Physical and Chemical Control of

Microorganisms

Controlling Microorganisms Reduce or destroy undesirable microbes in a given area

Physical Chemical Mechanical

Relative Resistance of Microbes

Highest resistance Bacterial endospores & prions

Moderate resistance Pseudomonas sp. Mycobacterium tuberculosis Staphylococcus aureus Protozoan cysts

Least resistance most vegetative cells fungal spores enveloped viruses yeast protozoan trophozoites

Terms for Microbial Control Sterile

Inanimate objects free of all life Sterilization

destroys all viable microbes including viruses & endospores

Disinfection destroy vegetative pathogens not endospores

Sanitization cleansing technique that mechanically removes microbes

to safe levels Degerming

removing organisms from an object’s surface

Terms for Microbial Control Microbicidal agents

Causes microbial death Bactericide Sporocide Fungicide Viricide

Microbistasis Prevents microbial growth Bacteriostatic Fungistatic

Factors That Affect Death Rate1. Number of microbes2. Nature of microbes in the population3. Temperature & pH of environment4. Concentration or dosage of agent5. Mode of action of the agent6. Presence of solvents, organic matter, or

inhibitors

Cellular Targets of Control

Mode of action of antimicrobials:

Cell wall Cell membrane Cellular synthetic

processes (DNA, RNA) Proteins

Practical Concerns for Microbial Control Does the application require sterilization? Is the item to be reused? Can the item withstand heat, pressure, radiation,

or chemicals? Is the method suitable? Will the agent penetrate to the necessary extent? Is the method cost- and labor-efficient & is it

safe?

Methods of Physical Control1. Heat – Moist verse Dry2. Cold temperatures3. Desiccation4. Radiation

1. Heat (Moist)Moist heat – use of hot

water or steam mode of action

denaturation of proteins destruction of membranes destruction of DNA

Sterilization Steam under pressure Autoclave

15psi/121oC/10-40 min Steam must reach surface

of item being sterilized!

Autoclave Tape

1. Heat (Moist) Intermittent sterilization

unpressurized steam 100oC 30-60 min for 3 days spores, which are unaffected, germinate during

the intervening periods and are subsequently destroyed

Disinfection boiling at 100oC for 30 minutes destroy non-spore-forming pathogens

Pasteurization

1. Heat (Moist)

Pasteurization heat applied to kill potential agents of

infection and spoilage without destroying the food flavor or value 63°C - 66°C for 30 minutes

batch method 71.6°C for 15 seconds

flash method Not sterilization

kills non-spore-forming pathogens and lowers overall microbe count

does not kill endospores or many nonpathogenic microbes

1. Heat (Dry)Dry heat - higher

temperatures than moist heat

incineration 600-1200oC combusts & dehydrates

cells dry ovens

150-180oC coagulate proteins

Bunsen burner 1870oC

Dehydrates cells and removes water

Can also sterilize

Thermal Death Measurements

Thermal death time (TDT) shortest length of time required to kill all

microbes at a specified temperature Thermal death point (TDP)

lowest temperature required to kill all microbes in a sample in 10 minutes

2. Cold Temperatures Microbistatic

slows the growth of microbes

refrigeration 0-15oC freezing <0oC used to preserve food,

media and cultures

3. Desiccation gradual removal of water from cells leads to metabolic inhibition not effective microbial control

many cells retain ability to grow when water is reintroduced

4. Radiation1. Ionizing radiation

deep penetrating power breaks DNA gamma rays, X-rays, cathode rays used to sterilize medical supplies & food

products

2. Nonionizing radiation little penetrating power to sterilize air, water &

solid surfaces UV light creates thymine pyrmidines

interfere with replication

Chemical Agents in Microbial Control

Chemicals that sterilize Disinfectants, antiseptics, sterilants

Chemicals that inhibit deterioration Degermers, and preservatives

Desirable qualities of chemicals: rapid action in low concentration solubility in water or alcohol, stable broad spectrum, low toxicity penetrating noncorrosive and nonstaining affordable and readily available

