Rest of micro lectures

fomites

Inanimate objects that may carry microbial contamination

Fomites may be treated

with more aggressive control methods, or for longer time, to achieve lower levels of contamination

Disinfection

Reduces microbial load of an inanimate item through application of heat or antimicrobial chemical (chlorine, bleach, phenols, glutaraldehyde)

Used on objects and surfaces

Sanitization

Reduces microbial load of inanimate item safe public health levels through application of heat or antimicrobial chemicals (detergents containing phosphates, industrial strength cleaners containg quaternary ammonium compounds)

Used on objects and surfaces

Sterilization

Completely eliminated all vegetative cells, endospores, and viruses from inanimate item (pressurized steam, autoclave, chemicals, radiation)

Used on objects and surfaces

Antisepsis

Reduces microbial load on skin or tissue through application of an antimicrobial chemical (Boric acid, isopropyl alcohol, hydrogen peroxide, iodine)

Apply to living tissue and samples

Degerming

Reduces microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals (soap, alcohol swab)

Apply to living tissue and samples

Aseptic Technique

involves set of protocols that maintain sterility or asepsis, thus preventing contamination of the patient with microbes

Failure of aseptic technique

may put a clinical patient at risk for sepsis, a systematic inflammatory response to a systemic infection

Kill microbes

suffix -cide or -cidal

bactericides kill bacteria

viricides kill viruses

fungicides kill fungi

complete sterility

inhibit growth

suffix - stat or -static

bacteriostatic for bacteria

fungistatic for fungi

in infection, may allow immune system opportunity to clear an infection

Control microbial growth: Physical

Heat, radiation

Control microbial growth: Chemical

Gas, liquid

Control microbial growth: Mechanical removal

Filtration

Control microbial growth: Biological

Virus, toxin

Individual methods are typically tailored to work on

either objects or

living organisms

Treatments

dont kill microorganisms instantly

Killing is a probabilistic process

Microbial populations usually die exponentially

Eg, fixed percentage dies per unit time

Can plot on microbial death curve

• straight line on semi-log plot

Efficacy of killing agent is measured by D-Value

Decimal reduction time

time to kill 90% = 1 Log10 Unit

Heat: Physical method

Function of temp. and time

Various microorganisms respond differently to high temps. Endospore formers such as Clostridium botulinum are more heat tolerant

Boiling does not kill all microbes

Incineration at very high temp. destroy all microorganisms

Heat: Autoclaves

rely on moist heat sterilization and raise temp. above the boiling point of water; considered most effective method of sterilization

Internal indicators are used to ensure sterilization

heat-sensitive autoclave tape

biological indicator spore test - endospores of the thermophile Geobacillus stearothermophilus to determine whether endospores were killed

Pasteurization

uses heat but does not render the food sterile; it reduces the number of spoilage-causing microbes while maintaining food quality.

LTH

low temp holding 65C 30 min

HTST

High temp short time

72oC - 15 seconds; lowers bacterial numbers while preserving the quality of the milk

UHT

Ultra high temp

138oC >2 seconds; UHT pasteurized milk can be stored for a long time in sealed containers without being refrigerated

Low temp inhibits microbial metabolism

slowing growth

Bacterial cultures and medical specimens

requiring long-term storage or transport are often frozen at ultra-low temperatures (dry ice -70°C or liquid nitrogen tanks -196°C)

Refrigeration

inhibits metabolism

Freezing

stops metabolism, may kill microbes

Pressure High

Denatures proteins and can cause cell lysis

Pressure: Food

kills microbes while maintaining food quality and extending shelf life. High pressure between 100 and 800 MPa (sea level is about 0.1 MPa) kills vegetative cells by denaturing proteins

Pressure: Clinical

hyperbaric oxygen therapy is used to treat infections; patient breathes pure oxygen at ~1 - 3 atmospheres (atm). Inhibits the growth of oxygen-sensitive or anaerobic bacteria

Removing water

slows or halts bacterial growth, without killing microbes.

Freeze-drying, or lyophilization

involves freezing and applying vacuum so that water is lost by sublimation. This combines both exposure to cold temperatures and desiccation

Water content/ water activity

can be lowered by adding solutes such as salts or sugars. At very high concentrations of salts or sugars, the amount of available water in microbial cells is reduced dramatically due to osmosis

Ionizing radiation

X-rays, gamma rays, and high-energy electron beams

• Penetrates cells, directly damages biological molecules. Causes DNA

mutations, leading to cell death.

