Controlling Microbial Growth

Fomite: Inanimate items that potentially inhabited by microbes

Disinfectants: Fast acting, stable, and inexpensive antimicrobial chemicals that inactivate most
microbes. Process of application is called disinfection. Doesn’t kill all microbes so fomites on
which it is used are considered disinfected NOT sterilized. Ex: Vinegar.

Antiseptics: Antimicrobial chemicals safe for use on living skin or tissues. Process of
application is called antisepsis. Selectively effective against microorganism and capable of
penetrating tissue without causing tissue damage. Ex: Isopropyl alcohol and hydrogen peroxide.
● Asepsis: A state of microbial sterility.
● Sepsis: Systemic inflammatory response to an infection which results in high fever,
increased heart and respiratory rates, shock, and possibly death.

Sterilants: Anything that effectively kills all microbes, viruses, and eventually endospores on a
given fomite. Process of application is called sterilization.

Degerming: Reduces microbial load on skin or tissue through application of an antimicrobial

chemical.

-icides kill their prefix

-statics inhibit the growth of their prefix

Microbial Death Curve: Used to measure microbial control. Shows that the rate of killing
remains constant as population dwindles. In other words, microbial death is logarithmically
linear / the same percent of the CURRENT population dies each minute. Percentage killed is
important.

Decimal Reduction Time (DRT/D-Value): The amount of time it takes to kill 90% of the
population / the amount of time it takes to reduce the population to 10% of its original size.

Biosafety Levels (BSL): A tier list of biohazards based on infectivity, ease of transmission,
potential of disease severity, and work being done on the agent. Danger and rarity increases
with level.
● BSL-4: Dangerous and exotic. High risk of aerosol-transmitted (airborne!!) infections.
Frequently lethal with very few treatments or vaccines. Very few labs play with these. Ex:
Ebola and Marburg viruses.
● BSL-3: Microbes are indeginous or exotic and can cause lethal diseases through
respiratory transmission. Ex: Mycobacterium tuberculosis.
● BSL-2: Microbes are typically indigenous and are associated with diseases of varying
severity. They pose moderate risk to workers and the environment. Ex: Staphylococcus
aureus.
 ● BSL-1: MIcrobes are not known to cause disease in healthy hosts and pose minimal risk
to workers and the environment.

Using Physical Methods to Control Microorganisms

Heat Sterilization: Killing a microbe by heating it to the point of denaturing its proteins and
liquifying its membranes.
● Dry-heat Sterilization: Denatures proteins, alters membranes, dehydrates, desiccates.
Microbes are cooked in a dry-heat sterilizer (often an oven) at 170C for about 2 hours.
● Moist-heat Sterilization: Denatures proteins and alters membranes. Boils microbes.
Ineffective against endospores and at high altitude.

Thermal Death Point (TDP): The lowest temperature at which all microbes are killed in a
ten-minute exposure.

Thermal Death Time (TDT): The length of time needed to kill all microorganisms in a given
sample.

Autoclaves: A machine that relies on moist-heat sterilization to kill microbes. Replaces air with
increasing amounts of steam raising the interior pressure and allowing temperatures to climb
well ABOVE boiling point, thereby killing vegetative cells, viruses, and even endospores. Does
not damage autoclaved items. Well, most of them.

Refrigeration and Freezing:

● Refrigerators are usually between 0 and 7C.
● Freezing below -2C usually stops microbial growth and kills some microbes.
● -70C is used for long term storage and transport of bacterial cultures and medical
specimens.
Pressure: Pressure between 100 and MPa is sufficient to kill vegetative cells by protein
denaturation. Endospores will survive though

Sonication: The use of ultrasound waves to disrupt cell structures with rapid pressure changes.

Desiccation/Dehydration: All cells require water. Removing water might not kill all microbes or
their endospores, but it will stop microbial growth.
● Lyophilization/Freeze Drying (LOW PRESSURE THING TOO): The fomite is rapidly
frozen and placed under a vacuum, causing it to lose its water through sublimation.
● Water Activity: The water content of foods and materials. Can be lowered by the
addition of solutes and sugars.

Radiation:
● Ionizing Radiation: X-rays, gamma rays, and high-energy electron beams ionize
molecules inside the cell, changing molecular properties and damaging structures. Can
 introduce double-strand breaks in DNA molecules and cause potentially lethal mutations
during the repair.
● Nonionizing Radiation: UV light does not penetrate cells or packaging, but can cause
mutations in microbes???

Filtration: Physically separates microbes from samples.

