Microbial Growth Control-week 7 Flashcards
1
Q
What are the basic principles of control?
A
- Microorganisms have specific environmental requirements
- Certain structures and/or processes are essential
- Any control strategy has to be tailored
- What(is the object being treated)
- How many (microorganisms are present)
- What types/species are present
- What is the end goal?
- Not all control methods are equally effective
- Not all microorganisms are equally susceptible
2
Q
What are the 5 examples of antiseptic agents?
A
Alcohol
Iodophors
Chlorexidine
Parachlorometaxylenol
Triclosan
3
Q
What microorganisms are susceptible to alcohol
?
A
Bacteria, myobacteria, fungi and viruses
Bacterial spores are not susceptible to alcohol
4
Q
Describe heat as a common treatment for controlling microbial growth
A
Moist heat:
- boiling
- Autoclaving
Dry heat
-ovens
Incineration
Uses includes
Glassware
Lab media
Surgical instruments
5
Q
Why does heat act as a medium for controlling microbial growth?
A
Coagulates proteins: hydrogen bonds are broken
Native protein becomes coagulated proteins
6
Q
What is an autoclave?
A
A variation of a pressure cooker
7
Q
What is the correlation between steam and temperature ?
A
Steam is put under pressure, to result in higher temperature
Steam @ 1 atm(normal pressure)=100 degrees celsius
Steam @ 2atm(+15 psi)= 121 degrees Celsius
This temperature for 15 minutes kills most microorganisms and their spores
8
Q
What are the advantages of using autoclave/steam/ pressure to kill microorganisms?
A
- Can be used on a range of items
- Effective; inexpensive
- Damages a wide range of microorganisms
9
Q
What types of radiation are used to control microbial growth?
A
Non-ionizing radiation: Ultraviolet(UV)
Ionizing: gamma or X-ray
10
Q
What is used on objects that cannot be autoclave?
A
Radiation
11
Q
What objects use radiation when controlling microbial growth?
A
- Heat sensitive plastics e.g., gloves, Petri dishes
- Food items, including spices
- Biological hoods/cabinets, surgical rooms
12
Q
Contrast the wavelengths of ionizing and non-ionizing
A
Non-ionizing: gamma and x-rays have 0.1-40 nm wavelength
Ionizing: UV range= 50-400nmwavelength
13
Q
Contrast ionizing and non-ionizing radiation in terms of chemical abilities and effect on DNA
A
Ionizing:
-Dislodges electrons from atoms
-Damages DNA and produces peroxides
Non-ionizing:
Absorbed by purines and pyrimidine bases and forms pyramiding dimers
14
Q
Contrast ionizing and non-ionizing in terms of penetration abilities
A
Ionizing : penetrates well
Nom-ionizing: doesn’t penetrate well: limited to surface sterilization
15
Q
When liquids cannot be autoclaved or radiated, how can microbial growth be controlled?
A
Filtration
16
Q
How can filtration be done to to control microbial growth?
A
Physical separation using a filter, with pores that block the microorganism
E.g. 0.25 micron filter: 1-2 micron bacteria are too large to pass through the holes of the filter
-Mainly for sterilization of small volumes of liquids that are heat-sensitive
17
Q
What are the basic principles of chemical control?
A
-Apply a chemical as a spray, solution, mist, etc.
- Certain chemicals can inhibit/halt, kill or lyse microorganisms
- effect can vary depending on:- microorganism
- type of compound
- concentration of the compound
- Some chemical control agents have very general mechanism of action
- e.g. damage cell membranes
- might also be toxic to human cells
- e.g. damage cell membranes
- Some are very specific
- e.g. target unique structures or processes
- less likely to be toxic to human cells
18
Q
How may a compound affect the growth of a microbe ?
A
Reactions include hydrolysis, oxidation, alkylation etc.
A. Reactions affecting proteins
B. Reactions affecting membranes
C. Reactions affecting cell components
19
Q
What is the mechanism of alcohol as a chemical control agent?
A
Lipid solvent and protein denaturant
20
Q
What is the mechanism of action of Hydrogen peroxide (3% solution) as an antiseptic)?
A
Oxidizing agent
21
Q
What is the mechanism of action of iodophors as a chemical control agent?
