Control of Microbial Growth

Chapter 5
 Hospitals
Minimizing microbial population very important
Due to danger of nosocomial infections
Patients are more susceptible to infection
Pathogens more likely found in hospital setting
Numerous organisms develop antimicrobial

resistance due to high concentrations of

antibiotics
Instruments must be
sterilized to avoid
introducing infection to
deep tissues
Dantas and Sommer American Scientist 2014
 Microbiology laboratories
Use rigorous methods of control
To eliminate (sterilize) microbial contamination
to experimental samples and environment
Aseptic technique and sterile media used for growth
Eliminates unwanted organisms
Contaminated material treated for disposal
Eliminate contamination of environment
 Food and food production facilities
Retention of quality enhanced through
prevention of microbial growth and
contamination
Achieved through physical removal and chemical
destroying organisms
Heat treatment most common and most reliable
mechanism
Irradiation approved to treat certain foods
Chemicals prevent spoilage
Risk of toxicity
 Daily life
Washing a scrubbing with soaps and detergents
achieves routing control
Hand washing single most important step to achieving control
Soap acts as wetting agent
Aids in mechanical removal of microorganisms
Cleans by reducing surface tension and loosening,
dispersing, and suspending soils.
Removes numerous organism from outer layer of skin
Normal flora usually unaffected because it resides in
deeper layers
 Approaches to Control
are highly variable
Control mechanisms either physical or chemical
May be a combination of both
Physical methods
Heat
Irradiation
Filtration
Mechanical removal
Chemical methods
Use a variety of antimicrobial chemicals
Chemical depends on circumstances and degree of control
required
 Approaches to Control
Principles of control
Sterilization
Removal of all microorganisms
Sterile item is absolutely free of microbes, endospores and
viruses
Can be achieved through filtration, heat, chemicals and
irradiation
Disinfection
Eliminates most pathogens
Some viable microbes may exist
Disinfectants = used on inanimate objects and surfaces
Antiseptics = used on living tissues
Pasteurization
Brief heat treatment used to reduce organisms that cause
food spoilage
Surfaces can also be pasteurized
 Principles of control
Decontamination
Treatment to reduce pathogens to level considered safe
Degerming
Mechanism uses to decrease number of microbes in an area
Particularly the skin
Sanitized
Implies a substantially reduced microbial population
This is not a specific level of control
Preservation
Process used to delay spoilage of perishable items
Often includes the addition of growth-inhibiting ingredients
 Approaches to Control
Water treatment facilities
Ensures drinking water is safe
Chlorine generally used to disinfect water
Can react with naturally occurring chemicals
Form disinfection by-products (DBP)
Some DBP linked to long-term health risks
Some organisms resistant to chemical
disinfectants
 Selection of effective antimicrobial procedure is
complicated
Ideal method does not exist
Each has drawbacks and procedural parameters

Choice of procedure depends on numerous

factors
Type of microbe
Extent of contamination
Number of organisms
Environment
Risk of infection
Composition of infected item
 Type of microorganism
Most critical consideration
Is organism resistant or susceptible to generally accepted
methods?

Resistant microbes include

Bacterial endospores
Resistant to heat, drying and numerous chemicals
Protozoan cysts and oocysts
Generally excreted in feces and cause diarrheal disease
Mycobacterium species
Cell wall structure initiates resistance
Pseudomonas species
Can grow in presence of many chemical disinfectants
Naked viruses
Lacking envelope are more resistant to chemical killing
Figure 1.1 A tuberculosis ward at a U.S. Army
base hospital in France during World War I
Evolution of drug resistance
http://www.npr.org/sections/goatsandsoda/2017/01/17/510227493/a-superbug-
that-resisted-26-antibiotics
 Drug Resistance
(see Chp 20)
Mechanisms of resistance
Drug inactivating enzymes
Some organisms produce enzymes
that chemically modify drug
Penicillinase breaks -lactam ring
of penicillin antibiotics
Alteration of target molecule
Minor structural changes in
antibiotic target can prevent binding
Changes in ribosomal RNA
prevent macrolides from binding to
ribosomal subunits

What evolutionary process does

this remind
you of?
 Question of the Day*:

