expression to conserve energy and raw materials, maintaining protein balance, and allowing adaptation.

  • E. coli can synthesise 2000-4000 peptides, expressing a fraction at any one time.

Induction and Repression

  • E.coli growing in the presence of lactose induces synthesis of b-galactosidase (3000 molecules) while other bacteria in the absence leads to very low levels (<3 molecules).

    • beta-galactosidase is an inducible enzyme

    • Inducible enzymes triggered by inducers like allolactose. (ตัวเหนี่ยวนำ)

  • Repressible enzymes produced unless their pathway's end product is present (e.g., amino acids for biosynthesis).

    • will repress the expression of the enzyme required for its biosynthesis

Plasmids

  • Small, double stranded circular DNA in many bacteria; can exist independently of the host chromosome.

  • Separate replicon

  • Usually contains <30 genes; numerous plasmids (>40) may be present; just one

  • Not essential for survival, but play significant roles.

Plasmid Classification

  • Episome: can exist integrated or independently with chromosomes.

  • Conjugative: contribute to pili formation and conjugation.

  • F factors(fertility): fertility factors involved in conjugation.

  • R factors (resistant): confer antibiotic resistance.

  • Col plasmids: encode bacteriocins.

  • Virulence plasmids: produce toxins, siderophores, and aid adherence.

  • Metabolic plasmids: encode degradative enzymes.

Transposons

  • DNA segments that move within chromosomes or plasmids.

  • Cannot replicate independently

  • May contain genes required for transposition

  • Can carry genes like toxins or antibiotic resistance.

Effects of Transposons

  • DNA rearrangements (deletions)

  • Mutations

  • Gene activation by containing promoters (activate genes) or stop codons (block translation or transcription).

  • Transfer of Genetic Material (recombination DNA)

    • Occurs via transformation, transduction, and conjugation.

Transformation

  • Bacteria can uptake DNA fragments from the environment into their genome (often from dead cells lysis).

  • Not all bacteria can do this

  • Usually between cells of the sam genera (Streptococcus, Staphylococcus, Bacillus)

  • Recognised in Griffith's 1940’s experiments (Streptococcus pneumoniae).

Transduction

  • Bacteriophages (phages) are viruses that infect bacteria

    • may induce a lytic cycle

  • producing phage particles within bacterium and its subsequent lysis releasing the newly formed progeny

  • Fragmenting bacterial DNA.

  • When phage particles are being assembled some fragments of bacterial (donor) DNA may become incorporated into the phage DNA

    • Subsequent infections of other bacterial (recipient) cells transfers to the DNA

    • Refer as generalised transduction

  • Temperate phages DO NOT cause cell lysis BUT incorporate their DNA into bacterial chromosome (prophage)

    • Replicates with bacterial chromosome (lysogeny)

    • In some cases the prophage may carry genes for toxin production (Corynebacterium diphtheriae)

  • Conditions may arise that cause reversion to the lytic cycle

    • Phage particles assembled and may contain fragments of the bacterial DNA

    • Fragments will be those adjacent (ชิด) to the prophage in the lysogenic stat

    • New phage particles infect more bacteria

    • Referred to as specialised transduction

Conjugation

  • Requires physical contact between donor and recipient calls (unlike transformation and transduction)

    • Formation of sex pilus between 2 cells, genetic material is transferred via plasmids (may also be a transposon)

  • F factor plasmids direct sex pilus formation (F+)

    • F+ may enter recipient cell and continue to replicate independently conferring F+ status on the recipient (F-)

    • F+ may integrate with recipient chromosomal DNA to generate an Hfr cell

      • Act as donor cells in conjugation and generate a recombinant F-cell

Mutation

  • Defined as a permanent, heritable change in the sequence of nucleotide bases in the DNA sequence,

  • May be large deletions or insertion mutations but most are point mutation

    • Point mutations affecting a single base pair.

Point Mutations Types

  • Silent-no visible: no effect due to code degeneracy.

  • Missense-single base substitution:changing an amino acid.

  • Nonsense-conversion: sense codon converts to a termination codon.

  • Frameshift-insertion: deletion of base pairs altering the reading frame.

Origin of Mutations

  • Spontaneous mutations-mistakes: errors in DNA replication (1:109 cell divisions in bacteria)

  • Mutagens-substances: that induce mutations through chemical damaging DNA

  • Radiation-ionising: produces DNA damage through errors in replication produces DNA damage through errors in replication, non-ionising radiation generates thymine dimers

Mutations and Antimicrobial Drug Resistance

  • Mutations can confer drug resistance to a bacterial cell

  • Subsequent exposure of cell population to drug creates selective pressure

  • Susceptible cells are killed and resistant cells survive and multiply

Reasons for Selection of Resistant Cells

  • Prolonged antibiotic use

  • Failure to complete treatment

  • Overuse/misuse of drugs.

Changes Leading to Antimicrobial Resistance (short answers)

1. Production of enzymes that inactivate or destroy the drug

  • Some bacteria produce enzymes that break down antibiotics, rendering them ineffective.

  • Example: β-lactamase enzymes degrade the β-lactam ring found in penicillins and cephalosporins, preventing these antibiotics from inhibiting bacterial cell wall synthesis.

2. Alterations in membrane structure and permeability

  • Bacteria can modify their outer membrane (especially Gram-negative bacteria) to reduce the drug’s ability to enter the cell.

  • This can involve:

    • Mutations in porin proteins (small channels in the membrane that allow antibiotics to enter).

    • Thicker cell walls (as seen in vancomycin-resistant bacteria).

  • Example: Pseudomonas aeruginosa reduces permeability to carbapenems.

3. Efflux pumps that expel the drug

  • Some bacteria have efflux pumps that actively transport the drug out of the cell, preventing it from reaching its target concentration.

  • This mechanism is common for:

    • Tetracyclines

    • Fluoroquinolones

    • Macrolides

  • Example: The Tet(A) and Tet(B) efflux pumps in tetracycline-resistant bacteria.

4. Alteration of drug binding sites

  • Bacteria can mutate or modify the target sites where antibiotics usually bind, reducing drug effectiveness.

  • Examples:

    • Penicillin-binding proteins (PBPs) altered → resistance to penicillins & cephalosporins (e.g., methicillin-resistant Staphylococcus aureus – MRSA).

    • Changes in ribosomal binding sites → resistance to aminoglycosides & macrolides (erythromycin).

5. Modification of metabolic pathways

  • Some bacteria bypass the normal metabolic pathway targeted by the antibiotic by using alternative enzymes or pathways.

  • Example: Sulphonamide resistance

    • Sulphonamides inhibit dihydropteroate synthase (an enzyme in folic acid synthesis).

    • Resistant bacteria produce an alternative enzyme that does not bind to sulphonamides, allowing folic acid synthesis to continue.

Transfer of Drug Resistance

  • Occurs by plasmids during conjugation, possible across species and closely related genera (e.g., Shigella and E. coli).

  • Transposons can also carry multiple resistance genes.

  • Animal feeds contain antibiotics and may contribute to the transfer of drug resistant organisms

Drug Resistance in Hospitals

  • The extensive use of antimicrobial drugs in hospitals provides a flourishing

  • Infection control procedures mandatory to prevent spread of resistant organisms to other patients

Drug Resistance in the Community

  • Caused by Overuse for minor infections

  • Misuse for viral infections with antibiotics

  • Alteration of pateint’s normal flora.

  • Increased resistant strains (e.g., Staphylococcus aureus).