A
Iodinated proteins
22
Q
What is the mechanism of action of chlorine compounds(e.g. sodium hypochlorite [bleach] )?
A
Oxidizing agent
23
Q
What is the mechanism of action of ethylene oxide gas as a chemical control agent?
A
Alkylating agent
24
Q
What is the mechanism of action of pine oils (e.g. pine-sol) as a chemical control agent?
A
Protein denaturant
25
What are the possible outcomes of antimicrobial growth that can be due to antimicrobial agents?
Static- total cell count and viable cell count plateau.
Cidal- total cell count plateaus while viable cell count decreases
Lytic- both total cell count and viable cell count decrease
26
What are the features of sterilants, disinfectants and sanitizers used?
Non-living surfaces
E.g. sodium hypochlorite[bleach], gluteraldehyde, ozone
-inhibit or kill microorganisms present on inert surfaces
```
-Nonspecific mechanisms
Damage cell membranes
Denature proteins
Inactivate enzymes
-Not usually suitable for use on living tissues
```
-can be combined with e.g. a surfactant(for better cleaning
27
What are applications of antiseptics?
1. Reduce numbers of microorganisms from mucous membranes before surgery e.g. iodine
2. Remove microorganisms from skin before an injection
E.g. alcohol
28
What are therapeutic or “in Vivo”antimicrobials?
Natural or synthetic compounds with activity against microorganisms, that can be used therapeutically to treat infection
- some can be used as creams, powders or lotions others are given orally or by injection
- Tend to have more specific targets(such as a specific enzyme) compared to chemical control agents that might target e.g., membranes
29
How are antimicrobials classified?
A. Molecular structure
B. Mechanism of action, e.g. -protein synthesis inhibitors
C. Spectrum(range) of activity
D.2 basic categories of antimicrobials:
1. Synthetic antimicrobial drugs
2. Naturally occurring antimicrobial drugs “antbiotics”
30
Give some examples of molecular structures of therapeutic antimicrobials
Tetracyclines e.g. tetracycline protein inhibitors
Glycopeptides e.g. Vancomycin
31
Describe how main targets/ mechanism of action may differ among therapeutical antimicrobials
1. Protein synthesis- 30s tetracyclines, 50s chloramphenicol
2. Cell wall synthesis- B lactams
3. Cell membrane function- Polymyxins
4. metabolism- trimethoprim
5. Nucleic acid synthesis- sulfonamides
32
What is selective toxicity?
Requirement for targeted effect on the microorganism, with minimal damage to host cells
33
Of all the drugs, only a fraction are clinically useful, why?
Side effects: toxic or harmful
-cellular damage to host
34
Describe synthetic antimicrobials
E.g, chemical dyes, manufactured compounds
Started with the work of Ehrlich and Domagk
Based around the idea of growth factor analogs:
- structurally like a growth factor, e.g. a purine
- But: subtle differences stop the microorganism from using it properly
E.g. sulfonamides, quinolone
35
Describe antibiotics
.g. Naturally produced by soil fungi, other bacteria
Can be subsequently modified, e.g. to make them acid-stable (can be swallowed) or extend the range of bacteria they work against
Various microbial targets, including:
I. Interfering with protein synthesis
II. Interfering with bacterial cell wall synthesis
36
What is a basic mechanism of antibiotics?
Basic mechanism: inhibit translation by interacting with ribosome
Examples of antibiotics in this category:
Amino glycosides-target 30S subunit: e.g., streptomycin-
Macrolides - target 50s subunit e.g. azithromycin
Tetracyclines-target 30s subunit e.g. tetracycline
37
Describe the antibiotics that interfere with peptidoglycan synthesis
The antibiotics are structurally similar (“analogs”) to D-ala-D-ala
- bind to and Schlage the transpeptidase enzyme
- Can’t function —> no cross-linking
- examples of antibiotics with this mechanism includes the B-lactate antobiotics
- penicillins group (e.g. Amoxicillin, Penicillin G)
- Cephalosporins
38
Do therapeutic medicine categories work for other compounds they are designated for?
Antibiotics don’t work against fungi, antifungals don’t work against viruses etc.
With one or two exceptions
39
What are the basic principle common with all therapeutic compounds?
