Post on 25-Dec-2015
The Selection of Microbial Control Methods
•Factors Affecting the Efficacy of Antimicrobial Methods▫Site to be treated
Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects
Method of microbial control based on site of medical procedure
3
The Selection of Microbial Control Methods
•Factors Affecting the Efficacy of Antimicrobial Methods▫Relative susceptibility of microorganisms
Germicides classified as high, intermediate, or low effectiveness High-level kill all pathogens, including
endospores Intermediate-level kill fungal spores, protozoan
cysts, viruses, and pathogenic bacteria Low-level kill vegetative bacteria, fungi,
protozoa, and some viruses
5
The Selection of Microbial Control Methods
•Methods for Evaluating Disinfectants and Antiseptics▫Phenol coefficient
Evaluates efficacy of disinfectants and antiseptics by comparing an agent’s ability to control microbes to phenol
Greater than 1.0 indicates agent is more effective than phenol
Has been replaced by newer methods
7
The Selection of Microbial Control Methods
• Methods for Evaluating Disinfectants and Antiseptics– Use-dilution test
• Metal cylinders dipped into broth cultures of bacteria• Contaminated cylinder immersed into dilution of
disinfectant• Cylinders removed and placed into tube of medium to
see how much bacteria survived• Most effective agents entirely prevent growth at
highest dilution• Current standard test in the U.S.• New standard procedure being developed
8
The Selection of Microbial Control Methods
•Methods for Evaluating Disinfectants and Antiseptics▫Kelsey-Sykes capacity test
Alternative assessment approved by the European Union
Bacterial suspensions added to the chemical being tested
Samples removed at predetermined times and incubated
Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective
9
The Selection of Microbial Control Methods
•Methods for Evaluating Disinfectants and Antiseptics▫In-use test
Swabs taken from objects before and after application of disinfectant or antiseptic
Swabs inoculated into growth medium and incubated
Medium monitored for growth Accurate determination of proper strength
and application procedure for each specific situation
10
Physical Methods of Microbial Control
•Heat-Related Methods▫Effects of high temperatures
Denature proteins Interfere with integrity of cytoplasmic
membrane and cell wall Disrupt structure and function of nucleic acids
▫Thermal death point Lowest temperature that kills all cells in broth
in 10 min▫Thermal death time
Time to sterilize volume of liquid at set temperature
11
Physical Methods of Microbial Control
• Heat-Related Methods▫Moist heat
Used to disinfect (remove organisms and spores), sanitize (kill organisms but not necessarily their spores), and sterilize (kill all organisms and spores)
Denatures proteins and destroys cytoplasmic membranes
More effective than dry heat Methods of microbial control using moist heat
Boiling Autoclaving Pasteurization Ultrahigh-temperature sterilization
12
Physical Methods of Microbial Control
•Heat-Related Methods▫Moist heat
Boiling Kills vegetative cells of bacteria and fungi,
protozoan trophozoites, and most viruses Boiling time is critical
▫Different elevations require different boiling times Endospores, protozoan cysts, and some viruses
can survive boiling
13
Physical Methods of Microbial Control
•Heat-Related Methods▫Moist heat
Autoclaving Pressure applied to boiling water prevents steam
from escaping Boiling temperature increases as pressure
increases Autoclave conditions – 121ºC, 15 psi, 15 min
14
Physical Methods of Microbial Control
•Heat-Related Methods▫Moist heat
Pasteurization Used for milk, ice cream, yogurt, and fruit
juices Not sterilization
▫Heat-tolerant microbes survive Pasteurization of milk
▫Batch method▫Flash pasteurization (High temp, short time)▫Ultrahigh-temperature pasteurization (very high
temp, very short time)
17
Pasteurization of milkBatch method
• The batch method uses a vat pasteurizer which consists of a jacketed vat surrounded by either circulating water, steam or heating coils of water or steam.
In the vat the milk is heated and held throughout the holding period while being agitated. The milk may be cooled in the vat or removed hot after the holding time is completed for every particle.
18
Pasteurization of milkFlash method• High Temperature Short Time (HTST)• Milk is heated to 72°C (161.6°F) for at least 15
seconds. • Used for perishable beverages like fruit and
vegetable juices, beer, and some dairy products. Compared to other pasteurization processes, it maintains color and flavor better.
