Lecture 7&8: Control of microbial populations

Question 1

Chemical method of ethylene oxide sterilisation

Answer 1

• strong alkylator reacting with guanine

Question 2

3 levels of disinfectants

Answer 2

1. High risk: in close contact with a break in skin; inactivates viruses, fungi, mycobacteria & spores
2. Intermediate: in contact with mucous membranes; inactivates viruses, fungi, mycobacteria
3. Low: in contact with healthy skin; inactivates sporulating bacteria and lipid-enveloped viruses

Question 3

Chemical modifications of various disinfectants

Answer 3

• Glutaraldehyde: cross-linking
• Peracetic acid: oxidising agents
• Alcohols: protein denaturation & coagulation
• Iodophors: oxidising agents
• QACs: surfactants

Question 4

Examples of biocides

Answer 4

• alcohols: bacteria, fungi, viruses, mycobacteria
• Chlorhexidine: bacteria, fungi*, mycobacteria, some viruses
• Iodine & Iodophors: bacteria, fungi, viruses, mycobacteria
• Triclosan: bacteria, fungi*, mycobacteria

Question 5

Spectrum of activity of antimicrobials

Answer 5

• Narrow: Metronidazole (effective for anaerobes)
• Broad: Aminoglycosides (both Gram +ve & -ve)

Question 6

Antibiotic families

Answer 6

Family groups of antibiotics with activity involving:
1. cell wall: penicillins, beta-lactams

• Carrier protein must be phosphorylated
• Cycloserine: analogue of D-alanine
• Bacitracin: inhibits de-phosphorylation so it cannot take up new carrier molecule
• Beta-lactams: glycopeptides including penicillins 5 member ring & cephalosporins 6 member ring, vancomycin

2. Protein synthesis: aminoglycosides (induce codon misreading), tetracylcines (block binding of tRNA to A site)
3. Metabolism: Sulfonamides
4. Nucleic acid synthesis: Quinolones (DNA replication), Metronidazole (DNA replication), Rifampin (RNA synthesis)

Question 7

Synergistic example

Answer 7

• Combination of:
• Sulfamethoxazole: inhibits Dihydropterate Synthase
• Trimethoprim: inhibits Dihydrofolate reductase
• useful in treating Enterococci (uses exogenous folic acid)
• also wide-range of Gram +ve and -ve bacteria

Question 8

Intrinsic & acquired antibiotic resistance

Answer 8

Intrinsic:

• Gram -ve cell wall confers resistance to glycopeptides
• efflux pump that pumps antibiotic

Acquired

• bacteria modifying MEC gene on a cassette to produce alternative forms of transpeptidases (PBP)
• bacteria to produce degradation enzymes ex. beta-lactamase enzymes cleaves beta-lactam. Note: in order to overcome this antibiotic resistance, add inhibitor beta-lactamase such as clavulinic acid
• Conjugation: R-plasmids
• Transformation:
• Transduction: phage
• Transposition

Question 9

Bacterial Antibiotic resistance mechanisms

Answer 9

• Beta-lactams: drug inactivation, altered uptake & target
• Glycopeptides: altered target
• Tetracyclines: drug inactivation
• Aminoglycosides: drug inactivation, altered uptake

Question 10

Conjugation events and outcomes

Answer 10

• Complete transfer
• Incomplete
• Complete transfer & integration: Hfr

Question 11

Transformation conferring antibiotic resistance

Answer 11

• loose DNA conferring resistance
• Haemophilus & Streptococci possess DNA binding proteins on surface; Haemophilus on acquire DNA from other haemophilus bacteria
• acquire during late-lag phase

Question 12

Transduction

Answer 12

2 types of transduction:

1. Generalised transduction: Virulent (Lytic) bacteriophage infects bacteria and bacteria produces new phage; Virulent -> lytic cycle only; increased pathogenicity
2. Specialized transduction: Temperate bacteriophage infects and genetic material incorporates into bacterial genome; Temperate -> lytic + lysogenic (prophage); rarely results in lysis of bacterial cell; increased pathogenicity

Question 13

Categories of antifungals

Answer 13

1. Direct membrane damage: Creates channels thru cell wall by binding to ergosterol; polyenes, ex.amphotericin B
2. Ergosterol biosynthesis inhibitors (indirect membrane damage): a. azoles b. terbinafine (inhibit squaline epoxidase)
3. Nucleic acid synthesis inhibitor/antimetabolite: ex. 5-fluorouracil
4. Beta-glucan synthesis inhibitors: fungal cell wall component inhibitor, echinocandins, ex. Caspofungin; block caspofungin

Question 14

Antifungal resistance mechanisms

Answer 14

Mutations in cytosine deaminase

Decreased rate of transport into fungal cell

Alteration of target enzyme (E.g., mutation, over- expression)

Alteration of Ergosterol biosynthetic pathway

Growth as biofilm