What are some common

antibiotics?
History and Development
of
Antimicrobial Drugs
Development of
new generation of
drugs
In 1960s scientists
alteration of drug
structure gave them
new properties
Penicillin G altered
to created
ampicillin
Broadened
spectrum of
antimicrobial
killing
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Mechanisms of resistance
Decreased uptake of the drug
Alterations in porin
proteins decrease
permeability of cells
Prevents certain drugs from entering
Increased elimination of the drug
Some organisms produce efflux
pumps
Increases overall capacity of organism
to eliminate drug
Enables organism to resist
higher concentrations of drug
Tetracycline resistance
Chapter 21 Where antibiotics work
But with the new iCHIP with diffusion of growth
factors can cross a permeable membrane of
single isolated cells (see Ling paper in Course
content)
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Acquisition of resistance
Can be due to spontaneous
mutation
Alteration of existing genes
Spontaneous mutation called
vertical evolution
Or acquisition of new genes
from the environment
Resistance acquired by
transfer of new genes called
horizontal
transfer
Mechanisms of antibiotic resistance
* Synthesis of the enzyme penicillinase or other beta-lactamases
provides protection from the beta-lactam antibiotics. These enzyme
break the beta-lactam ring at the position shown with the green arrow in
the diagram of penicillin G.
* Likewise synthesis of cephalosporinases defeats the cephalosporins.
* Some bacteria modify their DNA gyrase thus defeating quinolones.
* Some bacteria synthesize "pumps" in their plasma membrane
through which they remove antibiotics like tetracyclines from the interior
of the cell.
* Bacteria may methylate their ribosomes obscuring the target of
antibiotics (e.g., erythromycin) that ordinarily bind to and inactivate the
ribosome or conversely
* they may enzymatically modify the antibiotic (e.g., kanamycin) so it
can no longer "see" its ribosomal target.
* Bacteria may modify the structure of their peptidoglycan wall and
thus avoid the inhibitory effects of antibiotics like cycloserine.
http://www.cofc.edu/~delliss/virtuallabbook/DrugRes/AntibioticRes
Modern Germ warfare (or phobia)

Typically, people carry between 10,000 and 10 million bacteria on each hand.
Hand sanitizers can be as effective as hand washing only in certain situations.
The type of soil which may be present on hands can significantly alter their
effectiveness.
Solutions containing 60-95% alcohol are most effective. Higher concentrations
are less potent because proteins are not denatured easily in the absence of
water.

The CDC guidelines for hand hygiene in healthcare settings do not apply to those
in foodservice primarily because the types and levels of soil on the hands are
quite different.

Triclosan is the most commonly used chemical ingredient in antimicrobial soaps.

A key factor in its effectiveness is that it must be left on the skin long enough to
work, as in a good 30-45 second scrub. Companies have not published
information on what combination of triclosan concentrations and washing times
are most effective.
A recent study by * Heidler J and Halden RU (2007), show that conventional
sewage treatment was demonstrated to be much less effective in destroying the
antimicrobial than the aqueous-phase removal efficiency of the plant would make
believe.
http://www.colostate.edu/orgs/safefood/NEWSLTR/v8n3s06.html
April 2011
AE Waters et al, 2011
http://cid.oxfordjournals.org/content/early/2011/04/14/cid.cir181.full.pdf+html
http://www.tgen.org/news/index.cfm?newsid=1948
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Spontaneous mutation
Rare - 1 X 10-8
Such mutations have profound effect of
resistance of bacterial population
Example of spontaneous mutation
Resistance to streptomycin is result a change in
single base pair encoding protein to which
antibiotic binds
When antimicrobial has several different
targets it is more difficult for organism to
achieve resistance through spontaneous
mutation
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Acquisition of new genes through gene
transfer
Most common mechanism of transfer is
through conjugation
Transfer of R plasmid
Plasmid often carries several different resistance
genes
Each gene mediating resistance to a specific antibiotic
Organism acquires resistance to several different
drugs simultaneously
Horizontal
(or lateral)
Gene
Transfer
(HGT)
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Examples of emerging antimicrobial
resistance
Enterococci
Part of normal intestinal flora
Common cause of nosocomial infections
Intrinsically resistant to many common
antimicrobials
Some strains resistant to vancomycin
Termed VRE
Vancomycin resistant enterococcus
Many strains achieve resistance via transfer of
plasmid
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Staphylococcus aureus
Common cause of nosocomial infections
Becoming increasingly resistant
In past 50 years most strains acquired resistance to
penicillin
Due to acquisition of penicillinase genes
Until recently most infections could be treated with
methicillin (penicillinase resistant penicillin)
Many strains have become resistant
MRSA methicillin resistant Staphylococcus aureus
MRSA many of these strains still susceptible to
vancomycin
Some hospitals identified VISA
VISA vancomycin intermediate Staphylococcus aureus
Bacterial resistance to Antimicrobial Drugs