• It is done prior to filling into containers in order to kill spoilage microorganisms, to make the products safer and extend their shelf life. Flash pasteurization must be used in conjunction with sterile fill technology.
19
Pasteurization of milkUltrahigh-temperature method
• Heating for 1-2 seconds at a temperature exceeding 135°C (275°F), which is the temperature required to kill spores in milk.
• The most common UHT product is milk, but the process is also used for fruit juices, cream, soy milk, yogurt, wine, soups, and stews.
• Can cause browning and change the taste and smell of dairy products.
• UHT canned milk has a typical shelf life of six to nine months, until opened.
20
Physical Methods of Microbial Control
•Heat-Related Methods▫Moist heat
Ultrahigh-temperature sterilization 140ºC for 1 sec, then rapid cooling Treated liquids can be stored at room
temperature
21
Physical Methods of Microbial Control
•Heat-Related Methods▫Dry heat
Used for materials that cannot be sterilized with moist heat
Denatures proteins and oxidizes metabolic and structural chemicals
Requires higher temperatures for longer time than moist heat
Incineration is ultimate means of sterilization
22
Physical Methods of Microbial Control
•Refrigeration and Freezing▫Decrease microbial metabolism, growth, and
reproduction Chemical reactions occur slower at low
temperatures Liquid water not available
▫Psychrophilic microbes can multiply in refrigerated foods
▫Refrigeration halts growth of most pathogens▫Slow freezing more effective than quick
freezing▫Organisms vary in susceptibility to freezing
23
Physical Methods of Microbial Control
•Dessication and Lyophilization▫Dessication is drying (98% of the water is
removed) inhibits growth due to removal of water
▫Lyophilization (freeze-drying) Substance is rapidly frozen and sealed in a vacuum Substance may also be turned into a powder
▫Used for long-term preservation of microbial cultures Prevents formation of damaging ice crystals
24
The role of HEPA filters in biological safety cabinets
Figure 9.10
High-Efficiency Particulate Arresting (HEPA) air filters are used in medical facilities, automobiles, aircraft, and homes. The filter must remove 99.97% of all particles greater than 0.3 micrometer from the air that passes through.
27
Physical Methods of Microbial Control
•Osmotic Pressure▫High concentrations of salt or sugar in
foods to inhibit growth▫Cells in hypertonic solution of salt or sugar
lose water▫Fungi have greater ability than bacteria to
survive hypertonic environments
28
Physical Methods of Microbial Control
•Radiation▫Ionizing radiation
Wavelengths shorter than 1 nm Electron beams, gamma rays
Ejects electrons from atoms to create ions Ions disrupt hydrogen bonding, cause
oxidation, and create hydroxide ions Hydroxide ions denature other molecules
(DNA) Electron beams – effective at killing but do not
penetrate well Gamma rays – penetrate well but require hours
to kill microbes
29
Physical Methods of Microbial Control
•Radiation▫Nonionizing radiation
Wavelengths greater than 1 nm Excites electrons, causing them to make new
covalent bonds Affects 3-D structure of proteins and nucleic
acids UV light causes pyrimidine dimers in DNA UV light does not penetrate well Suitable for disinfecting air, transparent
fluids, and surfaces of objects
32
Physical Methods of Microbial Control
•Biosafety Levels▫Four levels of safety in labs dealing with
pathogens Biosafety Level 1 (BSL-1)
Handling pathogens that do not cause disease in healthy humans
Biosafety Level 2 (BSL-2) Handling of moderately hazardous agents
Biosafety Level 3 (BSL-3) Handling of microbes in safety cabinets
Biosafety Level 4 (BSL-4) Handling of microbes that cause severe or fatal
disease
33
Chemical Methods of Microbial Control
•Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA
•Effect varies with differing environmental conditions
•Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa
35
Chemical Methods of Microbial Control
•Phenol and Phenolics▫Intermediate- to low-level disinfectants▫Denature proteins and disrupt cell
membranes▫Effective in presence of organic matter▫Remain active for prolonged time▫Commonly used in health care settings,
labs, and homes ▫Have disagreeable odor and possible side
effects
36
Chemical Methods of Microbial Control
•Alcohols▫Intermediate-level disinfectants▫Denature proteins and disrupt cytoplasmic
membranes▫More effective than soap in removing
bacteria from hands▫Swabbing of skin with 70% ethanol prior to
injection
37
Chemical Methods of Microbial Control
•Halogens▫Intermediate-level antimicrobial chemicals▫Believed to damage enzymes via oxidation
or by denaturation▫Widely used in numerous applications
Iodine tablets, iodophores, chlorine treatment, bleach, chloramines, and bromine disinfection
38
Chemical Methods of Microbial Control
•Oxidizing Agents▫Peroxides, ozone, and peracetic acid ▫Kill by oxidation of microbial enzymes▫High-level disinfectants and antiseptics▫Hydrogen peroxide (H2O2) can disinfect
and sterilize surfaces Not useful for treating open wounds due to
catalase activity: the tissues convert it into H20 and 0ygen bubbles.
