Medical Microbiology Control of Microbial Growth Chapter 7

1. Medical MicrobiologyControl of Microbial GrowthChapter 7 Porter High School Dr. John M. Bartlett, D.C.

2. Terminology • Sterilization – removal or destruction of ALL forms of microbial life. • Prions highly resistant to heat • Heat is most commonly used agent • Sterilizing agent called sterilant • Liquids and gases can be sterilized by filtration • Food only subjected to enough heat to destroy endospores of Clostridium botulinum. • this limited heat treatment called commercial sterilization • If enough heat was used to kill everything, it would degrade food quality too much.

3. Terminology • Disinfection – only destroys harmful microorganisms. • Destruction of vegetative (non spore forming) pathogens. • Chemicals, UV light, steam • Chemical called disinfectant and used on inert surface. If used on living tissue, called antisepsis. Chemical used is called antiseptic. • Some chemicals are both antiseptic and disinfectant. • Alcohols • Anilides • Chlorhexidine. • Chlorine- and iodine-based compounds • Hydrogen peroxide • Phenols

4. TERMINOLOGY • Degerming – swabbing skin with alcohol before injection. Mostly results in mechanical removal of microbes. • Sanitization – lowers microbial counts to safe standards to minimize disease transmission. • High temp. washing with dishwasher • Treatments that ‘kill’ microbes have suffix -cide. • Biocide or germicide kilssmocroorganisms. • Fungicide kills fungi. • Virucide kills viruses • Treatments that inhibit growth and multiplication of bacteria have suffix –stat or –stasis. Bacteriostasis.

5. Terminology • Sepsis – from Greek word for decay or putrid indicates bacterial contamination, as in septic tanks for sewage treatment. • Aseptic – area or object free of pathogens.

6. Factors Influencing Antimicrobial Treatments • Number of microbes – more microbes to begin with means longer time it takes to eliminate entire population. • Environmental influences – presence of organic matter inhibits antimicrobials. Blood, vomit, feces, bacterial biofilms, etc. • Time of exposure – chemicals require longer exposure to affect resistant and endospore forming bacteria. • Microbial characteristics – gram(-)(+), mycobacteria, etc.

7. How do antimicrobial agents work? • Alter cell membrane • Damage to proteins and nucleic acids: • Enzymes • DNA

8. Physical methods of microbe control • Heat – common • Denatures proteins/enzymes • TDP or Thermal Death Point: lowest temp. at which all microorganisms in a particular liquid suspension will be killed in 10 minutes. • TDT or Thermal Death Time: minimal length of time for all bacteria in a particular liquid culture to be killed at a given temperature. • DRT or Decimal Reduction Time: time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed.

9. Physical methods of microbe control • Moist Heat Sterilization – denatures proteins • Boiling: kills vegetative forms of bacterial pathogens, almost all viruses, fungi and their spores in about 10 minutes or less. • Does not kill endospores and some viruses this quickly. • Hepatitis can survive 30 minutes of boiling and some endospore forming bacteria can survive 20 hours at boiling. • Reliable sterilization requires adding pressure in an autoclave

10. Physical methods of microbe control • Pasteurization – mild heating to kill organisms that cause food spoilage without damaging product taste. It also lowers overall bacterial numbers so food lasts longer. • HTST or high temperature short time pasteurization: 72 degrees C for 15 seconds. • UHT or ultra high temperature sterilization: 140 degrees C for 4 seconds. A type of commercial sterilization. Milk widely sold in Europe and lesser developed parts of world where refrigeration is not available.

11. Physical methods of microbe control • Dry Heat Sterilization – kills by oxidizing (burning) • Flaming loop in lab • Hot air sterilization: items sterilized in oven 170 degrees C for 2 hours.

12. Physical methods of microbe control • Filtration – examples are commercially available water filters. Passage of liquid or gas through a material with small enough pores to trap/retain microorganisms. • HEPA or high efficiency particulate filters filter air and can remove microorganisms larger than 3 micrometers in diameter. • Membrane filters can be made from cellulose esters and plastic polymers and are used in labs and industry.

13. Physical methods of microbe control • Low temperatures – refrigerators and freezers • Refrigerator temps 0-7 degreesC slows metabolic rate of most microbes so they do not reproduce or make toxins. • So ordinary refrigerators have ‘bacteriostatic’ effect. • Some bacteria can still grow at below freezing temperatures • Rapid freezing render microbes dormant, but do not necessarily kill them. • Slow freezing more harmful to bacteria due to ice crystal formation.

14. Physical methods of microbe control • High Pressure – alters molecular structures of proteins and carbohydrates. • Endospores resistant to high pressure so have to combine with high temps.

15. Physical methods of microbe control • Dessication – removal of water • Bacteria can’t grow without water, but can remain viable for years. Add water later and they start reproducing and growing. • Lyophilization: freeze-drying. Preserve microbes for lab use. • Different microbes respond differently to dessication: • Gonorrhea can withstand dessication for only about 1 hour. • TB can survive for months.