• X-rays and gamma rays penetrate paper and plastic to sterilize packaged

materials.

Nonionizing radiation

Ultraviolet (UV) light

• Less energetic, less penetrating. Used for surface disinfection.

• Causes thymine dimers to form between adjacent thymines in DNA.

Filtration

• Physical separation of microbes from air or liquid.

• Uses filters with pores of specific sizes

• Ideal when liquids contain heat-sensitive components

High efficiency particulate air (HEPA)

filters have pores ~0.3 μm – filter out bacteria, endospores, and many viruses

• Efficiency of HEPA filters 99.97% for particles of 0.3 μm diameter or more

Membrane filters

Porous membranes with defined pore sizes

Microbes removed by physical screening

Cellular microbes ≥ 0.2 μm

Viruses ≥ 0.1 μm

Face masks

• Both surgical masks and KN95 respirators reduce the

outward particle emission rates by 90% and 74%

• For coughing, which produced the highest rates of particle

emission for of all expiratory activities tested, wearing

homemade masks considerably reduced the fraction of

large particles (> 0.8 μm)

chemical agents

chemically modify biological molecules, and can cause damage to proteins or DNA

preservatives act

by lowering the pH

inhibit metabolic pathways

important in microbes

Disk-Diffusion Method

or chemical agents

• Filter disks containing chemical placed on an agar plate inoculated with bacterium

• Compound diffuses and causes a zones of inhibition of microbial growth.

• Size of zone correlates with potency of compound

In-Use Test

determine whether an actively used solution of disinfectant in a clinical setting is microbially contaminated

Koch’s postulates

were the foundation of modern medical microbiology

• causative microbe must be present in diseased organisms, but not healthy organisms

• must be able to isolate the causative organism in pure culture

• must be able to infect a healthy organism with the isolated culture

• must be able to re-isolate microbe from experimentally infected organism

Pathogens

cause infectious disease in host organisms.

The host is a source of nutrients.

primary pathogens

almost always cause disease

• some Salmonella enterica strains; enterohemorrhagic E. coli

opportunistic

many common pathogens

• normally exist outside of the host (e.g., commensals)

• cause infection under the right circumstances

• age, weakened immune system, injury

• Staphylococcus epidermidis

some pathogens are obligate

• cannot exist outside host in the natural environment

• (perhaps can be cultured in the lab)

• Chlamydia, Rickettsia, Mycobacteria

reservoir

a natural population outside of the host

• drinking water supply, soil, etc.

• an animal population – e.g., zoonoses like rabies and influenza

• in organisms that spread the infection – e.g., arthropod vectors like mosquitos carrying malaria

WHERE PATHOGENS OFTEN EXIST

extracellular

• pathogens exist on or in host fluids and tissues, but do not enter host cells

• may move through circulatory system or migrate through the matrix between host cells

• can directly encounter elements of the immune system

•examples:

• E. coli

• Staphylococcus aureus

• Helicobacter pylori

• Borrela burgdorferi

intracellular

• microbes enter and multiply within host cells

• allows them to evade many elements of host immune system

examples:

• Listeria monocytogenes

• Mycobacterium tuberculosis

• Salmonella enterica

• Legionella pneumophila

infections have

a characteristic pattern of progression

Incubation period

pathogen entry, before symptoms

Prodromal stage

first onset if symptoms

Period of illness

disease is most severe, symptoms apparent

Period of decline

body fights off infection

Convalescence

symptoms resolve

Symptoms and disease trajectory

may be diagnostic

infectious dose 50 (ID50)

number of pathogens that will infect 50% of hosts in a specified time

Infectivity

• varies with pathogen, strain, etc.

• this is why antisepsis and disinfection are sufficient to reduce incidence of many infections

The dose of pathogens needed to bring on disease varies greatly – can be as few as 1

virulence

the intensity of pathogenicity – degree of harm to host

• may correlate with pathogen lifestyle

• opportunistic pathogens more likely to kill host

• obligate pathogens less likely to severely (or rapidly) harm host

virulence factors

facilitate infection, tissue invasion, or harm

• encoded by genes in the chromosome or on plasmids

• may determine whether opportunistic pathogens can cause infection

• can be acquired by horizontal gene transfer

Genes encoding virulence factors are often found in the

chromosome clustered within

pathogenicity islands

pathogenicity islands

correlate with pathogenicity

• absent in related but non-pathogenic strains or species

• may be transferred by HGT

• sequencing of these regions can determine if strain is virulent

pathogenicity islands encode common phenotypes

• toxin secretion

• pilus, or other features for attachment to host

• iron uptake

• biofilm formation

adherence

once a pathogen has gained entry to host, it must adhere somewhere

• typically recognizes specific host molecules

• mediated by adhesins – typically pili or surface proteins

• in pathogenic E. coli, adhesins target bacteria to sites of infection

• diarrhea: fimbriae bind sugars on intestinal epithelium

• hemolytic uremia: pili bind sugars on kidney cells

• urinary tract infection: pili bind sugars on urethral epithelium

invasiveness

ability to spread to adjacent tissues

secreted exoenzymes may aid spreading:

