Lesson 3 Control of Microbial Growth January 22, 2015

Reproduction in Prokaryotes

• • Binary fission—asexual reproduction by a separation of the body into two new bodies – DNA replication – Cytokinesis—splitting of the cell Budding—asexual reproduction in which a new individual develops from some generative anatomical point of the parent organism – Yeasts (Candida albicans and Cryptococcus neoformans)

Figure 6.12a Binary fission in bacteria.

Cell elongates and DNA is replicated.

Cell wall and plasma membrane begin to constrict.

Cell wall Plasma membrane DNA (nucleoid) Cross-wall forms, completely separating the two DNA copies.

Cells separate.

(a)

A diagram of the sequence of cell division FtsZ protein

Budding

YEAST AND FUNGI

Phases of Bacterial Growth

• Bacteria have four distinctive growth phases – Lag—intense activity preparing for population growth but has little to no increase in population – Log—(logarithmic growth) period of growth where cellular reproduction is most active – Stationary—growth rate slows. Number of bacterial death balances the number of new cells. Population stabilizes – Death—number of deaths exceeds the number of new cells formed. Population can die out entirely

Figure 6.15 Understanding the Bacterial Growth Curve.

Lag Phase Intense activity preparing for population growth, but no increase in population.

Log Phase Logarithmic, or exponential, increase in population.

Stationary Phase Period of equilibrium; microbial deaths balance production of new cells.

The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast).

Death Phase Population Is decreasing at a logarithmic rate.

Staphylococcus spp.

Question

• If a culture of mesophilic bacteria were placed in a refrigerator, what phase of growth would it be in? Incubator (set at 37 degrees)?

Terminology of Microbial Control

• Sepsis refers to microbial contamination of the body – Septic shock occurs when the body responds to pathogen/toxin contaminating the blood – SIRS—Systemic Inflammatory Response Syndrome • Not specific to infection (burns, pancreatitis, trauma) • Asepsis is the absence of significant contamination • Aseptic surgery techniques prevent microbial contamination of wounds

The Terminology of Microbial Control

• Sterilization: removing ALL microbial life – Eradicates microbes AND their endospores – Any agent used to sterilized is a sterilant – Steam and pressure (autoclave) • Commercial sterilization: – How do you think can goods are sterilized?

– Heated enough to kill C. botulinum endospores • Other thermophilic endospores still persist

The Terminology of Microbial Control

• • Disinfection: destruction of vegetative (growing) pathogens – Endospores still persist – Can be mechanical or chemical – Chemicals, UV radiation, boiling water, and steam – Usually refers to inanimate objects. Antisepsis: the removal of pathogens from living tissue

The Terminology of Microbial Control

• Degerming: refers to the removal of microbes from a limited area – Alcohol swabbing before an injection • Mechanical and chemical – Server wiping down table at a restaurant • Sanitization is the practice of lowering microbial counts on public eateries – High-temperature dishwashing and dipping drinking glasses in chemical disinfectant

• Hotel Room Sanitation?!?!

• http://www.youtube.com/watch?v=kLssaWxip N8

• • Names of treatments that cause death of a microbe end in the suffix –cide – Biocide/germicide is the generic name for killing microbes • Bactericide • Fungicide • Virucide Names of treatments that only stop microbial growth end in the suffix –static/stasis – Once the bacteriostatic agent is removed, growth resumes

Factors that affect the efficacy of Antimicrobial Treatments

1. Number of microbes. More microbes; longer it takes to kill them 2. Environmental influences. 3. Time of exposure. Chemical antimicrobials require extended time to function to affect more resistant microbes 4. Microbial characteristics. Features of the microbe itself determine the effectiveness of antimicrobial agent (constituents of membrane, enzymes, endospores, etc.)

Rate of Microbial Death

• Bacterial populations usually die at a constant rate.