16. Physical methods of microbe control • Osmotic pressure – high concentrations of salts and sugars to preserve food. • Create hypertonic environment so water leaves the bacterial cell. • Molds and yeasts more capable of growing in these conditions. • Molds, rather than bacteria cause spoilage of fruits and grains

17. Physical methods of microbe control • Radiation – ionizing and nonionizing • Ionizing: gamma rays, x-rays. Short wavelength, so carries lots of energy. • Gamma rays have great penetration but require long times to sterilize large quantities of product. • X-rays have lower penetrating power but require only seconds of exposure • Enough ‘hits’ with ionozing radiation will cause enough mutations in DNA to kill the microbes. • Useful in food industry: low level ionizing used for spices, meats, vegetables. High energy used in pharmaceutical industry for disposable medical and dental supplies.

18. Physical methods of microbe control • Nonionizing radiation – longer wavelength • UV light most commonly used • Causes bonds to form between adjacent thymines in DNA in bacteria (thymine dimers) so DNA can’t replicate correctly. • Can be used in hospitals in germicidal lamps. Microbe has to have direct exposure, so any covering (paper, etc) protects it. • UV light can damage human eyes

19. Physical methods of microbe control • Microwaves – not very effective

20. Chemical Methods of Microbial Control • Used on living and non-living tissue/objects. • No single disinfectant is appropriate for all cases. • Label indicates groups of microorganisms it is effective against. • May have to dilute according to specifications. • Area may have to be scrubbed and rinsed if it is rough or porous. • May have to leave on surface for extended period of time for effectiveness.

22. Evaluating disinfectants • Use-Dilution tests: The AOAC Use Dilution method is specified by US EPA as a method which can be used to substantiate efficacy claims for disinfectants. It is particularly appropriate for dilutable disinfectants. • A 48 hour culture of an individual species of bacteria is dried onto a number of small, cylindrical, stainless steel test surfaces called penicylinders. • If the test includes "organic soil load," then the culture is amended with some percentage of organic matter (animal serum), before application to the test surfaces. • Each dry, contaminated test surface is transferred, individually, to a test tube filled with 10 mL of disinfectant using a wire hook. Depending on the purpose of the test, either 10 or 60 contaminated test surfaces (and corresponding tubes filled with disinfectant) are required.

23. Use-Dilution Test • Contaminated test surfaces incubate in the disinfectant for a specified contact time, typically near ambient temperature (~20-25 C). • After the contact time has elapsed, the treated test surfaces are transferred, individually at intervals, to test tubes containing a liquid growth medium that has been amended with chemical agents to immediately neutralize the action of the disinfectant. • After transfer from the disinfectant, the treated test surfaces are incubated in the neutralizing growth medium for 48 hours. • After incubation, the number of tubes showing growth of the target microorganism is recorded. • To "pass" a 60 carrier test, at least 59 of the 60 surfaces tested must demonstrate complete disinfection (no detectable growth of the target microorganism in the test tubes containing neutralizing growth medium). To "pass" a 10 carrier test, complete disinfection must take place on all test surfaces.

24. Use-Dilution Test • Strengths of the AOAC Use-Dilution Test Method: • The AOAC Use-Dilution Test is approved by the EPA for data submission in conjunction with registration of dilutable disinfectants sold in the US. bullet The AOAC Use-Dilution Test is a "high-level" test for disinfectants, meaning that an antimicrobial solution must have appreciable biocidal activity on a relatively short (<10 minutes) time frame to "pass" the test.

25. Use-Dilution Test • Weaknesses of the Test Method: • The AOAC Use-Dilution Test Method is notoriously variable, on the basis of statistics alone (a product that produces a "passing" 1+/60 on average will fail the test some appreciable percentage of the time). • The AOAC Use-Dilution Test is subject to additional variability resulting from ambiguities of the official method. For example, the official test method does not address important parameters such as humidity level during drying of the carriers and standardization of the concentration of bacteria on the test surfaces. • The AOAC Use-Dilution Test is far removed from "real-life" use of disinfectants. The most glaring disconnect is that contaminated surfaces are literally submerged in disinfectant for the entire contact time (up to 10 minutes). Unless end users of dilutable disinfectants are literally submerging their contaminated surfaces for the full contact time specified on product labels, then they may not be getting the reductions in microorganisms they expect from the disinfectant products they purchase. • In order to avoid false-positive results, the scientist performing the Use-Dilution Test must have experience and skill handling the wire hooks under considerable time pressure, and must be able to transfer the contaminated penicylinders between test tubes without touching the sides of the tubes or contaminating the test system in other ways.

26. Chemical Methods of Microbial Control • Evaluating a disinfectant • Disk-diffusion method Figure 7.6

27. Disinfectant evaluation • All are compared to phenol which is considered Gold Standard

28. Types of Disinfectants • Phenol • Phenolics. Lysol (cresol) • Bisphenols. Hexachlorophene, Triclosan • Disrupt plasma membranes Phenols not used much anymore re: irritating and smell bad. Phenolics have been modified to reduce these properties. Phenols last a long time on surfaces so they are suitable for disinfecting pus, saliva, and feces. Figure 7.7

29. Bisphenols • Derivatives of phenol: 2 phenols connected by a bridge • Hexachlorophene • also known as Nabac • A commercial preparation of the drug, pHisoHex, was widely used as a very effective antibacterial skin cleanser in the treatment of acne.