Using Chemicals to Control Microorganisms

A wide variety of chemicals can be used as disinfectants or antiseptics. When choosing one,
bear in mind the type of microbe targeted, the desired cleanliness of the item, the disinfectant’s
effect on the item's integrity, biological impact of the disinfectant, the price, and ease of use.

Phenolics: Phenolics all contain phenol -- a benzene ring with an -OH group -- as part of their
chemical structure. They, like alcohols, denature protein and disrupt membranes, but only
INHIBIT growth.

Heavy metals: Bind to proteins and inhibit enzyme activity.

● Mercury: Bacteriostatic but highly toxic to the central nervous, digestive, and renal
systems in high concentrations. Bad for the environment due to bioaccumulation.
Tinctures, solutions of mercury dissolved in alcohol, used to be used as syphilis
medication. This did not go well.
● Silver: Often used as an antiseptic and combined with antibiotics. Extremely affective,
but is suspected to enhance antibiotic resistance and can cause argyria (turns the
patient’s skin blue) if it bioaccumulates.
● Copper, Nickel, and ZInc: Algicides

Halogens: Oxidation and destabilization of cellular macromolecules. Ex: Iodine, Chlorine,

Fluorine.

Alcohols: Denature proteins and disrupt membranes. Ex: Ethanol, Isopropanol.

Surfactants: Surfactants are long, amphipathic chains of fatty acids. They lower surface tension
of water to help wash away microbes and disrupt cell membranes. Ex: Quaternary ammonium
salts.

Peroxygens: Strong oxidizing agents that can be used as disinfectants or antiseptics.

Hydrogen peroxide (H2O2) is often used in solution to disinfect surfaces and can be used as a
gaseous agent as well. Hydrogen peroxide works by producing free radicals that damage
cellular macromolecules. It is considered environmentally safe because it breaks down into
water and oxygen gas.

Testing the Effectiveness of Antiseptics and Disinfectants

Phenol coefficient: A measurement of the effectiveness of a disinfectant or antiseptic through
comparison with phenol. A chemical with a phenol coefficient of 1.0 is equal to phenol, whereas
a chemical with a phenol coefficient less than 1.0 is weaker than phenol, and one with a phenol
coefficient larger than 1.0 is more effective than phenol.

Disk-Diffusion Method: Different chemicals are applied to separate, sterile filter paper disks.
The disks are then placed on an inoculated agar plate, and the chemicals are left to diffuse out
of the disks.
● Lawn: The growing surface area of the agar plate covered in bacterial growth.
● Zone of Inhibition: A radius around the sterile paper disks in which bacteria cannot
grow. Looks like a transparent circle around the paper disks. More effective chemicals
have LARGER circles.

Use-Dilution Test: Used to determine a chemical’s disinfection effectiveness on a fomite. A

cylinder of stainless steel is dipped in a culture of the targeted microorganism and then dried.
The cylinder is then dipped in solutions of disinfectant at various concentrations for a specific
amount of time, before being transferred to a new test tube containing a sterile medium.
Turbidity is measured.

History of Chemotherapy and Antimicrobial Discovery

Fundamentals of Antimicrobial Chemotherapy

Bacteriostatic drug: causes a reversible inhibition of growth, with bacterial growth restarting
after elimination of the drug.

Bactericidal drug: kills their target bacteria.

Narrow-spectrum antimicrobial: targets only specific subsets of bacterial pathogens. (Used

when the pathogen has already been identified).

Broad-spectrum antimicrobial: targets a wide variety of bacterial pathogens. (Used when the
pathogen is yet to be identified).

Mechanisms of Antibacterial Drugs

Structures/functions that can be targeted:

- Penicillin-binding proteins;
- Peptidoglycan subunits and subunit transport;
- 30S and 50S ribosomal subunit;
 - Lipopolysaccharide;
- DNA and RNA;
- Folic acid and mycolic acid synthesis enzyme;
- Mycobacterial ATP synthase.

Mechanisms of Other Antimicrobial Drugs

Antifungal drugs: Most common is to cause the disruption of the cell membrane. They take
advantage of small differences between fungi and humans in the biochemical pathways that
synthesize sterols (Fungi use ergosterol, whereas humans use cholesterol). Other antifungals
may inhibit cell wall synthesis; bind ergosterol in the cell membrane and create pores that
disrupt the membrane, or even inhibit microtubules and cell division.

Antiprotozoan drugs: Can produce damaging reactive oxygen species. It can also inhibit: -
electron transport in mitochondria, - folic acid synthesis, - DNA synthesis, and - heme
detoxification.