Levels of Chemical Decontamination High-level germicides

kill endospores devices that are not heat sterilizable and intended to be

used in sterile environments (body tissue) Intermediate-level

kill fungal spores (not endospores), tubercle bacillus, and viruses

used to disinfect devices that will come in contact with mucous membranes but are not invasive

Low-level eliminate only vegetative bacteria, vegetative fungal cells,

and some viruses clean surfaces that touch skin but not mucous membranes

Factors that Affect Germicidal Activity of Chemicals

Nature of the material being treated Degree of contamination Time of exposure Strength and chemical action of the

germicide Dilutions

Volume of liquid chemical diluted in larger volume of solvent (water)

Chemical Control Of Microbial Agents

1. Halogens2. Phenolics3. Chlorhexidine4. Alcohols5. Hydrogen peroxide6. Detergents & soaps7. Heavy metals8. Aldehydes9. Gases and aerosols

1. Halogens

Chlorine Cl2, hypochlorites (chlorine bleach), chloramines

Denaturation of proteins by disrupting disulfide bonds Can be sporicidal

Iodine I2, iodophors (betadine)

Denature proteins Can be sporicidal Milder medical & dental degerming agents, disinfectants,

ointments

2. Phenolics Disrupt cell

membranes & precipitating proteins bactericidal, fungicidal,

virucidal, not sporicidal Lysol Triclosan

antibacterial additive to soaps

3. Chlorhexidine A surfactant & protein denaturant with

broad microbicidal properties Not sporicidal Used as skin degerming agents for

preoperative scrubs, skin cleaning & burns

4. Alcohols Ethyl, isopropyl in solutions of 50-90% Act as surfactants

dissolve membrane lipids and coagulating proteins of vegetative bacterial cells and fungi

Not sporicidal Good for enveloped viruses

5. Hydrogen Peroxide Weak (3%) to strong (35%) Produce highly reactive hydroxyl-free

radicals that damage protein & DNA while also decomposing to O2 gas toxic to anaerobes

Strong solutions are sporicidal in increasing concentrations

6. Detergents & Soaps Ammonia compounds

act as surfactants alter membrane

permeability of some bacteria & fungi

Not sporicidal Soaps

mechanically remove soil and grease containing microbes

Low concentrations Only have microbistatic

effects

7. Heavy Metals Solutions of silver &

mercury kill vegetative cells in

low concentrations by inactivating proteins

Oligodynamic action Not sporicidal

8. Aldehydes Glutaraldehyde & formaldehyde kill by

alkylating protein & DNA glutaraldehyde in 2% solution (Cidex) used

as sterilant for heat sensitive instruments formaldehyde

disinfectant, preservative, toxicity limits use

9. Gases & Aerosols Ethylene oxide, propylene oxide,

betapropiolactone & chlorine dioxide Strong alkylating agents, sporicidal

Mechanical Control Filtration

physical removal of microbes

passing a gas or liquid through filter

organisms above a certain size trapped in the pores

used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms

Air can be filtered using a high-efficiency particulate air (HEPA) filter

Antimicrobial Therapy

Origins of Antimicrobial Drugs Antibiotics

Common metabolic products of aerobic spore-forming bacteria & fungi

bacteria in genera Streptomyces & Bacillus molds in genera Penicillium & Cephalosporium

Inhibiting other microbes in the same habitat antibiotic producers have less competition for

nutrients & space

Ideal Antimicrobial Drug….. Selectively toxic to microbe

Not host cells Microbicidal, not microbistatic Soluble Potent No antimicrobial resistance Remains active Readily delivered to site of infection Expense Not allergen

Chemotherapy Antimicrobial

Control infection Antibiotic

Produced by the natural metabolic processes of microorganisms

Can inhibit or destroy other microorganisms

Semisynthetic Chemically modified drugs

in lab Synthetic

Synthesized compounds in lab

Chemotherapy Narrow spectrum

Effective against limited microbial types Target a specific cell component that is found only in

certain microbes Broad spectrum

Effective against wide variety microbial types Target cell components common to most pathogens

