Lesson 2 Flashcards
Chemicals produced by microorganisms that inhibit the growth of other
microorganisms
Antibiotics
Antibiotic has
Antibacterial agents
Antibiotic can be
Bactericidal or bacteriostatic
Antibiotic can be
Bactericidal or bacteriostatic
Classified as NARROW spectrum or BROAD Spectrum
Antibiotics
with limited coverage against some specific bacteria
Narrow spectrum
have a wide coverage to groups of bacteria
Broad spectrum
Antibiotics Routes of Administration
Oral
Intravenous (IV)
Intramuscular (IM)
occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials/antibiotics
Bacterial resistance
Bacteria develop resistrance to antibiotic through evolution by changing their structure or
components.
Intrinsic Resistance:
Bacteria develop resistance to antibiotics through a new genetic mutation that helps the
bacterium survive or by getting DNA from a bacterium that already is
resistant.
Acquired resistance
Example of intrinsic resitance
antibiotic that affects the wall-building mechanism of the
bacteria, such as penicillin, cannot affect bacteria that do not have a cell wall.
Type of AR where Resistant strains outgrow Susceptible strains and
new strains are R
Chromosomal Mutations
Type of AR where extrachromosomal elements of DNA that are assoc with virulence and antibiotic R
Plasmids
can transfer from plasmid to
plasmid or from DNA chromosome to plasmid
Transposons “jumping genes”
Bacterium DNA change and alter the production of protein, different bacterial components and
receptors, bacteria unrecognized by the antibiotic
Genetic change
Example of genetic change
Escherichia coli (E. coli) and Haemophilus influenza resistance to trimethoprim
Bacteria can share genetic components with other bacteria
and transfer the resistant DNA through a horizontal gene transfer.
DNA Transfer
How can a Bacteria develop R to antibiotics?
- Intrinsic resistance
- Acquired resistance
- Genetic change
- DNA Transfer
Factors for Antibiotic Resistance
•Natural Occurrence/Selection
• Self-medication
•Clinical Misuse/Overuse
• Environmental Pollution/Improper discarding of
unsused/used antibiotics
• Overuse of disinfectants
Laboratory test/procedure in microbiology to determine which
drug will inhibit/kill the microorganism and which drug is
resistant to the microorganism
Antimicrobial susceptibility test
The result will help the physician to decide which drug
(antibiotic) is effective in killing the bacteria causing the
infection as well as what bacteria is present or causing the
infection
Antimicrobial susceptibility test
Standards in analyzing the results, agar used, methods and
incubation is set by CLSI and EUCAST
Antimicrobial susceptibility test
CLSI means
Clinical and Laboratory Standards Institute
EUCAST means
European Committee on Antimicrobial Susceptibility Testing
AMST means
Academy for Medical Science Technology
AMST antimicrobial susceptibility test
Media
Disc
Inoculum
low in Ca and Mg ions that interfere activity of antibiotic
- gives favorable growth on fastidious bacteria)
Mueller-Hinton agar
Best media culture to use is
Mueller-Hinton Agar
Concentration of bacteria that will be added to the agar or broth
Inoculum
Standardize by comparing the turbidity to McFarlands
Inoculum
used as a reference to adjust the turbidity
of bacterial suspensions so that the number of
bacteria will be within a given range to standardize
microbial testing
McFarland standards
McFarland standard usually at
0.5
Commercially prepared _______ strips at different
concentrations
Antibiotics
Selection is base from the bacteria isolated and identified, and
from the availability of _________ discs/strips
Antibiotics
- Disc Diffusion method
- Qualitative test
KIRBY BAUER DISK DIFFUSION TEST
MIC method
– Quantitative test
Minimum inhibitory concentration
placing a strip impregnated with antimicrobials onto an agar
plate
E test
can be determined by culturing microorganisms in liquid media or on plates of solid growth medium
Mic method
VITEK 2, BD Phoenix, and Microscan systems, are
the most common methodology for AST
Automated systems
Antibiotic sensitivity test (diffusion)
Kirby-Bauer method
Strokes method
Antibiotic sensitivity test (dilution)
Tube dilution
Agar dilution
Antibiotic sensitivity test (diffusion and dilution)
E test
Antibiotic sensitivity test (diffusion) =
Qualitative methods
Antibiotic sensitivity test (dilution) =
Quantitative method
culture-based microbiology assay used in diagnostic and drug
discovery laboratories
Kirby-Bauer test method
performed by inoculating the surface of an agar plate with
bacteria isolated from a patient’s infection
Kirby-Bauer test method
circular area around the spot of the antibiotic
in which the bacteria colonies do not grow
Zone of inhibition
Qualitative method:
Sensitivity
Intermediate
Resistant
- Inoculated agar plate
- Addition of antibiotic discs
- Incubation
- Measurement of zone of inhibition
Kirby-Bauer disc diffusion method
Quantitative method to determine the lowest concentration of an
antibiotic to prevent visible in vitro growth of bacteria
Minimum Inhibitory Concentration (MIC)
the lowest
concentration of an antibacterial agent required to kill a
bacterium over a fixed, somewhat extended period
Minimum Bactericidal Concentration (MBC)
a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate.
