Disinfection, Sterilisation & Antibiotics Flashcards
Why control the growth of bacteria? (3)
- Prevent infection of humans, other animals and plants
- Prevent spoilage of food
- Prevent contamination of industrial processes & products
Define; -
- Sterilization
- Disinfection
- Preservation
- Antiseptic
Sterilization: An absolute term meaning destruction or removal of all life forms - esp. microorganisms
-something is either sterile or not
Disinfection: Killing or removal of organisms capable of causing infection (reduce load so that not a problem)
-focuses on pathogenic organisms
-usu on inanimate objects & surfaces w/ chem. agent
Preservation: Treatment or condition that prevents growth of microbes
-usu used for food
Antiseptic: Chem. agent (milder action) used on skin or in living tissues that kills or inhibits microbes
-has to be milder so doesnt damage living tissues
How bacteria die on exposure to physical or chemical factors
-D Value
- Populations of bacteria die at an exponential rate
- not all bacteria are equally sensitive due to different metabolic states, mutations etc.
- Most sensitive bacteria die first, most resistant bacteria die last
D Value: Time taken for a 10 fold reduction in a population
- shorter the time = more effective/efficient the treatment is
- is the way rate of death is measured
Physical Agents for sterilisation (4)
- Heat
i) Incineration
ii) Dry heat
iii) Wet heat - UV radiation
- Ionizing radiation
- Filtration
Sterilisation using heat
-effects of heat
Incineration - what it is and what it’s used for
-Heat degrades nucleic acids, causes disruption and melting of cell membranes and coagulation/denaturation of proteins
i) Incineration: Flame sterilisation, commonly used in laboratory for glass spreaders, wire loops and metal spatulas
- also used for clinical waste and farm buildings
Sterilization using heat; Dry heat
- What it is and does
- what it’s used for - how long & temp
- 2 requirement for it to work
-Dry heat oxidizes cytoplasm as it heats in the absence of water (i.e. in an oven)
*This method requires long times of exposure and very hot temperatures
-not that effective
Used for glassware in lab - 170 degrees Celsius for 2 hours
Sterilization using heat; Wet heat
- what is it
- 3 ways to achieve wet heat
-Moist heat is more efficient than dry heat because tends to coagulate microbial protein
Ways to achieve wet heat;
- Boiling
- Pasteurization
- Tyndallization
Wet heat; Boiling
- What it is effective & ineffective on
- Downfall
(100 degrees Celsius)
-Rapidly kills vegetative bacterial cells, altho viruses can survive longer and spores can survive very long periods (i.e. 20 hours)
*Boiling doesn’t sterilize - therefore inadequate for surgical instruments
Wet heat; Pasteurisation
- What it is used for
- 3 types
-Common for milk products; kills pathogens but not spores
i) LTH = low temp holding (63 deg. for 30 mins)
ii) HTST - “flasth methods” (7 deg for 15-30 secs)
iii) UHT - Ultra - heat treated (150 deg for 3-4 secs)
Wet heat; Tyndallization
- Process & what it targets
- what it doesn’t guarantee
- Is the process of heating material in hydrated state, let material cool so spores germinate and then heat up again before bacteria can sporate. Repeat
- sequential three day cycle of 100 deg for 20 mins used to treat soils (kills spores)
*Doesn’t guarantee sterility in many situations
How to achieve sterility w/ Wet Heat?
- What steam usu works agains
- 4 ways to increase the efficiency of killing by moist heat
- Way steam is altered to be most efficient
-Steam (100 deg) rapidly kills vegetative bacteria, but not viruses & spores
Efficiency of killing by moist heat affected by:
1. Hydration
2. Time of exposure
3. Temp of exposure
4. pH
- Steam under pressure leads to higher temperatures -> rapidly kills bacteria, destroys viruses and spores
- is the most efficient and effective process for routine sterilization
-Carried out using autoclave
Sterilization using UV radiation
- What dose is lethal
- effects
- what it is used for and what it can’t penetrate
- Lethal at 260nm
- Affects DNA: causes thymine dimers that result in mutations and misreading of genes
- Used to sterilize surfaces, atmospheres
- Doesn’t penetrate glass, plastic, paper, water
Sterilization using Ionising Radiation
- E.g.