Antihelminthic drugs: Can block neuronal transmission (causing paralysis and starvation), and
induce calcium influx. This type of drugs can also inhibit: - microtubule formation (reducing
glucose uptake), - ATP production, and - RNA synthesis.

Extra: Antiviral drugs: Can cause nucleoside analog inhibition of nucleic acid synthesis, and
non-nucleoside noncompetitive inhibition.They also inhibit: - escape of virus from endosomes, -
neuraminidase, - viral uncoating, - protease, - integrase, and - membrane fusion.

Drug Resistance

Drug resistance is the reduction of effectiveness of a medication (such as an antimicrobial or

an antineoplastic) in treating a disease or condition.

Some factors that can accelerate drug resistance include: the overuse and misuse of
antimicrobials; inappropriate use of antimicrobials; subtherapeutic dosing; and patient
noncompliance with the recommended course of treatment.

Exposure of a pathogen to an antimicrobial compound can select for chromosomal mutations

conferring resistance, which can be transferred to subsequent generations and eventually
become a predominant trait.

Many genes responsible for drug resistance are found on plasmids or in transposons that can
be transferred easily between microbes.
Inactivation (or Drug Modification): Resistance genes may code for enzymes that chemically
modify or destroy (through hydrolysis) an antimicrobial, thereby inactivating it. Example: -
Aminoglycoside resistance can occur through enzymatic transfer of chemical groups to the drug
molecule, impairing the binding of the drug to its bacterial target. - For Beta-lactams, bacterial
resistance can involve the enzymatic hydrolysis of the Beta-lactam bond within the Beta-lactam
ring of the drug molecule; once the Beta-lactam bond is broken, the drug loses its antibacterial
activity.

● Efflux: involves inhibiting the accumulation of an antimicrobial drug, which then prevents
the drug from reaching its cellular target. This strategy is common among gram-negative
pathogens and can involve changes in outer membrane lipid composition, porin channel
selectivity, and/or porin channel contractions. Also, many Gram - and Gram + pathogenic
bacteria produce efflux pumps that actively transport an antimicrobial drug out of the cell
and prevent the accumulation of drug to a level that would be antibacterial. Example:
resistance to Beta-lactams commonly occurs through active efflux out of the cell, and it is
rather common for a single efflux pump to have the ability to translocate multiple types of
antimicrobials.

Target modification: Because antimicrobial drugs have very specific targets, structural
changes to those targets can prevent drug binding, rendering the drug ineffective. Through
spontaneous mutations in the genes encoding antibacterial drug targets, bacteria have an
evolutionary advantage that allows them to develop resistance to drugs. Example: Genetic
changes impacting the active site of penicillin-binding proteins (PBPs) can inhibit the binding of
Beta-lactam drugs to and provide resistance to multiple drugs within class.

Enzymatic Bypass: - The microbe may overproduce the target enzyme such that there is a
sufficient amount of antimicrobial-free enzyme to carry out the proper enzymatic reaction. -
The bacterial cell may develop a bypass that circumvents the need for the functional target
enzyme. Both have been found as mechanisms of sulfonamide resistance. Example:
Vancomycin resistance among S. aureus has been shown to involve the decreased
cross-linkage of peptide chains in the bacterial cell wall, which provides an increase in

Testing the Effectiveness of Antimicrobials

The Kirby-Bauer disk diffusion test has been used as a starting point for determining the
susceptibility of specific microbes to various antimicrobial drugs. Antibacterial activity is
observed as a clear circular zone of inhibition around the grug-impregnated disk. The diameter
of the zone of inhibition, measured and compared to a standardized chart, determines the
susceptibility or resistance of the bacterial pathogen to the drug.

Microbial Inhibitory Concentration (MIC): Is determined by examining the tubes to find the
lowest drug concentration that inhibits visible growth. This is observed as turbidity in the broth.
MBC: Is the lowest drug concentration that kills ≧99.9% of the starting inoculum. Tubes with no
visible growth are inoculated onto agar media without antibiotics to determine the MBC.

Current Strategies for Antimicrobial Discovery

The recent development of the iChip allows researchers to investigate the

antimicrobial-producing capabilities of soil microbes that are difficult to grow by standard
cultivation techniques in the laboratory. Rather than growing them in the laboratory, they are
grown in situ (in the soil). The use of the iChip has resulted in the discovery of teixobactin, which
targets two distinct steps in gram + cell wall synthesis and for which antimicrobial resistance
appears not yet to have evolved.