▫Ozone treatment of drinking water▫Peracetic acid is an effective sporocide
used to sterilize equipment
41
Chemical Methods of Microbial Control
•Surfactants▫“Surface active” chemicals
Reduce surface tension of solvents▫Soaps and detergents
Soaps have hydrophilic and hydrophobic ends Good degerming agents but not antimicrobial
Detergents are positively charged organic surfactants▫Quats (Quaternary ammonium cations)
Low-level disinfectants; disrupts cell membranes Ideal for many medical and industrial applications Good against fungi, amoeba, and enveloped viruses,
but not endospores, Mycobacterium tuberculosis and non-enveloped viruses.
42
Chemical Methods of Microbial Control
•Heavy Metals▫Heavy-metal ions denature proteins▫Low-level bacteriostatic and fungistatic
agents▫1% silver nitrate to prevent blindness
caused by N. gonorrhoeae▫Thimerosal used to preserve vaccines▫Copper inhibits algal growth
43
Chemical Methods of Microbial Control
•Aldehydes▫Compounds containing terminal –CHO
groups▫Cross-link functional groups to denature
proteins and inactivate nucleic acids▫Glutaraldehyde disinfects and sterilizes▫Formalin used in embalming and
disinfection of rooms and instruments
44
Chemical Methods of Microbial Control
•Gaseous Agents▫Microbicidal and sporicidal gases used in
closed chambers to sterilize items▫Denature proteins and DNA by cross-linking
functional groups▫Used in hospitals and dental offices▫Disadvantages
Can be hazardous to people Often highly explosive Extremely poisonous Potentially carcinogenic
45
Chemical Methods of Microbial Control
•Enzymes▫Antimicrobial enzymes act against
microorganisms▫Human tears contain lysozyme
Digests peptidoglycan cell wall of bacteria▫Enzymes to control microbes in the
environment Lysozyme used to reduce the number of
bacteria in cheese Prionzyme can remove prions on medical
instruments
46
Chemical Methods of Microbial Control
•Antimicrobials▫Antibiotics, semi-synthetic, and synthetic
chemicals▫Typically used for treatment of disease▫Some used for antimicrobial control outside
the body
47
Chemical Methods of Microbial Control
•Development of Resistant Microbes▫Little evidence that products containing
antiseptic and disinfecting chemicals is beneficial to human or animal health
▫Use of such products promotes development of resistant microbes
48
Antimicrobial Agents
•Chemicals that affect physiology in any manner
•Chemotherapeutic agents▫Drugs that act against diseases
•Antimicrobial agents ▫Drugs that treat infections
49
The History of Antimicrobial Agents
•Semi-synthetics▫Chemically altered antibiotics that are
more effective than naturally occurring ones
•Synthetics▫Antimicrobials that are completely
synthesized in a lab
50
Mechanisms of Antimicrobial Action
•Key is selective toxicity•Antibacterial drugs constitute largest
number and diversity of antimicrobial agents
•Fewer drugs to treat eukaryotic infections (protozoa, fungi, helminthes)
•Even fewer antiviral drugs
51
Mechanisms of Antimicrobial Action
• Inhibition of bacterial wall synthesis• Disruption of existing cytoplasmic
membranes• Inhibition of Protein Synthesis• Inhibition of Nucleic Acid Synthesis• Inhibition of Metabolic Pathways• Prevention of Virus Attachment
52
Basic Principles of Microbial Control•Action of Antimicrobial Agents
▫Alteration of cell walls and membranes Cell wall maintains integrity of cell
Cells burst due to osmotic effects when damaged
Cytoplasmic membrane controls passage of chemicals into and out of cell Cellular contents leak out when damaged
Non-enveloped viruses have greater tolerance of harsh conditions
53
Mechanisms of Antimicrobial Action•Inhibition of Cell Wall Synthesis
▫Inhibition of bacterial wall synthesis Most common agents prevent cross-linkage of
NAM-NAG subunits Beta-lactams are most prominent in this
group Functional groups are beta-lactam rings Beta-lactams bind to enzymes that cross-link