E test
Etest (previously known as the
Epsilometer test
a computer system that perform organism detection and susceptibilities on specimens
Automation (automated system)
Methods of controlling microbial growth
Physical
Chemical
Under physical
- Sterilization by heat
- Sterilization by irradiation
- Sterilization by filtration
- Low temperature
Under physical by sterilization by heat
- Dry heat
- Moist heat
- Boiling
- Pasteurization
Under chemical
- Alcohol
- Oxidizing agents
- Halogens
- Alkalies
- Acids
- Gases
- Quarternary Ammonium Compounds
- Soaps
purpose is to destroy all microorganisms and their spores on inanimate objects
Sterilization
purpose is to destroy or irreversibly inactivate microorganisms (but
not their spores) on inanimate objects
Disinfectant
chemical germicide for use on the skin or tissues and should not be
substituted for disinfectant
Antiseptic
inhibits/ suppresses growth of bacteria
Bacteriostatic
kills bacteria
Bactericidal
Spores killed in 2hrs
160°C Hot air oven
Pathogenic bacteria killed in 3 sec
140°C Ultra Heat Treatment (UHT) method
Most bacteria killed in 15 min.
Most spores killed in 30 min.
121°C autoclave
Spores killed in 2 hrs
100°C boiling water
Pathogenic bacteria killed in 15 sec
72°C flash pasteurization
Pathogenic bacteria killed in 30 min
63°C Holding method pasteurization
Human body temp
37°C
Refrigerator temp
4°C
Home freezer temp
-18°C
Kills microorganisms by denaturing/coagulating their proteins and enzymes
Sterilization by heat
Sterilization by heat Indicators of Effectiveness:
- Thermal Death Time (TDT)
- Thermal Death Point (TDP)
- Decimal Reduction Time (DRT)
minimum time it takes to kill a population of
microbes at a specific temperature
Themal Death Time (TDT)
lowest temperature that is required to kill a
population of microbes when applied for a specific time.
Thermal Death Point (TDP)
Time in minutes at which 90% of bacteria is
killed within a given period of time. 🡪 canning industry
Decimal Reduction Time (DRT)
Heating to 100C at boiling point
• Kills vegetative forms of bacteria (water-borne), most virus, and fungi within
1 minute
Boiling
can survive up to 30 minutes of boiling
Hepatitis virus
can survive up to 20 minutes of boiling
Endospores
Temperatures of about 160C for 60minutes – necessary to kill most spores
• Use primarily on glassware, metals and fatty substance which are not
permeable to water
• Ex: Hot Air Oven
Dry heat
Most effective method of sterilization (Autoclave)
• Requires moist heat at 121C, 15-30min
• Kills microorganisms by coagulating their proteins
Moist heat
2 methods of mosit heat
• 1. Tyndallization
• 2. Autoclaving
the steaming process performed at 100°C done in steam sterilizer for 15-20
minutes followed by incubation at 37°C overnight and this cycle is repeated
for successive 3 days
• Use to kill spores
• Uses Arnold Sterilizer
Tyndallization
Tyndallization AKA
Fractional sterilization
Most reliable method of heat sterilization
• More modern
• Use to sterilize culture media and surgical supplies
Autoclave
Autoclave
121°C, 15 mins, 15 lbs pressure
process of food preservation in which packaged and unpacked foods (e.g.,
milk and fruit juices) are treated with mild heat, usually to less than 100 °C
(212 °F), to eliminate pathogens and extend shelf life
Pasteurization
destroys or deactivates microorganisms and enzymes that contribute to food
spoilage or the risk of disease
Pasteurization
Types of pasteurization
- High-temperature short time (HTST) pasteurization
- Ultra-high temperature (UHT) pasteurization
for milk; (71.5 °C
(160.7 °F) for 15 seconds) which ensures safety of milk and provides a
refrigerated shelf life of approximately two weeks
High-temperature short time (HTSHT) pasteurization
milk is pasteurized at 135 °C
(275 °F) for 1–2 seconds, which provides the same level of safety, but along
with the packaging, extends shelf life to three months under refrigeration
Ultra-high temperature (UHT) pasteurization
UV rays with shorter WL are more effective in killing bacteria
Irradiation
used to sterilize rooms
Mercury vapor lamps with WL 240-280
Use if heat is not feasible (some carbohydrates solutions, serum, body fluids)
• Removes microbes by passage of liquid or gas through a screen like material with
small pores
Filtration
A typical microfiltration membrane pore size range is
0.1 to 10 micrometer
most
commonly used being____which is sufficient to eliminate bacteria
and fungi
0.2 to 0.45 micrometer
use in operating rooms to
eliminate bacteria; mostly filters particles that are 0.3um; capture pollen, dirt, dust,
moisture, bacteria (0.2–2.0 μm), viruses (0.02–0.3 μm), and submicron liquid
aerosol (0.02–0.5 μm).
