Download The Control Of Microorganisms

The Control of
Microorganisms
LC D R B R I A N B E A R D E N , M S , P E
U S P U B L I C H E A LT H S E R V I C E / U S E PA R 9
M A R I A N A I S L A N D S WAT E R O P E R ATO R S
A S S O C I AT I O N
FEBRUARY 5, 2015
Topics
• Review of pathogens
• Disinfection terminology
• Physical methods of disinfection and sterilization
• Heat, radiation, filtration
• Chemical disinfection
• Oxidizers
• Metals
• Other chemicals
Source: Madigan, Martinko, Dunlap, & Clark (2008 )
Microorganisms of interest
HARDEST TO KILL
(most resistant)
• Protozoa:
• Cryptosporidium cysts
• Giardia cysts
• Bacterial endospores
• Viruses
EASIEST TO KILL
(least resistant)
• Bacteria
• Cryptoporidium:
• Giardia:
Source: Madigan, Martinko, Dunlap, & Clark (2008 )
• Bacterial endospores
• Diseases:
• Anthrax
• Tetanus
• botulism
Source: Madigan, Martinko, Dunlap, & Clark (2008 )
• Viruses
• Bacteria
Cholera: (Vibrio chlolerae)
Tuberculosis: (Mycobacterium
tuberculosis)
http://www.microbiologyinpictures.com/bacteria%20photo
s/mycobacterium%20tuberculosis%20photos/mycobacteriu
m%20tuberculosis%20030.jpg
http://remf.dartmouth.edu/Cholera_SEM/images/03_Cholera
%200395%20area1%2020kX.jpg
Terminology
• Sterilization
• The killing or removal of all microorganisms
• Inhibition
• The limitation of the growth of microorganisms
• Decontamination: The treatment of an object or surface to
make it safe to use. Example: wiping a table or cleaning dishes
after a meal.
• Disinfection: Can involve killing or just inhibiting the growth
of microorganisms. Not all are eliminated. Example: bleach.
Terminology: Chemical Methods
• Sterilants
• Destroy all forms of microbial life, including bacterial endospores
• Disinfectants
• Chemicals that kill most, but not all microorganisms.
• Used on inanimate objects – not people!
• Sanitizers
• Reduce (but may not eliminate) microorganisms to a level that is
considered safe
• Used to sanitize food equipment, household objects, and laundry
• Antiseptics & Germicides
• Kill or inhibit growth of microorganisms, but non-toxic enough to be
applied to living tissues (people can use these on themselves)
Physical Methods of Control
• Heat
• Ionizing Radiation
• Filtration
• Ultraviolet Radiation
Heat (Thermal Destruction)
• Kills by: “Denaturation” – macromolecules lose structure &
function. In other words, the basic components of the cell
(proteins, DNA, outer membrane) and damaged and no
longer function.
http://kimwootae.com.ne.kr/apbiology/Protein%20Denaturation.jpg
http://www.btci.org/k12/bft/pcr/PCR_st
udentscenario_files/denaturation.JPG
Heat (Thermal Destruction)
• AUTOCLAVE most common form of heat
sterilization
• 121°C for 4 to 5 minutes will kill endospores
• Entire object must remain in autoclave long enough to
achieve this – typically 10-15 minutes, more for larger,
more moist materials
• Pasteurization another form (milk)
• BOIL water to sterilize:
• 1 minute at sea level (100°C);
• 3 minutes at altitudes above 1 mile (95°C)
Heat (Thermal Destruction)
• Autoclave (lab)
http://www.medsupplier.com/productimages/Autoclaves/Midmark/midmark-ritter-m11autoclave-sterilizer-hi-res-3.jpg
• Autoclave (medical
waste)
http://www.packworld.com/sites/default/files/styles/lightbox/p
ublic/images/issues/10_10/Images%20Features/Autoclave.jpg?
itok=qISshWMT
http://features.cgsociety.org/gallerycri
ts/86785/86785_1194093509_large.jpg
Ionizing radiation
• Kills by: damage to macromolecules, esp. DNA
• Gamma rays, X-rays, electrons
• Used mostly in food processing; some experiments
with sludge
http://people.chem.duke.edu/~jds/cruise_chem/nuclear/pics/fruit.gif
Filtration
• Kills by: Physically removing pathogens
• Must be sized for target organisms
Pore size 5 µm:
Algae and aquatic bacteria
Pore size 0.2 µm:
Source: Madigan, Martinko, Dunlap, & Clark (2008 )
Leptospira interrogans
Ultraviolet (UV) Radiation
• Kills by: Damaging the DNA/RNA, which prevents
replication. Some wavelengths can do broader damage to
other cell structures
• Commonly used for water disinfection
http://www.oemcollect.com/uv2.jpg
Ultraviolet (UV) Radiation
Organism
Ultraviolet Dose (pW-s/cm2)
Required for 90% Reduction
Bacterial endospores
45,000 to 56,000
Adenovirus
23,600 to 56,000
Coxsackievirus
11,900 to 15,600
Hepatitis A virus
3,700 to 7,300
Cryptosporidium cysts
3,000
Giardia cysts
2,000
E. coli
Vibrio cholerae (Cholera)
1,300 to 3,000
650 to 3,400
• Note sensitivity of Crypto & Giardia to UV – cysts are
transparent and allow UV radiation to penetrate easily.
Ultraviolet (UV) Radiation
• Strange phenomenon: photoreactivation