NAM-NAG subunits Bacteria have weakened cell walls and
eventually lyse
54
Mechanisms of Antimicrobial Action
•Inhibition of Cell Wall Synthesis▫Inhibition of synthesis of bacterial walls
Semi-synthetic derivatives of beta-lactams More stable in acidic environments More readily absorbed Less susceptible to deactivation More active against more types of bacteria
Simplest beta-lactams – effective only against aerobic Gram-negatives
55
Mechanisms of Antimicrobial Action•Inhibition of Cell Wall Synthesis
▫Inhibition of synthesis of bacterial walls Vancomycin and cycloserine
Interfere with particular bridges that link NAM subunits in many Gram-positives
Bacitracin Blocks secretion of NAG and NAM from
cytoplasm Effective against Gram positives
Isoniazid and ethambutol Disrupt mycolic acid formation in mycobacterial
species
56
Mechanisms of Antimicrobial Action•Inhibition of Cell Wall Synthesis
▫Inhibition of synthesis of bacterial walls Prevent bacteria from increasing amount of
peptidoglycan Have no effect on existing peptidoglycan layer Effective only for growing cells
57
Mechanisms of Antimicrobial Action•Disruption of Cytoplasmic Membranes
▫Some drugs form channel through cytoplasmic membrane and damage its integrity
▫Amphotericin B attaches to ergosterol in fungal membranes Humans somewhat susceptible because
cholesterol similar to ergosterol Bacteria lack sterols; not susceptible
58
Mechanisms of Antimicrobial Action
•Disruption of Cytoplasmic Membranes▫Azoles and allyamines inhibit ergosterol
synthesis▫Polymyxin disrupts cytoplasmic
membranes of Gram-negatives Oral form is toxic to human kidneys, so only
used topically▫Some parasitic drugs act against
cytoplasmic membranes
59
Which topical ointment is best?• Neomycin is an aminoglycoside antibiotic (disrupts protein
synthesis). It has excellent activity against Gram-negative bacteria, and has partial activity against Gram-positive bacteria.
• Polymixin disrupts bacterial cell membranes by interacting with its phospholipids. They are selectively toxic for Gram-negative bacteria.
• Bacitracin disrupts cell wall synthesis. Its action is on Gram-positive organisms. It can cause contact dermatitis and cross-reacts with allergic sensitivity to sulfa-drugs.
• Which topical ointment is best: Neomycin or Triple Antibiotic (contains all three)
60
Basic Principles of Microbial Control•Action of Antimicrobial Agents
▫Damage to proteins and nucleic acids Protein function depends on 3-D shape
Extreme heat or certain chemicals denature proteins
Chemicals, radiation, and heat can alter or destroy nucleic acids Can produce fatal mutants Can halt protein synthesis through action on
RNA
61
Mechanisms of Antimicrobial Action
•Inhibition of Protein Synthesis▫Prokaryotic ribosomes are 70S (30S and
50S)▫Eukaryotic ribosomes are 80S (40S and
60S)▫Drugs can selectively target translation▫Mitochondria of animals and humans
contain 70S ribosomes Can be harmful
62
Mechanisms of Antimicrobial Action
•Inhibition of Protein Synthesis▫Aminoglycosides: excellent against Gram
negatives, partially effective against Gram positives amikacin (Amikin®) gentamicin (Garamycin®) kanamycin (Kantrex®) neomycin (Mycifradin®) streptomycin tobramycin (TOBI Solution®, TobraDex®)
63
Mechanisms of Antimicrobial Action
•Inhibition of Nucleic Acid Synthesis▫Several drugs block DNA replication or
mRNA transcription▫Drugs often affect both eukaryotic and
prokaryotic cells▫Not normally used to treat infections ▫Used in research and perhaps to slow
cancer cell replication
65
Acyclovir• Acyclovir is used to decrease pain and speed the healing of
herpes sores or blisters in people who have varicella (chickenpox), herpes zoster (shingles; a rash that can occur in people who have had chickenpox in the past), and first-time or repeat outbreaks of genital herpes (a herpes virus infection that causes sores to form around the genitals and rectum from time to time).