High Efficiency Particulate Air filter (HEPA)
usually inhibit or stop microbial growth and proliferation but often do not
kill bacteria (BACTERIOSTATIC)
• Refrigeration (4ºC) and freezing (-20ºC or less) are commonly used in the
food, pharmaceuticals and biotechnology industry
Low temperature
three types of alcohol
Ethanol
Methanol
Isopropanol
works by denaturing and coagulating proteins, disrupting their cell wall, and
killing them; dissolves lipid membranes
• highly efficient against viruses and can be used in adjunct with other
alcohols to obtain a powerful synergistic effect against microorganisms
Alcohol
• act by oxidizing the cell membrane of microorganisms, which results in a loss
of structure and leads to cell lysis and death
Oxidizing agents
destroy the cellular protein, nucleic acid, and cell wall or membrane of
microorganisms
• Disrupts oxidative phosphorylation, which is the most important process in cel
Halogens
Example of halogens
Iodide, Chlorines
Porous surface:
Hard Surfaces:
Concentrated infectious agents:
1:10 dilution
1:100 dilution
1:5 dilution
Example of oxidizing agent
H2O2, K-permanganate
Example of oxidizing agent
H2O2, K-permanganate
produce saponification of the fatty acids within cell membranes, resulting in
the loss of membrane integrity.
Alkalies
Examples of alkalies
KOH, NaOH
disrupts the amphoteric matter in microbial surface structures and increases
the permeability of cell membrane, subsequently metabolic processes are
hindered
Acids
Ex. Of acids
Nitric Acid, Sulfuric Acid
inactivates microorganisms by alkylating the amino and sulfhydral groups of
proteins and ring nitrogen atoms of purine bases
Gases
Example of gases
Formaldehyde
bactericidal and fungicidal activity
• permeate into the membrane and disrupt its physical and biochemical
properties
Quaternary Ammonium Compounds
Example of Quaternary Ammonium Compounds
hexadecyltrimethylammonium (‘cetrimide’), chlorhexidine, and benzalkonium
chloride
disrupt the chemical bonds that allow bacteria, viruses and grime to stick to
surfaces, lifting them off the skin
• Antimicrobial soaps 🡪 triclosan and triclocarban
Soap
Any chemical use to treat an infections either by inhibiting or killing
pathogens
• Antibacterial
• Antifungal
• Antiprotozoals
• Antiviral
Antimicrobial agents
substance produced by microorganism that is effective in killing or
inhibiting growth of microorganisms
Antibiotics
Antimicrobial Agents
• Ideal Qualities
- kill/inhibit pathogens
- Cause no damage to the host
- Cause no allergic reactions
- Stable when stored in solid or liquid form
- Remain in specific tissue of the body long enough to be effective
- Kills the pathogens before they mutate and become resistant to it
trap bacteria with the assistance of cilia
Mucus (respiratory tract)
present in respiratory secretions lyses bacterial cell wall
Lysozymes
possess hydrolytic enzymes that breakdown bacteria
Saliva
destroys bacteria that are acid labile
Gastric acid of the stomach
constant flushing action
Eyes tears
Normal flora of the ________
• _______ of the urine and constant flushing action
• ____________ – normal flora
Large intestines
Acidic pH
Vaginal lactic acid
total changes occurring in tissue factors upon injury
Inflammation
increased blood flow 🡪 WBC, other cells, plasma proteins (complement,
interferons), antibodies migrate to the injured site 🡪 arrest the
insulting foreign organism
Inflammatory response
Acquired in the hospital or other health care setting
Nosocomial infection
- ______ – result from organisms that are part of the patient’s NF
- _______ – from external sources (contamination, inanimate objects)
Endogenous
Exogenous
presence and multiplication of microorganisms within a host
with no clinical signs of infections 🡪 Reservoir
Colonization
the entrance and multiplication of a microorganism in a host
Infection
condition assoc with functional and structural harm to
the host 🡪 s/s
Infectious disease
Common Nosocomial Infections
UTI
• Surgical wound infections
• LRTI
• Bacteremia
• Aspiration Pneumonia