Interfere with a fundamental process or structure
40
What structures are affected by antifungals?
Fungal cell wall, cytoplasmic membrane
41
Why are there much less antivirals than antibiotics?
Treating viral infections requires compounds that have different targets relative to antibiotics
42
What is the rationale of inhibitors of reverse transcriptase?
Due to genetic variation material of virus, replication of nucleic acid requires the virus to encode for a specialized enzyme
43
Some antivirals inhibit reverse transcriptase, why is this a good strategy?
Reverse transcriptase is UNIQUE to certain viruses (e.g. HIV, HBV); no equivalent exists in the eukaryotic cells
44
What is the mechanism of inhibiting reverse trsnscriptase?
Nucleoside analog*
- Activated (phosphorylated) inside the infected cell
- Inhibit the viral polymerase (reverse transcriptase)
45
Give examples of antivirals which inhibit reverse transcriptase
Zidovudine(AZT)
Stavudine
46
What is the general challenge with viruses?
How to target the virus without (negatively) impacting the host cell that it is replicating in, using the enzymes of, etc.
Inhibiting reverse transcriptase is a common mechanism in antivirals. But other polymerases can be affected
47
What is the outline of inhibiting viral enzymes and RNA polymerase?
1. Acyclovir (acycloguanosine)converted by a viral thymidine kinase
2. This creates ACV-monophosphate which is converted to cellular thymidine kinases
3. This creates ACV-diphosphate
4. ACV-triphosphate then inhibits viral DNA polymerase
48
What is the mechanism usually used by antifungals?
Inhibitors of Ergosterol
49
What is the rationale of antifungals inhibiting ergosterol?
By impacting ergosterol, the fungal cell membrane is weakened and doesn’t function properly
50
Why is inhibiting ergosterol for antivirals, a good idea?
Ergosterol is UNIQUE to fungi; not present in mammalian membranes
51
Give an example of inhibiting ergosterol for antifungals
A) bind to ergosterol and cause formation of pores —> leakage of cell contents and cell death
B) interfere with pathway for ergosterol formation —> weakened fungal formation
52
Give examples of antifungals that inhibit ergosterol
1. Bind directly to ergosterol: polyenes
| 2. Inhibit fungal enzyme used in ergosterol synthesis: azoles
53
Outline how antifungals bind directly to ergosterol to inhibit it
1. Bind directly to ergosterol such as polyenes e.g. Amphotercin B
Antifungals that do this: polyenes e.g. amphotercin b
- normal role of fungal sterols = maintain structure and function of membrane
- binding of antifungal to sterols —>
- channel function
- leakage of f cytoplasmic contents
-death of the fungus
54
Outline how antifungals may inhibit ergosterol synthesis pathway
Antifungals such as Azoles e.g. fluconazole
- Target the enzyme that converts lanosterol into ergosterol
- Lanosterol builds up in fungal cell
- Cell membrane isn’t properly permeable
- Cell contents leak out
- fungi are killed
55
Why is microbial resistance/or lack of susceptibility a problem?
Drugs no longer work:
-higher, more toxic concentrations needed
- switch to a more expensive treatment
- longer period for treatment
- side effects
- most genes encoding for resistance are carried on mobile genetic elements such as plasmids and transposons
- enables rapid spread
-Single gene (1 antibiotic), or multiple —> resistance to many different antibiotics, or entire families(e.g., resistance to all B-lactate antibiotics)
56
What is resistance?
Normal therapeutic methods are no longer working
57
How can resistant bacteria increase in number due to antibiotics?
Resistant bacteria cannot be killed
Resistant bacteria multiply
Resistant genes are often encoded on transmissible genetic elements such as plasmids
58
How bacteria evade antibiotics?
-Prevent accumulation -decrease/block uptake
E.g. Multi-drug resistance pumps
- Change target site-Change in receptor site affinity, modification of target pathway
- Produce an inactivating enzyme- e.g. B-lactamases
Chloramphenicol acetyltransferases
59
Describe cross linkage in gram positive bacteria and the enzyme responsible
Cross linking of the peptidoglycan layers requires removal of terminal D-ala
Done by an enzyme: Transpeptidase/ penicillin- binding protein
60
What kind of bacteria is S. Aureus?
