R.O Lecture 5&6 Flashcards
What is the rate of microbial death affected by?
*Microbial characteristics/susceptibilities
*Environmental influences such as:
- Number of Microbes (Proportional death rates)
- Time required for sterilisation
Sterilisation
The killing or removal of all microorganisms in a material or object
Disinfection
Reduction in the number of pathogenic microogranisms on surfaces/objects to the point where they pose no danger of disease
Antiseptic
Chemical agent that is safe to use externally on living tissue to destry microbes or inhibit their growth
Bacteriostatic Agent
An agent that inhibits the growth of bacteria
Bactericide
Agent that kills bacteria (though not spores)
What are the ideal qualities for selecting a disinfectant?
- Fast-acting
- Non-toxic
- Non-damaging to material
- Wide spectrum
- Easy to prepare/stable
- Inexpensive
- Odour
Mechanisms of Action - Effects on Protein
Denaturation of Protein:
- Permanent/Temporary
- hydrolysis by acids/alkalis
- oxidation by H2O2, KMnO4, halogens
- alkylating agents (e.g. some dyes) etc.
Denaturation of Protein (Bacteriocidal)
Active Protein - Inactive Protein - No reconfiguration; permanentely denatured
Denaturation of Protein - Bacteriostatic
Active Protein - Inactive Protein - Reconfiguration, temporarily denatured
Mechanisms of Action - Effects on Membranes
- Denaturationof Protein Component
- Disruption of Lipids
- Surfactants (Alcohols, detergents, quats)
- Wetting agents
- Indirect effect
Mechanisms of Action for Nucleic Acids
Nucleic Acids: Damage from heat, radiation, chemicals
Mechanisms of Action for Energy-producing systems
E.g. Fermentation inhibition by lactic acid or propionic acid
Mechanism of Action - Cell Walls
Dyes (e.g. crystal violet) can interfere with cell wall formation
Control Methods can be…?
Chemical or Physical
Chemical Antimicrobial Agents - Soaps and Detergents
- Remove microbes, oily substances and dirt
- Anionic: Clothes laundering, household cleaning agents - less effective
- Cationic: Sanitize food utensils (kill some viruses)
Quaternary ammonium compounds (quats)
Mixtures can increase efficacy
Chemical Antimicrobial Agents - Phenols
Phenol and phenol derivatives (phenolics):
- Denature proteins/enzymes and disrupt membranes
- Action not impaired by organic material
- Halogen addition can increase effectiveness
- E.g. Amphyl and Lysol: retain properties for days,
safe on skin and medical instruments
Chemical Antimicrobial Agents - Halogens
- Particularly Iodine and Chlorine; alone (I2 or Cl2) or part of
compounds (NaOCl) - Agent used in drinking water and swimming pools (HClO)
- Can be inactivated by organic material
- Iodophors (Iodine combined with organic molecule): slow release, less irritating , surgical scrubs and skin antiseptic
Chemical Antimicrobial Agents - Alcohols
- Denature protein when mixed with
water - Dissolve lipids (cell membranes)
- Effective against bacteria & fungi, but not endospores unenveloped viruses
- Evaporates quickly (low exposure time)
- Used as skin antiseptic (isopropanol or ethanol; effective at 60-95% (v/v)
Chemical Antimicrobial Agents - Heavy Metals and their compounds
- Selenium, Mercury, Copper, and Silver
- Very effective in small quantities (oligodynamic action)
- Selenium sulphide: Kills fungi; Anti-dandruff shampoo
- Silver wound dressings; Calamine lotion
Chemical AntiMicrobial Control - Oxidising Agents
- Disrupt disulfide bonds - hydrogen peroxide is used to clean puncture wounds, potassium permangenate to disinfect instruments.
Chemical Antimicrobial Agents - Alkalating Agents
Disrupt structure of proteins and nucleic acids.
