BIO220 Lecture Exam 4 (Ch 11, 12, 13)

Disinfection

•a process to destroy vegetative pathogens, not endospores; inanimate (nonliving) objects

Sanitization

•any cleansing technique that mechanically removes microbes (inanimate objects)

Sterilization

a process that destroys all viable microbes, including viruses and endospores

Degermation

•reduces the number of microbes through mechanical means (animate - living things)

Antiseptic; antisepsis

•disinfectants applied directly to exposed body surfaces

-antisepsis is using antiseptic chemicals to prevent microbes from entering the sterile tissues.

Sepsis

the growth of microorganisms in the blood and other tissues

microbicidal

-cidal = killing

microbistatic

-static = preventing the microbes from growing/reproducing, but not killing

(ex. virustatic agents affect virus reproduction, bacteriostatic agents affect bacterial reproduction)

most resistant forms of microbes

-endospores

-prions

microbial death is determined by

permanent loss of reproductive capability, even under optimum growth conditions

Antimicrobial Agents' Modes of Action - on cell wall

-cell wall becomes fragile and cell lyses;

-some antimicrobial drugs, detergents, and alcohol

Antimicrobial Agents' Modes of Action - on cell membrane

-cell membrane loses integrity;

-detergent surfactants

Antimicrobial Agents' Modes of Action - on protein & nucleic acid synthesis

-prevention of replication, transcription, translation, peptide bond formation, protein synthesis;

-chloramphenicol, ultraviolet radiation, formaldehyde

Antimicrobial Agents' Modes of Action - on proteins

-disrupt or denature proteins;

-alcohols, phenols, acids, heat

surfactants

•Ex. Detergents, are molecules that can insert into the cell membrane, and therefore destroy the cell.

Thermal death time (TDT)

•shortest length of time required to kill all test microbes at a specified temperature

Thermal death point (TDP)

lowest temperature required to kill all microbes in a sample in 10 minutes

Methods of Physical Control of Microbes

1.Heat - moist and dry

2.Cold temperatures

3.Desiccation (drying)

4.Radiation

5.Filtration

Methods of Physical Control of Microbes - Heat

•Moist heat - lower temperatures and shorter exposure time because it is more penetrating; coagulation and denaturation of proteins

•Dry heat - moderate to high temperatures and longer exposure time; dehydration, alters protein structure; incineration

Most resistant to methods of control?

bacterial endospores (usually require temperatures above boiling)

Moist Heat Method - Pressurized Steam

Sterilization - most effective

•Autoclave 15 psi/121oC/10-40min

•Steam must reach surface of item being sterilized

•Item must not be heat or moisture sensitive

•Mode of action - denaturation of proteins, destruction of membranes and DNA

Moist Heat Method - Tyndallization

Nonpressurized Steam

• intermittent sterilization for substances that cannot withstand autoclaving

•Items exposed to free-flowing steam for 30-60 minutes, incubated for 23-24 hours and then subjected to steam again

•Repeat cycle for 3 days

•Used for some canned foods and laboratory media

•Disinfectant

Moist Heat Method -Boiling Water

•Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens

•Disinfection

Moist Heat Method - Pasterurization

•heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value

•63°C-66°C for 30 minutes (batch method)

•71.6°C for 15 seconds (flash method)

•Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes

Dry Heat Method

using higher temperatures than moist heat

•Incineration - flame or electric heating coil

-Ignites and reduces microbes and other substances

•Dry ovens - 150-180oC - coagulate proteins

Effect of cold on microbes

•Microbiostatic - slows the growth of microbes

•Refrigeration 0-15oC and freezing <0oC

•Used to preserve food, media, and cultures

Desiccation (drying)

•Gradual removal of water from cells, leads to metabolic inhibition

•Not effective microbial control - many cells retain ability to grow when water is reintroduced

•Lyophilization - freeze drying; preservation

Radiation

Ionizing (x-rays) - sterilizing,

nonionizing (UV light) - disinfecting

Filtration

•Physical removal of microbes by passing a gas or liquid through filter

•Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms

Germicidal: Halogens

-Chlorine - toxic, destroys proteins, used to treat water & inanimate objects

-Iodine/betadine - milder (interferes w/ protein bonds), used as disinfectants

-Both intermediate level of germicides

Germicidal: Phenolics

-Disrupt cell walls and membranes and precipitate proteins

-Low to intermediate level - bactericidal, fungicidal, virucidal, not sporicidal

-Lysol

-Triclosan - antibacterial additive to soaps

-Chloraseptic - active ingredient: Phenol 1.4%

Germicidal: Chlorhexidine

•A surfactant and protein denaturant with broad microbicidal properties

•Low to intermediate level

•Hibiclens, Hibitane

•Used as skin degerming agents for preoperative scrubs, skin cleaning, and burns

Germicidal: Alcohol

•Ethyl, isopropyl in solutions of 50-95%

•70% is better than 95% because 70% kills more microbes, where 95% has more of a preserving action.

•Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi

•Intermediate level

Germicidal: Hydrogen Peroxide

•Produce highly reactive hydroxyl-free radicals that damage protein and DNA while also decomposing to O2 gas - toxic to anaerobes

•Antiseptic at low concentrations; strong (high level of activity) solutions are sporicidal (sterilizing)

Germicidal: Detergents & Soaps

•Quaternary ammonia compounds (quats) act as surfactants that alter membrane permeability of some bacteria and fungi

•Very low level

•Soaps - mechanically remove soil and grease containing microbes (for skin and for inanimate objects)

Germicidal: Heavy Metals

•Solutions of silver and mercury kill vegetative cells in low concentrations by inactivating proteins

•Oligodynamic action

•Low level

•Merthiolate, silver nitrate, silver

Germicidal: Aldehydes

•Glutaraldehyde and formaldehyde kill by alkylating protein and DNA

•Glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments

•High level

•Formaldehyde - disinfectant, preservative, toxicity limits use

-Formalin - 37% aqueous solution

•Intermediate to high level

Use only on inanimate objects due to toxicity

Germicidal: Gases & Aerosols

•Ethylene oxide, propylene oxide

•Strong alkylating agents

•Affects proteins, DNA

•High level

•Sterilize and disinfect plastics and prepackaged devices, foods

Germicidal: Dyes

•Aniline dyes are very active against gram-positive species of bacteria and various fungi (cell walls) ex. methylene blue

•Sometimes used for antisepsis and wound treatment

•Low level, narrow spectrum of activity

Germicidal: Acids & Alkalies

•Low level of activity

-Organic acids prevent spore germination and bacterial and fungal growth

-Acetic acid inhibits bacterial growth

-Propionic acid retards molds

-Lactic acid prevents anaerobic bacterial growth

-Benzoic and sorbic acid inhibit yeast

Prophylaxis

use of a drug to prevent potential for infection of a person at risk

Antimicrobials

any drug that works against microbes

Antibiotics

Substances naturally produced by some microbes to inhibit others (usually used against bacteria, ex. penicillin)

-do not work against viral infections

Semisynthetic Drugs

Drugs produced by modifying natural antibiotic structures, and are therefore partially or semi-synthetic (ex. Amoxicillin and ampicillin are modifications of penicillin).

Synthetic Drugs

Drugs completely man-made in design (ex. Sulfa drugs - Bactrim)

Narrow Spectrum Antibiotics

Effective against only certain microbes, ex. only Gram + bacteria or only Gram - bacteria.

Broad Spectrum Antibiotics

Effective against a wide range of microbes, ex. both Gram + and Gram - bacteria, or both bacteria and protozoa.

Antibiotics are produced naturally by these organisms

aerobic bacteria and fungi

Cell Wall Inhibitors - block synthesis (peptidoglycan) & repair

Mostly narrow spectrum drugs including:

Penicillins (narrow spectrum)

Cephalosporins (relatively broad spectrum)

Vancomycin (narrow spectrum) - used to treat MRSA

Bacitracin (narrow spectrum)

Cell Membrane - cause damage & disruption in metabolism or lysis

Polymyxins (narrow spectrum)

DNA/RNA - Inhibit replication & transcription

ciprofloxacin (Quinolones) (broad spectrum)

Protein Synthesis Inhibitors

Broad Spectrum Antibiotics including:

Erythromycin

Clindamycin

Aminoglycosides (streptomycin, gentamycin)

Tetracyclines

Metabolic Products - Block pathways & inhibit metabolism

Sulfonamides (sulfa drugs)

Beta-lactam nucleus structure

3 carbon, 1 nitrogen ring

Beta-lactams prevalence as antimicrobial drugs

Greater than 1/2 of all antimicrobial drugs

Most prominent beta-lactams

penicillins

cephalosporins (1/3 of all antibiotics administered)

Drugs that Interfere with Protein Synthesis are generally

broad spectrum

broadest spectrum antibiotic

Erythromycin - Mycoplasma pneumonia, legionellosis, Chlamydia, pertussis, diphtheria and as a prophylactic prior to intestinal surgery, •For penicillin-resistant - gonococci, syphilis, acne

Fungal cells are eukaryotic - what does that mean for humans taking medication for a fungal infection?

a drug that is toxic to fungal cells is also toxic to human cells

Agents used to treat fungal infections

-Macrolide polyenes -

-Azoles - used to treat yeast infections, thrush, athlete's foot, jock itch, ringworm, etc.)