• Different antimicrobial agents have varying microbial death rates • If an antimicrobial agent “x” kills 90% of a microbial population in 1 minute. It will subsequently kill the same amount each additional minute

Table 7.2 Microbial Exponential Death Rate: An Example

•

Actions of Microbial Control Agents on Microbes

• Microbial agents primarily affect microorganisms via three mechanisms 1. Alteration of membrane permeability – Plasma membrane is the target of many microbial agents 2. Damage to ribosomes and/or proteins – Denaturing proteins or abrogating its production results in the inability to carry out metabolic reactions 3. Damage to nucleic acids – DNA/RNA carry information for replication/metabolism http://www.ncbi.nlm.nih.gov/books/NBK7986/

Control of Microbial Growth

• There are two means of controlling microbial growth 1. Physical 2. Chemical

Physical Methods

1. Heat 2. Filtration 3. Low Temperatures 4. High Pressure 5. Dessication 6. Osmotic Pressure 7. Radiation

Heat

• • • Heat kills microorganisms by inactivating their enzymes – Heat resistance varies among different microbes Thermal death point (TDP): lowest temperature at which all cells in a culture are killed in 10 min Thermal death time (TDT): time during which all cells in a culture are killed

Moist Heat Sterilization

• • • Moist heat kills microbes by denaturing proteins Boiling kills vegetative forms of bacteria, most viruses, fungi and their spores in 10’ – Some viruses and endospores may survive Autoclave: steam under pressure destroys endospores – Most effective use of moist-heat sterilization – Some materials can be damaged by autoclaving

Moist Heat Sterilization

• Pasteurization—reduces spoilage organisms and pathogens in milk/juices – Raises the temp right below boiling – DOES NOT ERADICATE ALL MICROBES!!!!

– High-temperature short-time: 72°C (161°F) for 15 sec • Milk in U.S. Must be refrigerated. Short storage life.

– Ultra-high-temperature: 140°C (284°F) for 1-2 sec • Milk became popular in Europe. Can be stored for several months without refrigeration.

–

Equivalent treatments

• As temperature increases, length of time to kill microbes decreases

Dry Heat Sterilization

• Kills microbes by oxidation effects – Dry heat – Flaming – Incineration – Hot-air sterilization (oven)

Equivalent Treatments Hot-Air

170˚C, 2 hr Dry Heat

Autoclave

121˚C, 15 min Moist Heat

Filtration

• Blocks the passage of microorganisms • HEPA (high-efficiency particulate air) removes microbes >0.3 µm. • Membrane filtration (liquid) removes microbes >0.22 µm

Figure 7.4 Filter sterilization with a disposable, presterilized plastic unit.

Flask of sample Cap Sterile filtrate Membrane filter Cotton plug in vacuum line ensures sterility Vacuum line

Physical Methods of Microbial Control

• • • • Low temperature inhibits microbial growth – Refrigeration (4°C) – – Deep-freezing (-20°) Lyophilization (freeze-drying) (-80°) • Uses liquid nitrogen High pressure denatures proteins – Some endospores are resistant. Combined with elevating temps and alternating pressure cycles Desiccation is the removal of water and prevents metabolism Osmotic pressure causes plasmolysis

Radiation

1. Ionizing radiation (X rays, gamma rays, electron beams) – Ionizes water to release OH• – Damages DNA, proteins, membranes 2. Non-ionizing radiation (UV, 260 nm) – Damages DNA by causing thymine dimers – Inhibit DNA replication

Figure 7.5 The radiant energy spectrum.

Chemical Methods of Microbial Control

• Chemical agents are used to control microbial growth on inanimate objects and living tissue • Only a few chemicals achieve “sterility” due to 1. Endospores 2. Characteristics of some bacteria resist destruction • NOTE: No single disinfectant is appropriate for all circumstances

Principles of Effective Disinfection

1. Concentration of disinfectant – Greater the concentration, more effective 2. Organic matter that is being disinfected 3. pH – Can affect the action of the chemicals 4. Time – Longer the exposure; more microbes are killed

Evaluating a Disinfectant

• • • •

Use-Dilution Test

Metal/glass cylinders are dipped in test bacteria (broth) are dried at 37°C for a brief period of time Dried cultures are placed in disinfectant of varying concentrations for 10 min at 20°C Bacteria from cylinders are transferred to culture media to determine whether bacteria survived treatment

Disk Diffusion Test

• Disk of filter paper is soaked in a chemical agent • Filters are placed on a “lawn” of bacteria to evaluate growth inhibition • The area where growth is inhibited is called zone

of inhibition

– Larger the zone, the more effective the chemical agent is at controlling microbes – Antibiotic discs

Figure 7.6 Evaluation of disinfectants by the disk-diffusion method.