Fomehaldehyde is used to inactivate viruses without destroying antigenic properties, glutaraldehyde to sterilize equipmentn
Chemical Microbial Agents - Dyes
- May interfere with replicatoin or block cell wall syntheisis
- Acridine is used to clean wounds, crystal violet to treat some protozoan and fungal infections.
Name the Physical Agents used as control methods
- Heat (Various Methods)
- Refrigeration
- Desiccation
- Irradiation
- Filtration
Heat
- Cheapest, most effective and widely used control
- Denatures enzymes
- Suitable for materials undamaged by heat
- Various approaches:
- Dry heat
- Moist heat
- Pasteurisation
Dry Heat
- Used to sterilise metal objects and glassware
- Flame: Innoculating loops, flasks/tube mouths
- Dry heat sterilisation for moisture-sensitive materials
- Longer heating times/temperatues than moist heat
Moist Heat
- Causes denaturation of proteins and may disrupt
membrane lipids-widely used - Boiling water can kill most vegetative bacteria and fungi
- Heating water under pressure (higher temps) in an
autoclave: 121 °C for 15-20 min can kill also spores
Pasteurisation
Invented by Pasteur; does not achieve sterility
Kills pathogenic organisms in raw products (milk etc)
- Flash method: 71.6 °C for 15 sec
- Holding method: 62.9 °C for 30 min
- Ultrahigh temperature processing (UHT):
74°C— 140°C— 74°C (5 sec) Can be stored at RT
Physical Agents - Lower temperatures
- Refrigeration: (2-8 degrees) Slows growth based on reduction in enzyme kinetics
- Freezing : (-20 degrees) slows metabolic activity to prevent food spoilage but does not kill organisms.
Physical Agents - Dessication
- Water absence inhibits enzymatic activity
- Usually bacteriostatic, but may be bactericidal depending
on the species - Used to preserve some foods
- Freeze-drying: Lyphilisation - used to preserve bacterial cultures.
Physical Agents - Radiation. Name the 3 main types
- Ultraviolet (UV) light
- Ionising Radiation
- Microwave Radiation
Ultraviolet (UV) light
Ultraviolet (UV) light: (40 – 390 nm) Works best at 200nm;
Used for sterilising surfaces
Damages DNA and proteins; DNA repair can aid survival
Ionising Radiation
Ionising Radiation: X-rays and gamma rays (< 40 nm) – dislodge electrons from atoms, creating ions and radicals.
Radicals kill by interacting with DNA, proteins, lipids etc.
Microwave Radiation
(1mm – 1m) Acts on water molecules which release
heat – not effective on spores
Physical Agent - Filtration
- Passage of liquid/gas through small pores; traps in a sizespecific manner
- Membrane filters: Specified pore sizes (0.025 – 25 µm)
- Suitable for heat-sensitive materials (e.g. media
supplements, drugs, vitamins) - HEPA (High-efficiency particulate air) filters in labs
What pore sizes of membrane filters are used most often?
- 0.45 - a few bacteria, viruses molecules
- 0.22 - viruses, molecules
- 0.10 - medium-sizes to small viruses molecules
Physical Agent - Osmotic Pressure
High salt/sugar concentrations draw water from cells
Bacteriostatic – metabolism cannot progress
Food preservation: Curing, pickling, jams etc.
Sound
- Ultrasonic waves can cause bacteria to cavitate –
denatures proteins and disintegrates bacteria - Can be used to lyse bacteria to release components for
study, but not practical for sterilisation.
Physical Antimicrobial Agent - Strong Physical Light
- Oxidation of light-sensitive materials - can be used with dyes to destroy bacteria and viruses: may help sanitize clothing.
What are the methods for evaluating a disinfectant?
- Phenol coefficient
- Filter Paper Method
- Use-dilution Test
How do we decide whethere a disinfectant is effective?