• Acyclovir is also sometimes used to prevent outbreaks of herpes sores in people who are infected with the virus.
• Acyclovir disrupts nucleic acid function. It works by stopping the spread of the herpes virus in the body. Acyclovir will not cure herpes or protect others from catching it.
67
Mechanisms of Antimicrobial Action•Inhibition of Nucleic Acid Synthesis
▫Quinolones and fluoroquinolones Act against prokaryotic DNA gyrase (enzyme
that is needed for DNA to unwind during replication)
▫Inhibitors of RNA polymerase (enzyme used during transcription)
▫Reverse transcriptase inhibitors Act against an enzyme HIV uses in its
replication cycle Does not harm people because humans lack
reverse transcriptase
68
Mechanisms of Antimicrobial Action
•Inhibition of Nucleic Acid Synthesis▫Nucleotide analogs
Interfere with function of nucleic acids Distort shapes of nucleic acid molecules and
prevent further replication, transcription, or translation
Most often used against viruses Effective against rapidly dividing cancer cells
69
Mechanisms of Antimicrobial Action•Inhibition of Metabolic Pathways
▫Antimetabolic agents can be effective when pathogen and host metabolic processes differ
▫Quinolones interfere with the metabolism of malaria parasites
▫Heavy metals inactivate enzymes▫Some agents disrupt glucose uptake by
many protozoa and parasitic worms▫Some drugs block activation of viruses
71
Mechanisms of Antimicrobial Action•Inhibition of Metabolic Pathways
▫Antiviral agents can target unique aspects of viral metabolism Amantadine, rimantadine, and weak organic
bases prevent viral uncoating▫Protease inhibitors interfere with an enzyme
that HIV needs in its replication cycle
72
Mechanisms of Antimicrobial Action
•Prevention of Virus Attachment▫Attachment antagonists block viral
attachment or receptor proteins▫New area of antimicrobial drug
development
73
Clinical Considerations in Prescribing Antimicrobial Drugs
• Ideal Antimicrobial Agent▫ Readily available▫ Inexpensive▫Fast-acting▫ Chemically stable during storage▫ Easily administered▫ Nontoxic and nonallergenic▫ Selectively toxic against wide range of pathogens▫ Capable of controlling microbial growth while being
harmless to humans, animals, and objects
74
Clinical Considerations in Prescribing Antimicrobial Drugs
•Spectrum of Action▫Number of different pathogens a drug acts
against Narrow-spectrum effective against few organisms
(Gram positive bacteria only) Broad-spectrum effective against many organisms
(Gram positive and Gram negative bacteria) May allow for secondary or superinfections to develop Killing of normal flora reduces microbial antagonism
75
Clinical Considerations in Prescribing Antimicrobial Drugs
•Efficacy▫Ascertained by
Diffusion susceptibility test Minimum inhibitory concentration test Minimum bactericidal concentration test
77
Clinical Considerations in Prescribing Antimicrobial Drugs
•Routes of Administration▫Topical application of drug for external
infections▫Oral route requires no needles and is self-
administered▫Intramuscular (IM) administration delivers
drug via needle into muscle▫Intravenous (IV) administration delivers
drug directly to bloodstream▫Must know how antimicrobial agent will be
distributed to infected tissues
81
Clinical Considerations in Prescribing Antimicrobial Drugs
•Safety and Side Effects▫Toxicity
Cause of many adverse reactions poorly understood
Drugs may be toxic to kidneys, liver, or nerves
Consideration needed when prescribing drugs to pregnant women
▫Allergies Allergic reactions are rare but may be life
threatening Anaphylactic shock
83
Clinical Considerations in Prescribing Antimicrobial Drugs
•Safety and Side Effects▫Disruption of normal microbiota
May result in secondary infections Overgrowth of normal flora causing
superinfections Of greatest concern for hospitalized patients