- What it is good for, not good for
- What it is used for
- Gamma rays
- Excellent for sterilizing agent for bacteria and spores, but not effective for viruses
- Used to sterilize plastics, petri dishes, gloves, syringes; cold sterilization for antibiotics, hormones
Sterilization using Filtration
- What is it & how achieved
- What it is effective and not so effective for
- What it used to sterilize
- Physical removal of microbes using cellulose acetate filters
- achieved by air flow in laminar flow cabinets
- Effective process for bacteria and spores but not so effective for viruses
- Used to sterilize heat sensitive solutions such as serums (vaccines, antibiotics, vitamins)
Techniques for Preservation (3)
- inhibition of bacterial growth*
1. Low temp (freezing, refridgeration)- microbiologists use ultra cold storage to store microbes for years
- Dehydration: Makes water unavailable to bacteria - bacteria need water to grow
- e.g. jams (high sugar content), salted foods - both have high osmotic potential
- Preservatives
- e.g. Chemicals that inhibit bacteria, nitrite in ham, bacon and cured meats, sulfites in dried fruits, wine
- microbiologists use ultra cold storage to store microbes for years
Chemical methods for Sterilization
- comparison to physical sterilization
- e.g.
- Are slower and less effective than physical sterilization and w/ side effects such as mess, staining, toxicity
e. g. Vapourised and gaseous agents, Disinfectants and antiseptics and antimicrobial agents
Disinfectants and antiseptics
- Soaps
- Alcohols - most effective conc., effects
- Aldehydes - effects, cons, e.g.
- Soaps (i.e. used in handwashing) - key action is more physical (mechanical removal of bacteria)
- antiseptics such as triclosan can be added to inhibit or kill bacteria
- Alcohols: 70 % (v/v) conc. most effective (30% water helps absorption into membranes
- causes membrane damage, protein denaturation
- not effective for spores
- Aldehydes: Very toxic compounds; strong irritants, strong reducing agents (adverse effects on living)
- inactivate enyzmes, proteins, nucleic acid, will kill spores
i. e. formaldehyde: need long exposure as is poor at penetrating
- inactivate enyzmes, proteins, nucleic acid, will kill spores
Disinfectants & Antibiotics; Ethylene oxide gas
- challenge
- what it is used for
-Is a toxic gas; useful for sterilizing large materials (i.e. buildings)
-key challenge: removing gas
Used for;
-Plastics
-medical equip
-surgical supplies
-vaccines
-buildings
Disinfectants & antibiotics; Halogens (3 types)
*Are oxidising agents - oxidise cell components
- Chlorine - water supplies
- Hypochlorite (i.e. household bleach) - cheap, corrosive
- used for: dairy utensils, pools, nappies
- Iodine (used as antiseptic) - binds to proteins and is an oxidant
- e.g. betadine
Disinfectants & antibiotics; Phenolics
- features
- 1 eg
- Are very toxic compounds - are irritants
- denature proteins, disrupt membranes
- Neurotoxin when absorbed
e.g. hexachlorophene good for Staphylococcus
Disinfectants & antibiotics; Heavy Metals; Cationic Detergents; Acids & alkalis
Heavy metals: inactivate proteins
e.g. silver, mercury, copper
Cationic detergents: quaternary ammonium compounds - destroys cell membranes, denatures proteins
-e.g. Benzalkonium chloride
-low toxicity, biocidal effect, better at killing gram neg. bacteria
Acids & Alkalis: detergents and germinides
Factors that affect chemical killing (interactions determine effectiveness of treatment) (7)
- Nature of chemical used - mechanism of action
- Population size - larger populations take longer to kill