Agents that prevent growth at the lowest concentrations
are considered the most effective disinfectants
Phenol coefficient
- Comparison to the ‘original’ disinfectant
- Some disadvantages
Use-dilution tests
- Test bacteria coated onto carrier rings
- Incubated in test solutions (time based on product)
- Cultured in broth to detect surviving microbes
Filter Paper Method
Efficacy of a Chemical Agent applied to a filter paper disc
In vitro results may not translate in practice
Name the methods for Measuring sensitivity to an antimicrobial agent.
(antibiotic susceptibility testing)
- Disc-diffusion (Kirby-Baeur) method
- E (epilsometer test)
- Dilution Method
Disc-diffusion (Kirbay-Bauer) method
(antibiotic susceptibility testing)
- Lawn of target bacteria spread over plate
- Specific concentrations of antimicrobial agents on filter paper discs
- Zones of inhibition measured (diameter)
- Issues due to bacteriostatic vs. bactericidal actions
E (epsilometer test)
antibiotic susceptibility testing
- E (epsilometer) test determines sensitivity to antibiotic and also
minimum inhibitory concentrations (MIC) – antimicrobial gradient - Gradient of antibiotic concentrations on a strip (high to low)
- Intersection of zone of inhibition with the strip used to determine the MIC
The dilution method
(antibiotic susceptibilty testing)
- Bacteria inoculated into serial dilutions of an antimicrobial agent
MIC can be determined - Measuring sensitivity to an antimicrobial agent
(antibiotic susceptibility testing) - Can combine with second test to determine if bacteriostatic or
bacteriocidal (minimum bactericidal concentration; MBC)
Antimicrobial agent/drug
Chemical substance used to treat diseases cause by pathogenic mcrobes
Antibiotic
Chemical substance (drug) produced by microogransims, with the capacity to control growth/kill microbes.
Bacteriostatic or bacteriocidal
Synthetic or natural product (bacteria/fungi)
Who discovered antibiotics? Exaplain the discovery.
- Alexander Fleming (1929)
- Penicillium notatum inhibited S.aureus
- Recognised growth inhibition potential - Penicillin
- Mass production by 1940s (WW2)
- Post-war studies led to discovery of new antibiotics
Properties of useful chemoterapeutic agents
- Selectively toxic to pathogens; non-toxic to host and minimal effect on normal microbial flora – determine chemotherapeutic index
- Should not stimulate an allergic reaction (hypersensitivity)
- Host should not destroy or neutralise drug before effective
- Pathogens should not easily become resistant to the drug
- Agent should reach the site of infection (solubility)
Chemotherapeutic index
Chemotherapeutic index. The ratio of the minimal effective dose of a chemotherapeutic agent to the maximal tolerated dose.
Spectrum of activity
Look at slide 22
Range of different microogranisms treatable with an agent
Broad Spectrum
Antimicrobial Agents
Broad spectrum: Active across a wide range of taxonomic
groups; useful for untargeted treatment without pathogen
identification
Narrow Spectrum
Antimicrobial Agents
- Narrow spectrum: Specifically targets small number of
organisms; may protect host microflora; reduces development
of drug resistance
Name the 5 major modes of action of common antimicrobial agents
- Inhibition of Cell Wall Synthesis
- Disruption of Cell Membrane Function
- Inhibition of Protein Synthesis
- Inhibition of Nucleic Acid Syntheisis
- Action as Antimetabolites
Name the antibiotics used for the Inhibition of cell Wall Synthesis
- Penicillins
- Cephalosporins
- Bacitracin
- Vancomycin
Penicllins - Inhibition of Cell Wall Syntheisis
Mechanisms of antimicrobial agent activity
- Penicillins: Bactericidal; All contain a β-lactam ring;
- Natural penicillins (G, V) are penicillinase sensitive and
narrow spectrum. - Semi-synthetic penicillins are modified to increase penicillinase resistance and broaden spectrum (e.g.methicillin, ampicillin, amoxicillin)
- Interfere with PG cross-linking: Targets PBP
Cephalosporins - Inhibition of cell wall synthesis
Mechanims of Antimicrobial Agent Activity
- Cephalosporins - effective against Gram-negatices
- Resistant to Penicilliniases
- More expensive; mainly IV/IM
- Interfere with PG cross-linking Targets PBP
Bacitracin- Inhibition of cell wall syntheis
Mechanisms of Antimicrobial Agent Activity
- Bacitracin: Polypeptide antibiotic
- Effective against Gram positives
- Topical application
- Interfere with linear PG strand formation
Vancomycin
- Vancomycin: Glycopeptide antibiotics;
- Narrow spectrum;
- toxic
- Penicillinase-resistant
- Interfere with linear PG strand formation
Penicillin and Cephalosporin both bind to target enzymes (PBP) via B-lactam ring. Penicillin has 1 R site, Cephalosporin has 2. Why is this significant?