Selectively Toxic Should kill or inhibit microbial cells without

simultaneously damaging host tissues Complete selective toxicity

Difficult to achieve Characteristics of the infectious agent become

more similar to the vertebrate host cell More side effects are seen

Selective toxicity toxic dose of a drug

The concentration causing harm to the host therapeutic dose

the concentration eliminating pathogens in the host Together, the toxic and therapeutic doses are used

to formulate the chemotherapeutic index

Targets of Antimicrobial Drugs

1. Inhibition of cell wall synthesis

2. Inhibition of nucleic acid synthesis, structure or function

3. Inhibition of protein synthesis

4. Disruption of cell membrane structure or function

1. Bacterial Cell Wall Most bacterial cell walls

contain peptidoglycan Penicillin and

cephalosporin block synthesis of peptidoglycan Causes the cell wall to lyse

Penicillins do not penetrate the outer membrane less effective against

gram-negative bacteria Broad spectrum penicillins

and cephalosporins cross the cell walls of

gram-negative bacteria

2. Inhibit Nucleic Acid Synthesis May block synthesis of

nucleotides, inhibit replication, or stop transcription

sulfonamides and trimethoprim block enzymes required

for tetrahydrofolate synthesis

needed for DNA & RNA synthesis

3. Drugs that Block Protein Synthesis Ribosomes

eukaryotes differ in size and structure from prokaryotes

Aminoglycosides (streptomycin, gentamicin) insert on sites cause

misreading of mRNA Tetracyclines

block attachment of tRNA and stop further synthesis

4. Disrupt Cell Membrane Function Damaged membrane

dies from disruption in metabolism or lysis

These drugs have specificity for a particular microbial group based on differences in

types of lipids in their cell membranes

Polymyxins interact with phospholipids cause leakage, particularly

in gram-negative bacteria Amphotericin B and

nystatin form complexes with

sterols on fungal membranes

causes leakage

Drug Resistance Microorganisms begin to tolerate an

amount of drug that would ordinarily be inhibitory due to genetic versatility or variation intrinsic and acquired

Intrinsic verse Acquired Intrinsic resistance

Microbe must be resistant to antibiotic that they produce

Acquired resistance:1. spontaneous mutations in critical

chromosomal genes2. acquisition of new genes or sets of genes via

transfer from another species originates from resistance factors (plasmids) encoded

with drug resistance, transposons

Mechanisms of Drug Resistance Drug inactivation by

acquired enzymatic activity penicillinases

Decreased permeability to drug or increased elimination of drug from cell acquired or mutation

Change in drug receptors mutation or acquisition

Change in metabolic patterns mutation of original

enzyme

Antibiotic Resistance in Medical Community Improper or excessive use of antibiotics

causes antibiotic resistance Unnecessarily large antibiotic doses

Allow resistant strains to overgrow susceptible ones If resistant strains spread to other patients, a

superinfection occurs Antibiotics are available over the counter in

developing countries allows for overuse and incorrect use

Antibiotic use is widespread in livestock feeds can be transmitted to humans through meat

consumption

Side Effects of Drugs 5% of all persons

taking antimicrobials will experience a serious adverse reaction to the drug

Toxicity to organs Allergic responses Suppression and

alteration of microflora

Considerations in Selecting an Antimicrobial Drug

1. Identify the microorganism causing the infection Specimens should be taken before

antimicrobials initiated

2. Test the microorganism’s susceptibility (sensitivity) to various drugs in vitro when indicated (Next slide)

3. Overall medical condition of the patient

Testing for Drug Susceptibility Essential for groups of

bacteria commonly showing resistance

Kirby-Bauer disk diffusion test

Dilution tests Minimum inhibitory

concentration (MIC) smallest concentration of drug that

visibly inhibits growth In vitro activity of a drug is not

always correlated with in vivo effect If therapy fails, a different drug,

combination of drugs, or different administration must be considered

Best to chose a drug with highest level of selectivity but lowest level toxicity

What about viruses?!?!? Do not destroy their target pathogen Instead they inhibit their development

Inhibit virus before enters cell Viral-associated proteins

Stop it from entering the cell Stop it from reproducing Prevent from exiting the cell