85
Resistance to Antimicrobial Drugs•The Development of Resistance in
Populations▫Some pathogens are naturally resistant ▫Resistance by bacteria acquired in two
ways New mutations of chromosomal genes Acquisition of resistance genes (R-plasmids)
via transformation, transduction, and conjugation
86
Resistance to Antimicrobial Drugs•Mechanisms of Resistance
▫At least six mechanisms of microbial resistance Production of enzyme that destroys or
deactivates drug Slow or prevent entry of drug into the cell Alter target of drug so it binds less effectively Alter their metabolic chemistry Pump antimicrobial drug out of the cell before it
can act Mycobacterium tuberculosis produces MfpA
protein Binds DNA gyrase preventing the binding of
fluoroquinolone drugs
88
Resistance to Antimicrobial Drugs•Multiple Resistance and Cross
Resistance▫Pathogen can acquire resistance to more
than one drug▫Common when R-plasmids exchanged▫Develop in hospitals and nursing homes
Constant use of drugs eliminates sensitive cells▫Superbugs▫Cross resistance
90
Resistance to Antimicrobial Drugs•Retarding Resistance
▫Maintain high concentration of drug in patient for sufficient time Kills all sensitive cells and inhibits others so
immune system can destroy▫Use antimicrobial agents in combination
Synergism vs. antagonism
91
Resistance to Antimicrobial Drugs•Retarding Resistance
▫Use antimicrobials only when necessary▫Develop new variations of existing drugs
Second-generation drugs Third-generation drugs
▫Search for new antibiotics, semi-synthetics, and synthetics Bacteriocins Design drugs complementary to the shape of
microbial proteins to inhibit them
93
Vaccination•Vaccine – use the immune system to protect
against infectious disease•Types of vaccines
▫attenuated (weakened) microbe; virulence factors are removed
▫heat-killed / chemically killed microbe▫toxoids
•Passive versus Adaptive vaccination▫passive – immune system products from another
mothers milk (presence of IgA) gamma-globulin (anti-bee venom, anti-hepatitis A, etc)
▫active – stimulate individuals immune system to produce memory cells
94
U.S. cases against diseases for which there are vaccines.
96
SSPE: sub-acute sclerosingpanencephalitis (late stagemeasles)
•Why Your Cellphone Has More Bacteria Than a Toilet Seat
•By Susan E. Matthews, MyHealthNewsDaily Staff Writer | LiveScience.com – 3 hrs ago
•http://news.yahoo.com/why-cellphone-mor
e-bacteria-toilet-seat-124147769.html
97
•Cellphones carry 10 times more bacteria than most toilet seats, so it shouldn't be surprising that a man in Uganda reportedly contracted Ebola after stealing one.
•He stole the phone from a quarantined ward of a hospital, near the site of a recent Ebola outbreak.
•While toilets tend to get cleaned frequently, because people associate the bathroom with germs, cellphones and other commonly handled objects — like remote controls— are often left out of the cleaning routine.
•Cellphones pick up germs all the time; some people talk on their phone on toilets.
98
•However, the amount of germs on a phone isn't a problem — it’s the sharing of phones between people. Without sharing, each phone carries just one set of germs, and won't get its owner sick.
•The problem with phones is that we're in constant contact with them, and they spend a lot of time in close proximity to our faces and mouths.
•And, because it's an electronic device, most people are hesitant about cleaning them.
99
•This is also this case with remote controls, which, are also often used by people when they're sick.
•Remotes are more frequently shared, too, so they're usually even worse than phones for spreading germs.
•Other common culprits that are hotspots of unseen disease include office phones, shopping carts and the first-floor buttons of elevators.
100