Gram positive
61
How can an organism get around being affected by an antibiotic such as penicillin?
Change the pathway, e.g. use a different or modified transpeptidase enzyme
- don’t have peptidoglycan
- Produce an enzyme that stops it from working
- grow very slowly or not at all
- if not synthesizing peptidoglycan, it won’t be affected
- pump the drug out before it accumulates to a high enough level inside the cell
62
How do some bacteria have resistance to B-lactamase?
- Some bacteria secrete a B-lactamase (an enzyme that lyses the B-lactam antibiotic)
- Inactivation of the antibiotic by the enzyme —> no effect on the transpeptidase enzyme —> normal cross-linking and bacterial growth
63
Why do we have problems of resistance?
1. Inclusion of antibiotics in animal feed as a growth supplement
2. Inappropriate use/prescription of antibiotics by physicians
3. Poor patient adherence to treatment regimes
4. Self medication with black market medications
5. Use of standard/improperly stored antibiotics
6. Natural bacterial response to a harmful selective pressure
64
Why is it important assess antimicrobial activity in clinical settings?
-prevents unnecessary use of ineffective, expensive and/or compounds with side effects
- Helps guide treatment decisions
- tests that help determine what works and at what concentration:
- MIC
- Disk diffusion assay
- PCR for specific resistance genes
65
Describe MIC( minimum inhibitor concentration) broth dilution method
MIC= 1st tube without visible growth
Plate out onto agar
MBC- plate without visible colonies
66
Describe eTest strips
- Another way to determine the MIC
- Plastic strip with graded concentrations of antibiotic
- Area where growth touches the strip is the MIC
67
Describe the Disk-Diffusion (Kirby-Bauer Technique)
-Bacteria are inoculated onto surface of a special type of media
- Discs containing set concentrations of antibiotic are added:
- antibiotic diffuses out from the disc:
- close to the disc= highest concentration
- further away= lower concentration
-plates are incubated
-Zones of inhibition (clearing) measured
-the larger the zone, the more sensitive the bacteria are
Because lower concentrations of antibiotics are inhibiting them
68
What are the multiple factors that determine whether microorganisms are impacted by a treatment?
Population size: larger the population, the longer it will take for the effect to be shown
Population composition: microbes differ in terms of susceptibility and also, presence of endospores increase time required
Time exposure
D. Environmental conditions-temperature, presence of organic matter(blood, pus, etc) and pH
69
What does sterilization mean?
Making something free of living microorganisms
70
What is a disinfectant?
A chemical liquid that destroys all pathogenic organisms except spores(May or may not be destroyed)
71
What is pasteurization?
The partial sterilization of a product, like wine or millk, to make it safe for consumption and improve its keeping quality
72
What does antisepsis mean?
Prevention of infection by inhibiting growth. Implies scrupulously clean of all living organisms
73
What disinfectants can destroy endospores?
Hydrogen peroxide, formaldehyde, chlorine, gluteraldehyde
74
What are the 2 most resistant microganksms?
Prions the most, endospores, the least
75
What microbes are the most sensitive?
Gram positive the second most sensitive
Envelopes virus the most
76
How are inert surfaces cleansed?
Physical- non ionizing radiation,
Chemical- disinfectants
77
How are lab and surgical equipment cleansed?
Physical- autoclave/heat and ethylene oxide
Chemical-certain disinfectants
78
How are lab media cleansed?
Autoclave and filtration
79
How is skin cleansed?
Chemical- antiseptics
Therapeutic- topical antimicrobials
80
How are mucus membranes cleansed?
Antiseptics and antimicrobials
81
How are tissues and organs cleansed?
Antimicrobials
82
What is the function and active components in listerine?
Active components- eucalyptol, menthol, methyl salicylate and thymol
Action- bacterial cell wall destruction
Bacterial enzymatic inhibition
Extraction of bacterial LPS1
83
What are the active components and function of Savlon?
Active components- cetrimide, chloroexidine, gluconqte
Function- quarternary ammonium compound
84
What are the active components of detox and it’s main functions?
Active components- chloroxyleno
Action-disrupt cell membranes
In activation of proteins
