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 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://remf.dartmouth.edu/cholera_sem/images/03_cholera %200395%20area1%2020kX.jpg http://www.microbiologyinpictures.com/bacteria%20photo s/mycobacterium%20tuberculosis%20photos/mycobacteriu m%20tuberculosis%20030.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) Autoclave (medical waste) http://www.medsupplier.com/product- images/autoclaves/midmark/midmark-ritter-m11- autoclave-sterilizer-hi-res-3.jpg 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: 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
Ultraviolet (UV) Radiation Organism Ultraviolet Dose (pw-s/cm 2 ) 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 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)
Organism Strong Oxidants Comparison of strength (by C t values) Cryptosporidium cysts 9,740-11,300 C t Values for 99% inactivation Chlorine Chloramines Chlorine dioxide 11,400-64,600 Ozone 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 Killing Power Crypto & Giardia Attribute 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.