• Some UV-damaged bacteria can repair DNA
when exposed to sunlight
• Total and fecal coliform are capable
• Fecal streptococci are not
• To prevent:
• Requires sufficient UV dose
• Prevent direct exposure of disinfected water to
sunlight
• Use medium pressure or pulsed UV lamp (damages
more parts of the cell than just DNA)
http://www.nature.com/nature/journal/v421/n6921/images/nature014
08-f1.2.jpg
Chemical Methods of Control
• Strong oxidants:
• Chlorine, chloramines, chlorine dioxide, ozone
• Bromine and Iodine
• Metal ions
• Spray & wipe disinfectants
• Alcohol
Strong Oxidants
• Kill by: reacting with
any organic
molecule.
• Bacterial inactivation:
damage to cell
membrane,
impairment of cellular
functions,
destruction/damage of
DNA
• Virus inactivation:
reaction with outer
coating of virus
(capsid), reaction with
RNA/DNA
Source: Pepper, Gerba, & Gentry, (2014)
Strong oxidants
MOST POWERFUL
• Ozone
• Chlorine Dioxide
• Chlorine
LEAST POWERFUL
• Chloramines
Strong Oxidants
• Conditions that can interfere:
• pH
• Particulate matter & turbidity
• Organic matter
Controls strength of some oxidants
Reacts with , blocks, or “uses
 up” oxidants
• Disinfection byproducts (“DBPs”):
• All oxidants react with many substances in water to create
byproducts that are harmful to humans and regulated by
EPA:
•
•
•
•
THMs (trihalomethanes)
HAAs (haloacetic acids)
Chlorite
Bromate
Comparison of effects of agents
Source: Pepper, Gerba, & Gentry, (2014)
Chlorine
• “Free Chlorine”
= HOCl + OCl• HOCl (hypochlorous acid)
more effective than OCl(hypochlorite ion)
• Effectiveness depends on
pH: less effective at high
pH
• “Combined Chlorine”
results from reaction with
ammonia (chloramines)
Source: Pepper, Gerba, & Gentry, (2014)
Chloramines
• Chlorine reacts with Ammonia to form chloramines
• Less powerful but longer lasting than free chlorine
• Created intentionally to provide longer-lasting
residual in some water distribution systems
• “secondary” disinfection, following “primary” disinfection,
such as by ozone
• Occurs naturally when ammonia present in water
(especially in wastewater) – results in need to
achieve “breakpoint chlorination”
Chloramines
• Breakpoint chlorination – satisfaction of organic
and ammonia demand required before free
chlorine can be achieved
Source: Pepper, Gerba, & Gentry, (2014)
Chlorine Dioxide
• Very strong oxidizer
• pH does not affect disinfection strength
• does not create THMs (important when there is
organic matter in water), but does create chlorite
as a DBP
• Dangerous – potentially explosive. Must be
generated on-site by combining chlorine gas and
sodium hypochlorite
Ozone
• Made by passing electric discharge (arc) through a
stream of air or oxygen
• Does not produce THMs, but can make other
byproducts that have health concerns (aldehydes
and bromates)
• Effectiveness not influenced by pH or ammonia
• Much more powerful oxidant than Chlorine – lower
C·t values
• Can even kill Cryptosporidium with C·t of 1 to 3
Ozone – kills Cryptosporidium
Source: Pepper, Gerba, & Gentry, (2014)
Strong Oxidants – Comparison of
strength (by C·t values)
Organism
C·t Values for 99% inactivation
Chlorine
Chloramines
Chlorine
dioxide
Ozone
Cryptosporidium cysts
9,740 11,300
11,400 64,600
1,000
3.3 - 40
Giardia cysts
54 - 192
430 - 1,400
2.7 to 10.7
053 - 1.94
--
--
25
--
0.15
360 - 990
0.28
0.02
0.15 - 2.16
--
--
0.64 - 2.6
592
1.7
--
Bacterial endospores
Adenovirus
Coxsackie virus
Hepatitis A
Polio virus
1.7
1420
0.2 to 6.7
0.2
E. coli
0.6
113
0.48
0.006 to 0.02
Source: Pepper, Gerba, & Gentry, (2014)
Bromine & Iodine
• Bromine:
• used in hot tubs and spas
• Not as fast acting as Chlorine (larger C·t values)
• Effective against bacteria, viruses, and protozoa
• Iodine
• Used in small applications such as for camping
and survival
• Not effective against all protozoa:
cryptosporidium cysts are very resistant to iodine
• Physiologically active; not recommended for
very long periods
Metal Ions
• Metals that exhibit antimicrobial activity:
• Copper, Silver, Zinc, Lead, Cadmium, Nickel, Cobalt
• Only Silver (Ag) and Copper (Cu) used for disinfection,
due to toxicity of other metals
• Cu & Ag used as swimming pool and hot tub
disinfectants
• Cu used in hospital distribution systems to control
legionella growth
• Ag used in home faucet filters to prevent growth in
activated charcoal
• Action is slow, but effective for long periods of time in
water
Summary of water disinfectant
attributes
Disinfection method
Attribute
Killing
Power
Crypto &
Giardia
Residual
Toxicity/
Lack of
DBPs
Filtration (membrane)