PBP- transpeptidase
- Peniclllin has one R site, which means it can only bind to one target in bacteria. This limits its spectrum of activity and makes it more effective against certain types of bacteria than others.
- Cephalosporsins, have two R sites, which gives them a broader spectrum of actiity and makes them more effecive against a wider range of bacteria than penicllin. These two R sites also make cephalosprins more resistant to certai bacterial enzymes than can break down penicllin.
look at slide 25 for the common method of administration of each.
Describe how the disruption of cell membrane Functions.
Mechanism of antimicrobial agent activity
Alter permeability of bacterial cell membranes; Polypeptide
antibiotics; clinical application limited to certain members due to
similarities between host and bacterial cell membranes
Polymyxins - Disruption of cell membrane function
- Polymyxins: (A, B, C, D, E) used only when pathogen is
resistant to other less toxic antibiotics; Gram negatives; Topical
Nystatin - disruption of cell membrane function
combine with sterols; therefore effective against
mycoplasmas (and also systemic fungal infections).
Colicins - disruption of cell membrane function
- Bacteriocin of E.coli; encoded by Col plasmid, which
also codes for immunity protein. - Kills bacterial cells not carrying the plasmid e.g. cells of same species.
Bacteriocin
- Similar to antibiotics
- Produced only by bacteria
- Specific to certain bacterial strains , wheras antibiotics can be effective against a broad range of bacteria.
Inhibition of Protein SYntheisis
Takes advantage of differences between bacterial and eukaryotic ribosome - selective toxicity
Aminoglycosides - inhibition of protein syntheisis
- Amino sugars linked by glycoside bonds. Broad
spectrum. - Bacteriocidal; bacteriostatic at lower doses. Work
synergistically with other compounds (e.g. penicillin) - Streptomycin (1940s); now has high resistance levels; toxic.
- Other compounds (kanamycin, gentamicin etc.)
Tetracylines - inibition of protein syntheisis
Tetracyclines: Interfere with tRNA attachment. Bacteriostatic; very
broad spectrum; can inhibit intestinal microflora
Chloramphenicol - Inhibition of Protein Synthesis
Bacteriostatic; broad spectrum ; inhibits peptide bond formation. Damages bone marrow.
Macrolides - Inhibition of protein syntheisis
Macrolides: Erythromycin – Bacteriostatic. Can not penetrate
Gram negative cell walls. Used as an alternative to Penicillin
Inhibition of nucleic acid syntheisis
Can be Toxic to host cells due to common targets.
* Rifampin: Inhibits mRNA synthesis by binding RNA polymerase;
High absorbance into tissues and cells; Bactericidal; Broad
Spectrum; Interacts with other drugs; Treatment of mycobacteria
infections-leprosy and tuberculosis
* Quinolones: Blocks bacterial enzyme that unwinds DNA prior to
replication; Broad spectrum; Used for UTIs; Can affect cartilage
development
Actions as Antimetabolites
Compounds that interfere with metabolic reactions by:
1) Competitive inhibition of enzymes or
2) Erroneous incorporation into important molecules