http://www.medicinenet.com/mrsa_infection/article.htm#
 Determining Susceptibility of
Bacterial to Antimicrobial Drug
Streptococcus pneumoniae
Has remained sensitive to penicillin
Some strains have now gained resistance
Resistance due to modification in genes coding for
penicillin-binding proteins
Changes due to acquisition of chromosomal DNA
from other strains of Streptococcus
Generally via DNA mediated transformation
 Selection of Antimicrobial
Procedure
Number of organisms
initially present
Time it takes to kill it directly
affected by population size
Large population = more time
Commercial effectiveness is
gauged by decimal
reduction time
A.k.a D value
Time required to kill 90% of
population under specific
conditions
Washing reduces time required
to reach disinfection or
sterilization
 Selection of Antimicrobial
Procedure
Environmental conditions
Environmental conditions strongly influence
effectiveness
pH, temperature and presence of organic materials
can increase or decrease effectiveness
Most chemicals are more effective at higher
temperatures and lower pH
Effectiveness can be hampered by the presence of
organism molecules
Can interfere with penetration of antimicrobial agent
 Selection of Antimicrobial
Procedure
Potential risk of infection
Medical items categorized according to
potential risk of disease transmission
Critical items = come in contact with body tissues
Needles and scalpels
Semicritical instruments = contact mucous
membranes but do not penetrate body tissues
Endoscope
Non-critical instruments = contact unbroken skin
only
Show little risk of transmission
Stethoscope
 Selection of Antimicrobial
Procedure
Composition of the item
Some sterilization and disinfection methods
inappropriate for certain items
Heat inappropriate for plastics and other heat
sensitive items
 Heat as Control
Heat treatment most useful for microbial
control
Relatively fast, reliable, safe and inexpensive
Heat can be used to sterilize or disinfect
Methods include
Moist heat
Dry heat
 Heat as Control
Moist heat
Destroys through irreversible coagulation of
proteins
Moist heat includes
Boiling
Pasteurization
Pressurized steam
 Heat as Control
Boiling (100 C)
Destroys most microorganisms and viruses
Not effective means of sterilization
Does not destroy endospores
Pasteurization
Pasteur developed to avoid spoilage of wine
Does not sterilize but significantly reduces organisms
Used to increase shelf life of food
Most protocols employ HTST method
Heated to 72C and held for 15 seconds
Other protocol UHT
Heated to 140C - 150C, held for
several seconds then rapidly cooled
Heat as Control
Pressurized steam
Autoclave used to sterilize using
pressurized steam
Heated water steam
increased pressure
Preferred method of sterilization
Achieves sterilization at 121C
and 15psi in 15 minutes
Effective against endospores
Flash autoclaving sterilizes at
135C and 15psi in 3 minutes
Prions destroyed at 132C and
15psi for 4.5 hours
Heat as Control
Pressurized steam
Autoclave used to sterilize using
pressurized steam
Heated water steam increased
pressure
Preferred method of sterilization
 Heat as Control
Dry heat
Not as effective as moist heat
Sterilization requires longer times and higher temperatures
200C for 1.5 hours vs. 121C for 15 minutes
Incineration method of dry heat sterilization
Oxidizes cell to ashes
Used to destroy medical
waste and animal carcasses
Flaming laboratory
inoculation loop
incinerates organism
Results in sterile loop
 Other Physical Methods
of Control
Heat sensitive materials require other
methods of microbial control
Filtration
Irradiation
High-pressure treatment
 Filtration
Membrane filtration
used to remove
microbes from fluids
and air
Liquid filtration
Used for heat sensitive
fluids
Membrane filters allow
liquids to flow through
Traps microbes on
filter
Depth filters trap
microbes using Filtration of air
electrical charge High efficiency particulate
air (HEPA) filter remove
nearly all microbes from
air
Filter has 0.22 - 0.45m
pores to trap organisms
http://cedarmountainair.com/your_
air.html
 Physical Methods of Control
Radiation
Electromagnetic radiation
Energy released from
waves
Based on wavelength and
frequency
Shorter wavelength,
higher frequency =
more energy
Range of wavelength is
electromagnetic spectrum
Radiation can be ionizing or
non-ionizing
 Physical Methods of Control
Ionizing radiation
Radiation able to strip electrons from atoms
Three sources
Gamma radiation
X-rays
Electron accelerators