Common Pathogens causing NI
S. aureus
• E. coli
• P. aeruginosa
• Coagulase negative Staph
• Enterococcus
• Klebsiella
Susceptible Patients to NI
Elderly
• Pregnant women
• Premature Infants
• Surgical and Burn Px
• Diabetic and Cancer Px
• On medications with steroids, chemotherapy, radiation,
• Immunosuppressed px
• Undergoing renal dialysis, catheterization, or intubated
Routes of Infection
Direct
Indirect
across the placenta (Syphillis) or through the vaginal canal
(Gonorrhea)
Congenital
across the placenta (Syphillis) or through the vaginal canal
(Gonorrhea)
Congenital
common colds, skin infections, GI pathogens
Hand to hand contact
Strep throat, common colds, URTI
Droplets
Direct Route
- Droplets
- Sexual
- Congenital
- Hand to hand contact
Fomites – includes inanimate objects
• Ingestion of contaminated food and water
• Airborne
• Animal or arthropod vectors
Indirect route
describe the handling of clinical
specimens where BLOOD and other body fluids should be treated as
INFECTIOUS
Universal precautions
Guidelines to ensure safety in the laboratory 🡪
OHSA, CDC, CAP, JCAHO
Publication of standards for Bloodborne Pathogens
(OSHA)
a significant part of the Universal
Precaution
Ppe
(GHS)
Globally Harmonized System of Classification
and Labeling of Chemicals
General Considerations for Safety
No food and Drink
• No smoking
• Cosmetics are prohibited
• Use proper and recommended protective
eyewear
• Use Face shields with googles
• Use proper and appropriate mask
• Hair should be worn so as to prevent in
contact with surfaces
• Handwashing
• Accessible eyewash stations
• Emergency Showers
• Sharp object should be handled
with care
• Avoid spillage of blood and other
liquid specimens
an enclosed, ventilated laboratory workspace for safely working with
materials contaminated with (or potentially contaminated with) pathogens
requiring a defined biosafety level.
Biological safety cabinets
primary purpose of a ___ is to serve as a means to protect the laboratory
worker and the surrounding environment from pathogens.
Biological safety cabinets
provides protection for the user and surrounding environment, but no
protection for the sample being manipulated
• Open-front, negative pressure, ventilated cabinets
• Unsterilized room air enters and circulates within the cabinet and exhaust air
from the cabinet is filtered via HEPA filter
Class I BSC
Sterilize both the air entering and circulating the cabinet and exhaust air
• USED BY MOST HOSPITAL MICROBIOLOGICAL
LABORATORIES
• Also known as LAMINAR FLOW
Class II BSC
Provides the highest level of safety
• All air entering and leaving the cabinet is STERILIZED with HEPA filter
• System is entirely closed and all infectious material are handled with rubber
gloves that are sealed to the cabinet
Class III BSC
Lowest safety lecvel
Not known to cause disease in afult human
Non-pathogenic microbe
BSL-1
Moderate danger if inhale, swallow, or expose to skin
Influenza
BSL-2
Moderate danger if inhale, swallow, or expose to skin
BSL-2
Severe or potentially lethal disease
HIV, H5N1 Flu
BSL-3
Highest safety level
Life threathening disease
Ebola, SARs, CoV2
BSL-4
BSL means
Biosafety level
Protein antigen
Secondary immune response
IgG
Polysaccharude antigen
Primary immune response
IgM
Polysaccharude antigen
Primary immune response
IgM
Preventipn of bacterial or viral infection
Found in tears, milk, saliva
IgA
B cell receptor
Plays a role in Autoallergic diseases
IgD
Major role in allergic response
IgE
Dilates blood vessel
Histamine
Increase vascular permeability and enhance release of other mediators from WBC
Kinins
Affects WBC mobility and metabolism
Leukotrienes
Formed in hypothalamus
Induce fever
Prostaglandins
Liver protein play role im acute response
C-reactive protein
Stimulates cell immune response
Interleukin-1
Causes proliferation of activated T and B cells
Interleukin-2
Stimulate WBC promoting growth and differentation
Cytokines
Promote growth of T and B cells
Gamma interferon
Promote growth of T and B cells
Gamma interferon