the control of microorganisms · •the killing or removal of all microorganisms •inhibition...

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 F E B R U A R Y 5 , 2 0 1 5

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

• Protozoa: • Cryptosporidium cysts

• Giardia cysts

• Bacterial endospores

• Viruses

• Bacteria

HARDEST TO KILL

(most resistant)

EASIEST TO KILL

(least resistant)

• Cryptoporidium:

• Giardia:

Source: Madigan, Martinko, Dunlap, & Clark (2008 )

• Bacterial endospores

• Diseases:

• Anthrax

• Tetanus

• botulism

Source: Madigan, Martinko, Dunlap, & Clark (2008 )

• Viruses

• Bacteria

http://remf.dartmouth.edu/Cholera_SEM/images/03_Cholera%200395%20area1%2020kX.jpg

http://www.microbiologyinpictures.com/bacteria%20photos/mycobacterium%20tuberculosis%20photos/mycobacterium%20tuberculosis%20030.jpg

Tuberculosis: (Mycobacterium tuberculosis) Cholera: (Vibrio chlolerae)

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_studentscenario_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/product-images/Autoclaves/Midmark/midmark-ritter-m11-autoclave-sterilizer-hi-res-3.jpg

• Autoclave (medical waste)

http://www.packworld.com/sites/default/files/styles/lightbox/public/images/issues/10_10/Images%20Features/Autoclave.jpg?itok=qISshWMT

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

http://features.cgsociety.org/gallerycrits/86785/86785_1194093509_large.jpg

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: Leptospira interrogans

Source: Madigan, Martinko, Dunlap, & Clark (2008 )

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

• Note sensitivity of Crypto & Giardia to UV – cysts are transparent and allow UV radiation to penetrate easily.

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 1,300 to 3,000

Vibrio cholerae (Cholera) 650 to 3,400

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/nature01408-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

• Ozone

• Chlorine Dioxide

• Chlorine

• Chloramines

MOST POWERFUL

LEAST POWERFUL

Strong Oxidants

• Conditions that can interfere:

• pH

• Particulate matter & turbidity

• Organic matter

• 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

Controls strength of some oxidants

Reacts with , blocks, or “uses up” oxidants

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

Bacterial endospores -- -- 25 --

Adenovirus 0.15 360 - 990 0.28 0.02

Coxsackie virus 0.15 - 2.16 -- -- 0.64 - 2.6

Hepatitis A 592 1.7 --

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 non-enveloped 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

• Protozoa: • Cryptosporidium cysts

• Giardia cysts

• Bacterial endospores

• Viruses

• Bacteria

HARDEST TO KILL

(most resistant)

EASIEST TO KILL

(least resistant)

Summary

• Only sterilization results in 100% removal of microorganisms

• Heat

• Filtration

• Radiation

• But only chemical disinfection can prevent re-growth 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.