31. Hexachlorophene • In 1969, hexachlorophene became suspected of causing cancer, and studies determined that oral ingestion of hexachlorophene led to weakness and paralysis in laboratory rats. In 1973, after studies found relatively high concentrations of hexachlorophene in the blood of neonates washed with a 3% solution it was withdrawn from over-the-counter sales, though still available by prescription. A 1978 study undertaken by the U.S. National Institutes of Health, indicated that hexachlorophene does not cause cancer. The MSDS still lists this compound as an experimental teratogen.

33. Bisphenol derivatives • Triclosan: antimocrobial soaps and at least one toothpaste. • Use is so widespread that bacterial resistance is now evident. • Effective vs. gram-positive bacteria and yeasts and some gram-negatives.

34. Triclosan • For some consumer products, there is clear evidence that triclosan provides a benefit. In 1997, FDA reviewed extensive effectiveness data on triclosan in Colgate Total toothpaste. The evidence showed that triclosan in this product was effective in preventing gingivitis. • At this time, the agency does not have evidence that triclosan in antibacterial soaps and body washes provides any benefit over washing with regular soap and water.

35. Triclosan • It is marketed under the trade name Microban® when used in plastics and clothing, and Biofresh® when used in acrylic fibers. • Triclosan works by blocking the active site of the enoyl-acyl carrier protein reductase enzyme (ENR), which is an essential enzyme in fatty acid synthesis in bacteria. By blocking the active site, triclosan inhibits the enzyme, and therefore prevents the bacteria from synthesizing fatty acid, which is necessary for building cell membranes and for reproducing

37. Biguanides • Broad spectrum • Act on cell membrane • Especially effective on gram-positive. • Effective on gram-negative except for most pseudomonas. • Chlorhexadine: used on skin and mucous membranes. • combined with detergent or alcohol is used for preoperative skin prep. • Alexidine similar but faster acting.

38. Chlorhexidine • Also useful against fungi and enveloped viruses. • It is often used as an active ingredient in mouthwash designed to reduce dental plaque and oral bacteria.

40. Halogens • Iodine and Chlorine • Iodine one of oldest and most effective antiseptics • active against most bacteria, many endospores, fungi, some viruses. • Impairs protein synthesis and alters cell membrane • Usually used in an alcoholic solution (called tincture of iodine) as a pre- and post-operative antiseptic. • No longer recommended to disinfect minor wounds because it induces scar tissue formation and increases healing time. • Povidone-iodine (PVP-I) is a stable chemical complex of polyvinylpyrrolidone (povidone, PVP) and elemental iodine. • Do not stain and are less irritating

42. Chlorine • Chlorine is the most widely used wastewater disinfectant in the U.S., and it kills most bacteria, viruses, and other microorganisms that cause disease. • Sodium hypochlorite is bleach.

43. Alcohols • Effectively kill bacteria and fungi but not endospores and nonenveloped viruses. • Denatures proteins and disrupts lipid membranes. • Act and then evaporate quickly • Swab before injection is mainly removing bacteria and skin cells via friction • Unsatisfactory antiseptics for wounds. • Cause coagulation of layer of protein under which bacteria continue to grow.

45. Types of Disinfectants • Alcohols. Ethanol, isopropanol • Denature proteins, dissolve lipids Table 7.6

46. Alcohols • Ethanol: 60-95% concentrations all effective. If 100% does not denature proteins because denaturation requires water. • Isopropanol is slightly more effective than ethanol. • Less volatile, cheaper

47. Types of Disinfectants • Heavy Metals. Ag, Hg, Cu • Oligodynamicaction means it takes a very small amount of the metal. • Denature proteins. • Silver nitrate – used to be law to treat newborns eyes with 1% silver nitrate soln. to kill gonorrhea bacteria causing opthalmianenatorium. Now we use antibiotics due to cases of blindness. • Silver being used in dressings to prevent infection such as MRSA. • Silver nanoparticles are being used in plastics to help preserve food better and in clothing to minimize odors.

48. Heavy Metals • Combo of silver and sulfadiazine used for burn treatment. • Mercury not as commonly used now due to toxicity. • Copper in form of copper sulfate used to destroy green algae. • Zinc is anti-algal and is also used in mouthwashes and dandruff shampoos.

49. Surface active agents • Surfactants – decrease surface tension among molecules of a liquid. • Soaps and detergents: little action as antiseptics but function in mechanical removal of mocrobes. Soaps break oily film on skin into tiny droplets (emulsification) so all cell and microbe debris can be washed away. • Acid-Anionic sanitizers: sanitize equipment, esp. in dairy industry. • Negatively charged portion reacts with plasma membrane. • Act on wide spectrum of microbes and are nontoxic.

50. Quaternary Ammonium Compounds (Quats) • Most widely used. • Action related to positively charged portion of the molecule (NH4+) • Strongly effective vs gram-positive and not real effective vs gram-negative. • Very fungicidal, amoebicidal, and virucidal. • Do not kill endospores or mycobacteria