MicroBio Mod. 5

Control of Microbial Growth and Antibiotics

Sterilization

Removal of all microbial life

Commercial sterilization

Removal of endospores + microbial life

• canned food, etc

Disinfection

Removal of growing, vegetating pathogens from inanimate objects by chemical means

Antisepsis

Removal of pathogens from living tissue

• like disinfection

Sanitization

To lower microbial counts to a safe enough level for public health

• cleaning a public bathroom or restaurant silverware

Sepsis

Microbial contamination

Asepsis

The absence of significant contamination

Degerming

Removal of microbes from a limited area

• from skin using soap and water

• the skin around where a needle will go

Thermal death point (TDP)

lowest temperature at which all cells in a culture are killed in 10 mins

Thermal death time (TDT)

time to kill all cells in a culture

Decimal reduction time (DRT)

minutes to kill 90% of a population at a given temperature

Moist heat vs dry heat

Moist heat:

• denatures proteins

• most effective

Dry heat sterilization:

• kills by oxidation

• needs to be really hot (incineration, flaming, hot-air sterilization)

• less effective

Autoclave (type of moist heat)

Steam under pressure

• goes above boiling point

• kills vegetative cells and endospores in about 15 minutes

• drawbacks: kills other molecules too

Pasteurization

Reduces spoilage organisms and pathogens by heating at a low temp for a longer time

• used for things that cannot withstand high temp and pressure

Equivalent treatments:

• 63 degrees C for 30 minutes

• high-temp short-time (HTST) 72 degrees C for 15 seconds

• ultra-high-temp (UHT) 140 degrees C for less than 1 second

Refrigerating + Freezing

Inhibit microbial growth

• bacteriostatic

Radiation

Damages DNA

• thymidine/pyrimidine dimers

• ionizing radiation (x rays, gamma rays, electron beams)

• nonionizing radiation (UV)

Alcohol

Denatures proteins

Dissolves lipids

• great for degerming

• needs to be diluted with water to be antimicrobial

• 95-60% is more effective at lower exposure times

Examples:

• ethanol, isopropanol

Effective against vegetative cells (Bacteria)

• not great for spores or viruses without envelopes

Phenol

Disrupts plasma membrane

Phenols - phenol chemically modified to be more effective

• Lysol

Bisphenols - disrupt plasma membranes

• hexachlorophene

• triclosan

Biguanides

Disrupt plasma membranes

Chlorhexidine

• used for surgical scrub/pre-op

Aldehydes

Inactivate proteins by crosslinking with functional groups (-NH2, -OH, -COOH, -SH)

• glutaraldehyde, formaldehyde

Peroxigens

Great at disinfecting inanimate objects

Not a great antiseptic because it gets easily broken down to oxygen and water by enzymes in our bodies (catalase)

• oxidizing agents

• hydrogen peroxide

O3, H2O2, benzoyl peroxide

Halogens

Disrupts protein configuration by oxidation

• bleach

• chlorine

• iodine - also disrupts plasma membrane

Heavy metals

Denature proteins

• Ag, Hg, Cu

• oligodynamic action - low concentrations still exert antimicrobial activity

Surfactants: Soap

Degerming

• breaks bonds that hold molecules together/on our skin, allowing them to be washed off

Surfactants: Acid-anionic detergents

More effective than soap at breaking surface tension/disrupting microbial interactions

• negative charge/anionic

Surfactants: Quaternary ammonium compounds

Bactericidal because they change membrane permeability allowing loss of ions (like potassium) and more to leak out of the cell.