• break down extracellular matrix

• collagenase breaks down collagen

• hyaluronidase breaks down hyaluronic acid

• degrade carbohydrate-protein matrix between cells

• can also disrupt host cell surface

entry to blood or lymph: bacteremia

presence of bacteria in the blood

entry to blood or lymph: septicemia

pathogens or their toxins in the blood

Exotoxins

factors secreted by bacteria to cause damage to the host

• may induce tissue damage, aiding invasion

• may cause host cell lysis, releasing nutrients

channel-forming toxins

• self-assemble into pores in host-cell plasma membrane

• cause host cell lysis

AB toxins

• A and B components form a complex

• B component attaches to host cell, triggers endocytosis of AB complex

• A component is released, causes toxicity

(many ways to cause cell death)

anthrax

Anthrax toxin

A and B toxin components produced and secreted separately by Bacillus anthrax

Toxin has 3 constituent proteins

• (B) PA – protective antigen

• (A) EF – edema factor

• (A) LF – lethal factor

PA

assembles into a pore on the surface of the host cell

Either “A” toxin docks onto the PA pore

d is carried into the cell

lipopolysacchraride

endotoxin

LPS decorating the outer membrane of Gram-negative bacteria can be toxic

• LPS is not secreted, but either shed or released from dying bacteria

lipid A

Toxic

• it potently triggers a blood clotting factor, causing blood clotting

• septic shock

• also triggers fever, other symptoms

endotoxin must be

in the body to trigger these effects

• digestive tract is full of Gram-negative bacteria that do not trigger septic shock

intravenous drugs

must be free of even trace amounts of lipid A

Endotoxin: limulus amebocyte lysate assay

• Horseshoe crab blood cells contain a clotting factor (coagulogen) that responds to endotoxin

• In the presence of endotoxin, coagulogen clots into a gel

• Turns out this is an incredibly sensitive assay

coagulation

endotoxin contamination

no coagulation:

no contamination

Many extracellular pathogens can form biofilms

• in lungs of cystic fibrosis patients

• on implants and catheters

• in chronic wounds

Genes for biofilm formation are associated with

virulence

Pathogen biofilms:

• are associated with chronic infection

• increases virulence

• reduce sensitivity to antibiotics

• form a structural barrier from host immune cells

Biofilms may aid

attachment and invasion

Biofilms may also protect bacteria from immune cells

Immune system clears

most microbes

Many successful pathogens evade immune system

infect immune system cells

diminishing function

• Legionella, Mycobacteria

directly infect adjacent cells

evade antimicrobial peptides and immune cells

• Listeria, Rickettsia

capsules

prevent phagocytosis

• Streptococcus

minimize expression of antigens

Borrelia, Neisseria gonorrhoeae

Listeria monocytogenes

• Gram positive, intracellular pathogen

• virulent food-born pathogen

• meningitis, sepsis, stillbirth

Listeria monocytogenes binds to cell surface

and stimulates endocytosis, even in non-lymphocytes

• evades extracellular immune system components

• secretes enzymes that allow it to destroy vacuolar membrane, escape into cytosol

hijacks the actin cytoskeleton

• stimulates actin polymerization

• propels bacterium around in host cell

termed actin gliding

• actin gliding can project Listeria into adjacent cell

Legionella pneumophila

• Gram negative, intracellular pathogen

• accidental human pathogen – normally infects amoeba and protists

• humans are a dead-end host

• Legionella responds to macrophages as if they were amoeba

Legionella pneumophila endocytosed by host cell

• does not escape vacuolar compartment

• creates a special type 4 secretion system

• secretes hundreds of effector proteins that modulate host cell

• disrupts normal vacuolar trafficking to lysosome

• recruits proteins and lipids to disguise surface of its vacuole

• creates a safe niche for itself inside host cell, where it multiplies