Zone of inhibition Chlorine Hexachlorophene O-phenylphenol Quat

Staphylococcus aureus

(gram-positive) Chlorine Hexachlorophene O-phenylphenol Quat

Escherichia coli

(gram-negative) Chlorine Hexachlorophene O-phenylphenol Quat

Pseudomonas aeruginosa

(gram-negative) Disk Diffusion Method

Chemicals in Disinfectants

Phenol and Phenolics

• Also referred to as carbolic acid • Disrupt plasma membranes and results in cellular leakage • Found in some disinfectants, mouth washes, and throat sprays (1% concentration) – Over the counter throat lozenges are lower concentration • Therefore are not antimicrobial

Bisphenols

• Derivative of phenol – Two phenols bridge together • Hexachlorophene, triclosan – Disrupt plasma membranes and inhibits enzyme needed for synthesis of fatty acids (lipids) • Used in nurseries – Affects the growth of gram positive staph and strep in newborns • Over use results in resistance

Figure 7.7cd The structure of phenolics and bisphenols.

(c) Hexachlorophene (a bisphenol) (d) Triclosan (a bisphenol)

Biguanides

• Broad spectrum of activity – Taken into the plasma membrane and abrogates permeability – Binds to DNA thus affecting transcription • Especially effective against Gram positive bacteria – Not as effective against Gram negative bacteria and viruses • Chlorhexidine disrupts plasma membranes • Used for surgical hand scrubs and pre-operative skin prep in patients

Halogens

• • Iodine – Active against all bacteria – – Tinctures: Iodine in aqueous alcohol Iodophors: Iodine in solubilizing agent such as a surfactant – Alter protein synthesis and membranes Chlorine – Bleach: hypochlorous acid (HOCl) – Chloramine: chlorine + ammonia • Water treatment facilities – Mode of action is widely debated

Alcohols

• • Kills bacteria and fungi – Ineffective at killing endospores and non-enveloped viruses Ethanol, isopropanol – Denature proteins, dissolve lipids, disrupts membranes • • Requires water to facilitate movement across membrane 70% more effective than 100% – Certain pathogens that lack a lipid envelope are resistant to alcohols •

Clostridium difficile

Heavy Metals

• • • Ag (silver), Hg (mercury), and Cu (copper) – Silver sulfadiazine used as a topical cream on burns – Copper sulfate is an algicide (kills algae) • Used in pools Oligodynamic action—refers to the antimicrobial effect of heavy metals – Method of action is unknown Silverware (100%) self-sanitize!!!!

Figure 7.8 Oligodynamic action of heavy metals.

Surface-Active Agents (Surfactants)

Surfactants decrease surface tension among molecules of a liquid Soap (emulsifies oil) “little antiseptic value” (Axe, Dove) Acid-anionic detergents (attacks membrane) (dishwashing liquid) Degerming Sanitizing Quaternary ammonium compounds (cationic detergents) “Quats” More effective on gram(+) Bactericidal, denature proteins, disrupt plasma membrane

Food Preservatives

• • • Organic acids – – Control molds and bacteria in foods and cosmetics Inhibit metabolism – Sorbic acid (canned goods), benzoic acid (facial cleaners), and calcium propionate (bread) Sodium nitrite is added to meats to prevent endospore germination Antibiotics – Nisin (bacteriocin) and natamycin prevent spoilage of cheese

Aldehydes

• Inactivate proteins by cross-linking with the functional groups (–NH 2 , –OH, –COOH, –SH) • Use: Sterilization of medical equipment – Glutaraldehyde and formaldehyde

Peroxygens

• Oxidizing agents • Use: contaminated surfaces – O 3 , H 2 O 2 , peracetic acid • Especially effective against anaerobic bacteria

Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.