Causes damage to DNA and potentially to plasma

membrane (but see Deinococcus species)
Used to sterilize heat resistant materials
Medical equipment, surgical supplies, medications
Some endospores can be resistant
 Physical Methods of Control

Ultraviolet radiation
Non-ionizing radiation
Only type to destroy microbes directly
Damages DNA
Causes thymine dimers
Used to destroy microbes in air, drinking
water and surfaces
Limitation
Poor penetrating power
Thin films or coverings can limit effect
 Other Physical Methods
of Control
High pressure processing
Used in pasteurization of commercial foods
Does not use high temperatures
Employs high pressure
Up to 130,000 psi
Destroys microbes by
denaturing proteins and
altering cell membrane
permeability

http://wwwtc.pbs.org/wgbh/nova/abyss/frontier/images/b
athysphere.jpeg Deep sea bathysphere
 Chemicals as Control
Chemicals can be
used to disinfect and
sterilize
Called germicidal
chemicals
Reacts with vital cell
sites
Proteins
DNA
Cell membrane
 Chemicals as Control
Potency of chemicals Sterilants =
Formulations generally Destroy all microorganisms
contain more than one High-level disinfectants
antimicrobial agent
Destroys viruses and vegetative
Regulated by
cells,
FDA
Antiseptics
Not endospores
EPA Intermediate-level disinfectants
Disinfectants Kills vegetative cells fungi, most
Germicidal agents grouped viruses,
according to potency Not endospores
Low-level disinfectants
Removes fungi, vegetative bacteria
and enveloped viruses
Not mycobacteria, naked viruses or
endospores
 Chemicals as Control
Selecting appropriate chemical
Points to consider
Toxicity
Benefits must be weighed against risk of use
Activity in presence of organic material
Many germicides inactivated in presence of organic matter
Compatibility with material being treated
Liquids cannot be used on electrical equipment
Residue
Residues can be toxic or corrosive
Cost and availability
Storage and stability
Concentrated stock relieves some storage issues
Environmental risk
Is germicidal agent harmful to environment
 Chemicals as Control
Classes of chemicals
Germicides represent a number or chemical families
Alcohols
Aldehydes
Biguanides
Ethylene oxide
Halogens
Metals
Ozone
Peroxides
Phenolics
Quaternary ammonium compounds
 Chemicals as Control
Alcohols
Solutions of 60% - 80% isopropyl or ethyl alcohol kill
vegetative bacteria and fungi
Not effective against endospores and some naked viruses
Mode of action
Coagulation of proteins and essential enzymes
Damage to lipid membranes
Commonly used as antiseptic and disinfectant
Limitations
Evaporates quickly limiting contact time
May damage material such as rubber and some plastics
 Chemicals as Control
Aldehydes
Destroy organisms by inactivating proteins
and DNA
2% glutaraldehyde solution most widely used
liquid sterilant
Orthophthalaldehyde studied as alternative
Formalin used to kill bacteria and inactivate
viruses
Also used for specimen preservation
Formalin is solution made from formaldehyde
 Chemicals as Control
Biguanides
Most effective member of group is
chlorhexidine
Extensively used in antiseptics (mouthwash)
Relative low toxicity
Destroys wide range of organisms
 Chemicals as Control
Ethylene oxide
Useful gaseous sterilant
Destroys microbes including endospores and
viruses
Mode of action
Reacts with proteins
Useful in sterilizing heat or moisture sensitive
items
Limitations
Mutagenic and potentially carcinogenic
 Chemicals as Control
Halogens
Common disinfectants
Mode of action
Oxidizing proteins and other cell components
Includes chlorine and iodine
Chlorine
Destroys all types of organisms and viruses
Used as disinfectant
Caustic to skin and mucous membranes
Chlorine dioxide replacing chlorine in many applications
Iodine
Kills vegetative cells
Not reliable with endospores
Used in tincture or iodophore on skin
 Chemicals as Control
Metal compounds
Compounds combine with enzymes and
proteins
Interfering with function
High concentrations of many metals toxic to
human tissue
Silver still used as disinfectant
Creams containing silver sulfadiazine used to prevent
secondary infections
Also available on bandages for wound care
Once used for newborn eye infections
 Chemicals as Control
Ozone - O3 (reactive oxygen species)
Free radicals, Unstable form of oxygen
Powerful oxidizing agent
Used as alternative to chlorine
As disinfectant for drinking and waste water
 Chemical Bonds and the
Formation of Molecules
Atoms are most stable when the outer orbital
contains the maximum number of electrons
2, 8, etc.
To fill outer orbitals atoms form bonds with other
atoms to fill outer orbitals
Bonds are formed with the sharing or the gain or loss
of electrons
Molecules are formed when atoms bond together
 REVIEW:
Covalent Bonds
Achieve stability through the
sharing of electrons
between atoms
Creates a strong bond
Difficult to break
Requires significant energy
usually in the form of heat
Never break spontaneously at
physiological temperatures
Enzyme required to break at
lower temperature
Bonds can be polar or non-
polar
 Chemicals as Control
Peroxygens
Includes hydrogen peroxide and peracetic
acid
Powerful oxidizing agents
Readily biodegradable
Less toxic than ethylene oxide and
glutaraldehyde
 Chemicals as Control
Hydrogen peroxide (H2O2)
Effectiveness depends on surface being treated
Living tissue produce catalase enzyme
Breaks down hydrogen peroxide to oxygen and water
More effective on inanimate object
Useful as disinfectant
Leaves no residue
Doesnt damage most materials
Hot solutions used in food industry
Vapor-phase can be used as sterilant
Peracetic acid
More potent then hydrogen peroxide
Effective on organic material
Can be used on wide range of material
 Chemicals as Control
Phenolics
A.k.a carbolic acid
One of the earliest disinfectants
Now has limited use
Active ingredient in Lysol
Mode of action
Destroy plasma membrane
Denature proteins
Kills most vegetative cells
Can kill mycobacterium at high concentrations
Not reliable on all groups of viruses
Triclosan and hexachlorophene phenols used in
soaps and lotions
Triclosan
Triclosan is a chlorophenol, a class of chemicals which is suspected of
causing cancer in humans