- Contact time - death proportional to contact time
- Cell physiology - young cells more sensitive (log phase vs stationary phase)
- Temp - rate of killing increases w/ temp
- Local enviro - organic matter decreases effectiveness (chem. binds to organic matter - effectively decreases the conc. of chemical available to bind to bacteria)
- Conc. of chemical
Criteria for effective Antibiotics (7)
- Active at high dilution
- selective toxicity (affects microbes, not host)
- Non-allergic to host cells
- Stable in storage and in body
- Soluble in body fluids (blood - means it can be injected)
- Retained in body at effective conc (otherwise excreted rapidly in urine)
- Target micro-organism and slow for microorganisms to develop resistance
Action of antibiotics (2)
Bacteria either have a;
- Bacteriostatic effect (Only inhibits growth of bacteria - does NOT kill)
- Bactericidal effect (kills bacteria)
6 ways antibiotics work
- Have v. specific modes of action. Interefere w/ structure, replication or metabolism
1. Cell wall synthesis (penicillin, cephalosporins)
2. mRNA translation (aminoglycosides)
3. Plasma membrane disruption (polymyxins)
4. DNA synthesis (quinolones -nalidixic acid)
5. Gene transcription (rifampin, nalidixic acid)
6. Enzyme activity (sulphonamides)
Antibiotic mode of action; Cell wall synthesis
- penicillin: stops cell making peptidoglycn for new walls
- only active on growing cells
- more effective of G +ves
- beta-lactam ring susceptible to degradation by bacterial beta-lactamase enzymes
- is a bacteriocidal
- cephalosporins; similar mode of action to penicillins, tho increased spectrum of activity
- also susceptible to beta-lactamase enzymes- new generations of drugs progessively available - less susceptible to enzymes (tho as antibiotics evolve, so do bacterial enzymes)
Natural Penicillins - advantages and disadvantages
Advantages;
- Low toxicity
- potent activity
- easy administration (injection, oral)
Disadvantages;
- Narrow spectrum
- acid sensitivity
- Easily excreted
- Resistance - through penicillinase (breaks down penicillin)
Ways to overcome the disadvantages of Penicillin (2)
- Produce different penicillins - grow fungus on different substrates to get different variable or R group on penicillin backbone (changes the chemical nature of penicillin)
- Semi-synthetic penicillins (most of penicillins today)
-Diff R groups added to natural penicillin
ampicillin - acid resistant, broad spectrum
Antibiotic mode of action; Inhibition of protein synthesis
- 4 classes
-bind to ribosomes to inhibit translation of mRNA to proteins
-only active on growing cells (i.e. those in exponential phase)
-can be bactericidal or bacteriostatic
4 classes;
1. Aminoglycosides
2. Tetracyclines
3. Macrolides
4. Chloramphenicol
Classes of antibiotics inhibiting protein synthesis; Aminoglycosides
- how it works
- e.g.
- attach to 30 S ribosomal subunit - causes misreading of mRNA and wrong protein produced
- bactericidal effect
- effective against gram +ve (get taken up easier)
- toxic to humans - auditory nerve, kidneys
e.g. streptomycin, kanamycin, gentamicin, neomycin
Classes of antibiotics inhibiting protein synthesis; Tetracyclines
- method
- side effects
- Attach to 30 S ribosomal subunit
- block attachment of tRNA to A site - no protein produced
- bacteria are inhibited
- Bacteriostatic effect - bact. aren’t killed
- broad spectrum (+ve & -ve)
- Side effects: inhibit normal micro-flora, liver damage, discoloured teeth in children
Classes of antibiotics inhibiting protein synthesis;Macrolides
- method
- common side effects
- e.g.