Boiling




UV Radiation




Chlorine




Chloramines




Chlorine dioxide




Ozone




Bromine




Iodine




Silver & Copper




Spray & Wipe Disinfectants
• Quats (quaternary ammonium compounds)
• Antibacterial handsoaps, antiseptic wipes, mouthwashes,
household & workplace cleaners
• Common compounds: benzalkonium chloride;
cetylpyridinium chloride
• Kills common bacteria, but not endospores
• Effective against enveloped viruses (influenza, Ebola?)
• Some specific formulations effective against nonenveloped viruses
• Some microorganisms may develop increased
tolerance to quats over time
• Tolerance does not mean the same thing as reistance!
Quats remain effective at higher doses
Spray & Wipe Disinfectants
• Triclosan
• Anti-bacterial and antifungal agent
• Hand soaps, mouthwashes, shampoos, toothpastes, and
also incorporated into materials such as cutting boards
• Mild to skin
• Some cases of increased tolerance, but still useful
• Some bacteria have built-in resistance to triclosan
Alcohol
• Kills by: dissolving/damaging cell membrane,
damaging cellular proteins
• Used in: medical and laboratory sterilization, hand
sanitizers
http://www.bizpacreview.com/wp-content/uploads/2015/01/sanitizer-2.jpg
Summary
• Methods of disinfection:
• Physical
• Heat
• Radiation
• Filtration
• Chemical
• No method of disinfection is “perfect”
• Physical methods can removal all pathogens, but do not
prevent re-growth
• Chemical methods vary in effectiveness and create
byproducts or side effects
Summary
HARDEST TO KILL
(most resistant)
• Protozoa:
• Cryptosporidium cysts
• Giardia cysts
• Bacterial endospores
• Viruses
EASIEST TO KILL
(least resistant)
• Bacteria
Summary
• Only sterilization results in 100% removal of
microorganisms
• Heat
• Filtration
• Radiation
• But only chemical disinfection can prevent regrowth of microorganisms
Final word
• Microbial resistance: does the widespread use of
disinfectants risk the development of “superbugs”,
as we often hear about in reference to antibiotic
drugs?
NO - the methods of disinfection and sterilization in use
today act very aggressively and non-specifically on all of the
organic molecules which make up a microorganism, not like
antibiotics which act with very specific mechanisms, against
which microorganisms can evolve defenses.
Chlorine has been in use for over 100 years now, with no
change in microbial resistance to it.
References
Madigan, M. T., Martinko, J. M., Dunlap, P. V., &
Clark, D. P. (2008). Brock Biology of Microorganisms
(12th ed.). Benjamin Cummings.
Pepper, I. L., Gerba, C. P., & Gentry, T. J. (2014).
Environmental Microbiology (Third.). Academic
Press.