It is an ingredient in many detergents, dish-washing liquids, soaps,

deodorants, cosmetics, lotions, anti-microbial creams, various toothpastes,
and an additive in various plastics and textiles. However, the safety of
triclosan has been questioned in regard to environmental and human health.
While the companies that manufacture products containing this chemical
claim that it is safe, the United States Environmental Protection Agency (EPA)
has registered it as a pesticide. The chemical formulation and molecular
structure of this compound are similar to some of the most toxic chemicals on
earth, relating it to dioxins and PCBs. The EPA gives triclosan high scores
both as a human health risk and as an environmental risk.

Recent studies show that even after sewage/sludge treatment, measurable

levels of triclosan are still present. Processed sludge then gets dispersed as
fertilizer.
Are we growing new super resistant bugs?
 Chemicals as Control
Quaternary ammonium compounds
A.k.a Quats
Cationic detergents
Nontoxic
Used to disinfect food preparation surfaces
Mode of action
Reduces surface tension
Aids in removal of dirt and organic matter
Facilitates mechanical removal of organisms
Positive charge attracts Quats to negative charge of cell
surface
Reacts with membrane
Destroys vegetative bacteria and enveloped viruses
Not effective on endospores, mycobacteria and naked
viruses
 Preservation of Perishable
Products
Preservation extends shelf-life of many
products
Chemicals are often added to prevent or slow
growth of microbes
Other methods include
Low temperature storage
Freezing
Reducing available water
 Chemical preservatives
Numerous chemicals are used as preservatives
Formaldehyde, Quats, and phenols
Weak organic acids often used as food preservatives
Benzoic, ascorbic and propionic acids
Used in bread, cheese and juice
Mode of action
Alter cell membrane function
Interfere with energy transformation
Nitrates and nitrites used in processed meats
Inhibits germination of endospores and growth of vegetative
cells
Have been shown to be potent carcinogen
 Low temperature storage
Microbial growth is temperature dependent
Low temperatures slow down or stop enzymatic reactions of
mesophiles and thermophiles
Some psychrophiles still able to grow
Freezing means of food preservation
Essentially stops microbial growth
Irreversibly damages cell, membrane breakage by ice
crystals
Kills up to 50% of microbes
Some cells still pose potential
threat
 Chemicals as Control
Reducing water availability
Decreasing water availability accomplished by salting
or drying food
Addition of salt (or sugar) increases environmental solutes
Causes cellular plasmolysis
Numerous bacteria can continue to grow in high salt
environments
Staphylococcus aureus can survive in high salt concentrations
Desiccation or drying is often supplemented by other
methods
Salting
Lyophilization (freeze drying)
Widely used to preserve foods like coffee, milk and meats
December 16, 2011