- attach to 23S rRNA of the 50 S ribosomal subunit
- inhibits peptide chain elongation therefore protein not produced
- bacteriostatic effect (once antibiotic taken away, bact can grow)
- broad spectrum
- Common side effects - anemia, kidneys, allergic response
e.g. erythromycin, clindamycin
Classes of antibiotics inhibiting protein synthesis; Chloramphenicol
- method
- side effects
- Attach to 23S rRNA on 50S subunit
- inhibits peptidy transferase activity therefore no protein produced
- bacteria inhibited = bacteriostatic effect
- broad spectrum
- side effects: can inhibit formation of blood cells in bone marrow = aplastic anemia
Antibiotic Sensitivity Testing
- Inoculate “lawn of pure culture on agar plate
- filter discs with different antibiotics
- Zone of inhibition = level of sensitivity (size of death zone around disc directly related to antibiotic efficiency)
*Kirby-Bauer method (relates mm of zone to how sensitive bacteria is)
Minimum Inhibitory concentration (MIC)
- Can be done as an agar dilution test or as a broth dilution test
- make dilutions of antibiotic in broth
- add equal bacteria inocula to each dilution
- note last tube in which bacterial growth is inhibited = M.I.C. (is just before where conc of antibiotic is too low)
*Aim for 2-4 x MIC in blood or 10-20 x MIC in urine
General Considerations for antibiotics (common sense)
- Antimicrobials assist natural defense mechanisms, NOT replace them
- must always be used at full therapeutic doses for adequate period
- narrow spectrum antibiotics preferable to those w/ broad spectrum for sensitive organisms
- bactericidal preferable to bacteriostatic
- not be used for mild infections
- avoid use for routine prophylaxis - do not use as alternative for surgical asepsis and other forms of disease control
- not v. effective against abscesses or foreign bodies & prolonged treatment is required for intracellular parasites
- Antibiotic w/holding times important when treating production animals (all about when animal can enter food chain)
Potential adverse effects of antibiotics (2 types)
- Direct Toxic effects
- local irritation at site of administration
- systemic toxic effects on tissues distant from site of administration
- Indirect adverse effects
- effects on normal microbial flora
- immune responses possibly depressed
- hypersensitivity to drug
- masking of the disease
Antibiotic Resistence
-4 examples of infectious organisms that are drug resistant
*Major world wide problem
Many infectious organisms are drug resistant, i.e.
- Mycobacterium tuberculosis - tuberculosis
- Staphylococcus aureus - golden staph
- Neisseria gonoffhoeae - gonorrhea
- Shigella sonnei - dyssentry
How do Bacteria resist drugs?
- Enzyme breakdown (i.e. penicillinase)
- Modify antibiotic so it is no longer in a chemical form that is effective
- Prevent drug uptake
- Drug efflux (export drug out)
- Produce more affected enzyme (that is effected by enzyme)
- Change receptor for drug (i.e. if part of seq of ribosome that binds to antibiotic changed, antibiotic can no longer bind)
Why does resistance develop?
- Misuse of antibiotics
- use of sub-lethal doses (doesn’t kill whole popuation)
- Incorrect antibiotic
- incorrect method of administration
- stopping treatment too soon
- bacteriostatic antibiotic inhibits bacteria only - bacteria are removed by natural immune system
- if treatment stopped too soon, body may not have gotten rid of all bacteria yet
How do bacteria get resistant? (2 methods)
Acquire antibiotic resistance through:
- Mutation: spontaneously producing new genes
- Exchange of DNA: Reason for rapid spread of resistance genes
- bacteria have the capacity to transfer genes b/w themselves (i.e. via plasmids)
Why does resistance spread?
- Resistant bacteria survive during drug use, then multiply & increase
- if plasmid borne resistance - they transfer the resistance genes to other bacteria
Combining drugs - can we do it? -2 types of drug interactions
- Sometimes
- Synergistic interaction = 2 drugs in combo may be better/more efficient at killing bacteria
- i.e. penicillin & streptomycin for endocarditis - penicillin damages cell wall then streptomycin taken up faster
- Antagonistic interaction = when 2 drugs in combination can be harmful
- i.e. penicillin & tetracycline - tetracycline inhibits growth of bacteria, prevents penicillin damaging cell wall (penicillin requires growing bacteria) - after treatment infectious organism can grow
Drug development - steps (5)
- is a very long & expensive process
1. Screen exotic organisms against test microbes
2. Isolate active chemical
3. Determine & evaluate chem. structure
4. Animal tests
5. Clinical trials - can be a 5-20 year process
Possible alternatives to antibiotics
-Growth promotion (banned in EU)
-Prophylaxis
-Treatment
Alternatives;
-acidifiers, probiotics, prebiotics, herbs & spices, esential oils, honey for wounds
- improved management, hygiene, diets, etc.
- Bacteriophage therapy, bacterocins, new antimicrobials
- new vaccines
The future of antibiotics
-Increased misuse of antibiotics leads to increased resistance