• positively charged/catatonic

Conditions effecting rate of microbial death

Number of microbes

• more at once are harder to kill

Environment

• organic matter, temperature, biofilms

• potentially inhibit chemical disinfection

Time of exposure

• how long they must be in contact with the chemical disinfectant for disinfection to be effective

Microbial characteristics

• differences in cell wall, presence of glycocalyx, endospore potential

Identity of microbe

Concentration of disinfectant

• some work better diluted

Environment pH

Contact

• ease of how it contacts the microorganism

Chemicals used for food preparation

Organic acids/nitrates:

• readily metabolized by our bodies, so its safe for humans

• inhibit microbe metabolism (bacteriostatic)

• sorbic acid, benzoic acid, calcium propionate

• change pH, impact metabolism, disrupt membrane integrity

• control molds and bacteria in food and cosmetics

Nitrite:

• prevents endospore germination by disrupting cell metabolism and spore formulation

• reacts with enzymes interacting with iron (in the blood in meat products)

Chemotherapy

the use of drugs to treat disease

• paul ehrlich’’s magic bullet that would kill bacteria within a host without harming the host

Antimicrobial drug

interfere with the growth of microbes within a host
* paul ehrlich was the 1st to discover one that was a derivative of arsenic used to treat syphyllus

Antibiotic

substance produced by by a microbe that, in small amounts, inhibits another microbe

Selective toxicity

a drug that kills harmful microbes without damaging the host

Narrow spectrum

Work only on certain types of organisms

Specifically dictated by cell wall structure:

• mycobacteria

• gram-negative

• gram-positive

Broad spectrum

affects larger groupings of microbes/many gram positive and gram negative organisms

• also eliminates your normal microbiota/healthy bacteria

• creates opportunity for a superinfection

Superinfection

infection with other organisms not sensitive to the drug

• opportunists or drug resistant strains

• opportunists - the normally harmless bacteria that don’t cause disease take advantage of weak immune system

Bacteriocidal

kills bacteria directly

Bacteriostatic

stops growth of bacteria long enough for the immune system to get rid of them

Testing antibiotic effectiveness: Kirby Baur disk-diffusion test

• a piece of filler paper is saturated at a certain spot with the antibiotic and applied to a plate with growing microbes

• incubation will show either the presence of absence of a zone of inhibition

• the larger the zone, the more effective the antibiotic

Testing antibiotic effectiveness: Broth dilution test

Allows for measurement of smallest effective dose

• MIC (Minimal inhibitory concentration)

• MBC (Minimal bactericidal concentration)

Place an organism in decreasing concentration of an antibiotic

• growth = not effective dose

• no growth - effective dose

Place a sample from a well with no growth after treatment with the antibiotic into a new broth culture without the antibiotic

• growth = microbes were only inhibited by the antibiotic

• no growth = microbes were killed by the antibiotic

Antibiotic resistance: mutations

1. enzymatic destruction of the drug

2. prevention of penetration of drug

• from changes to cell wall structure

3. alteration of drugs target site

• changes of internal structures (proteins, enzymes)

4. rapid ejection of the drug

Resistance genes are often on plasmids or transposons that can be duplicated and transferred between bacteria.

Antibiotic resistance: misuse

• using outdated, weakened antibiotics

• using antibiotics for the common cold or other inappropriate conditions

• use of antibiotics in animal feed

• failure to complete prescribed regimen

• using someone else’s leftover prescription

Inhibitors of cell wall synthesis

Cephalosporins:

• same as penicillin (targets peptidoglycan synthesis)

• weakening of the cell wall can potentially lead to lysis

Polypeptide antibiotics

• bacitracin

• vancomycin

• protein in nature instead of chemical

• affect peptidoglycan synthesis at an earlier stage

Antimycobacterial Antibiotics

• isoniazid

• ethambutol

• target mycolic acid synthesis in mycobacterium

Injury to the plasma membrane

Polymyxin B

• membrane disruptor

• alters the outer membrane permeability of bacterial cells

• targets gram negative bacteria

• results in destabilization of LPS layer of the outer membrane layer

Inhibitors of nucleic acid synthesis

Rifamycins

• Rifampin - similar to a macrolide

• inhibits RNA synthesis by stopping RNA polymerase

Quinolones and Fluoroquinolones

• ciprofloxacin - broad spectrum that inhibits DNA synthesis

Competitive inhibitors of the synthesis of essential metabolites

Sulfonamides

• target the enzymes in the metabolic pathway that leads to folate creation, shutting the pathway down

• kills the cell

• broad spectrum

• unique to prokaryotes