Drug resistance - how does it happen?

-spontaneous mutations

-acquisition of new genes or sets of genes via transfer from another species

Drug Resistance - drug inactivation

Inactivation of a drug like penicillin by penicillinase, an enzyme that cleaves a portion of the molecule and renders it inactive

Drug Resistance - Decreased permeability

The receptor that transports the drug is altered, so that the drug cannot enter the cell.

Drug Resistance - Activation of drug pumps

specialized membrane proteins are activated and continually pump the drug out of the cell

Drug Resistance - Change in drug binding site

binding site on target (ribosome) is altered so drug has no effect

Drug Resistance - Use of alternate metabolic pathway

The drug has blocked the usual metabolic pathway (green), so the microbe circumvents it by using an alternate, unblocked pathway that achieves the required outcome (red).

Natural selection & drug resistance

Large populations of microbes likely to include drug resistant cells due to prior mutations or transfer of plasmids - no growth advantage until exposed to drug

If exposed, sensitive cells are inhibited or destroyed while resistance cells will survive and proliferate.

Eventually population will be resistant - natural selection

Side effects of antimicrobials

-direct damage to tissues due to drug toxicity

-allergic reactions

-superinfections possible

-resistance

How a superinfection can develop

antimicrobials prescribed for one infection can end up causing a much worse infection elsewhere in the body

Tests for drug susceptibility

Kirby-Bauer disk diffusion test

E-test - gives MIC

Diffusion test- gives MIC

Kirby-Bauer disk diffusion test

simple, rapid method for determining susceptibility and resistance of a bacterial pathogen to antibacterial drugs. The test involves drug-impregnated disks placed on an agar plate inoculated with a bacterial lawn

Etest

uses a plastic-coated strip to test the MIC

broth tube dilution test

determines rate at which selected bacteria are destroyed by various chemical agents

Which parts of the human body should be sterile (microbe-free)?

internal organs, tissues, and fluids

Which sites of the body DO harbor normal flora/microbes?

skin & its continuous mucous membranes

upper respiratory tract

GI tract (parts)

outer opening of urethra

external genitalia

vagina

external ear & canal

external eye (lids, lashes)

Which microbes are considered normal flora?

Staphylococcus

Streptococcus

Escherichia coli

Candida

Enterobacter

Normal flora of skin

Staphylococcus

Streptococcus

Candida

Normal flora of GI tract

Escherichia

Enterobacter

Candida

Which microbial flora is found in the most places on the human body?

Candida

exogenous agents

originate from source outside the body

endogenous agents

already exist on or in the body (normal flora)

endogenous infections

occur when normal flora is introduced to a site that was previously sterile

Portals of entry

characteristic route a microbe follows to enter the tissues of the body: skin, GI tract, respiratory tract, urogenital tract, transplacental

Ways microbes attach/adhere to hosts

fimbria or flagella/cilia

capsules or slime layers

spikes (viruses)

Infectious Dose (ID)

the minimum number of microbes necessary to cause an infection to proceed

Initial response of host defenses?

phagocytes (WBCs)

endotoxin

a toxin that is present inside a bacterial cell and is released when the cell is damaged

causes fever, aches, fatigue

exotoxin

toxin molecule secreted by a living bacterial cell into the infected tissue; have strong specificity for target cells

exotoxin - hemolysins

lyse red blood cells

A-B toxins

contain an enzyme component (A part) and a binding component (B part)

necrosis

tissue damage or death

Process of infection & disease (4 stages)

incubation period

prodromal stage

period of invasion

convalescent period

incubation period

the period between exposure to an infection and the appearance of the first symptoms

prodromal stage

vague feelings of discomfort; nonspecific complaints

period of invasion

multiplies at high levels, becomes well-established; more specific signs and symptoms

convalescent period

as person begins to respond to the infection, symptoms decline

localized infection

microbes enter the body and remains confined to a specific tissue

systemic infection

infection spreads to several sites and tissue fluids usually in the bloodstream

focal infection

when infectious agent breaks loose from a local infection and is carried to other tissues

(ex. heart patient with abscessed tooth à infection spreads to heart and causes endocarditis)

acute infection

comes on rapidly, with severe but short-lived effects (ex. flu, stomach virus)