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MICROBIAL CONTROL
Ms. Nerralyn N. Muni R.M.T.
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Biosafety Levels
LevelLevel RiskRisk Types of Types of
AgentsAgents
Example ofExample of
AgentsAgents
Precautions requiredPrecautions required
II MinimalMinimal
biohazardbiohazard
Those notThose not
known toknown to
cause diseasecause disease
in healthyin healthyadultsadults
Bacillus subtilis,Bacillus subtilis,
MycobacteriumMycobacterium
gordonaegordonae, soil, soil
microbesmicrobes
No special equipmentNo special equipment
required. Work can berequired. Work can be
done on opendone on open
benchtops.benchtops.
IIII ModerateModerate
biohazardbiohazard
CommonCommon
pathogenspathogens
found infound inhumanhuman
blood, bodyblood, body
fluids, andfluids, and
tissuestissues
Escherichia coli,Escherichia coli,
SalmonellaSalmonella, HIV,, HIV,
hepatitis B virus,hepatitis B virus,influenzainfluenza
Procedures with theProcedures with the
potential for aerosolspotential for aerosols
or splashes must beor splashes must beconducted in aconducted in a
biologic safety cabinet.biologic safety cabinet.
Access toAccess to
laboratory must belaboratory must be
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Biosafety Levels Continued
LevelLevel RiskRisk Types of Types of
AgentsAgents
Example ofExample of
AgentsAgents
Precautions requiredPrecautions required
Limited when work isLimited when work is
being conducted. Anbeing conducted. An
autoclave must beautoclave must beavailableavailable
IIIIII HighHigh
biohazardbiohazard
Agents thatAgents that
may causemay cause
serious orserious or
lethal diseaselethal diseasefromfrom
exposure viaexposure via
inhalation.inhalation.
EffectiveEffective
Bacillus anthracisBacillus anthracis
Francisella,Francisella,
Brucella,Brucella,
MycobacteriumMycobacteriumtuberculosis,tuberculosis,
Rickettsia rickettsiiRickettsia rickettsii,,
Manipulation ofManipulation of
infectious materialsinfectious materials
must be conducted inmust be conducted in
class II biologic safetyclass II biologic safetycabinet by personnelcabinet by personnel
wearing personalwearing personal
protective clothing.protective clothing.
Access toAccess to
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Biosafety Levels Continued
LevelLevel RiskRisk Types of Types of
AgentsAgents
Example ofExample of
AgentsAgents
Precautions requiredPrecautions required
TreatmentTreatment
availableavailable
Coxiella burnetii,Coxiella burnetii,
mold stages ofmold stages of
systematic fungisystematic fungi
laboratory must belaboratory must be
controlled.controlled.
Specialized ventilationSpecialized ventilationminimizing release ofminimizing release of
infectious aerosolsinfectious aerosols
requiredrequired
IVIV ExtremeExtreme
biohazardbiohazard
Agents thatAgents that
pose a highpose a highrisk of liferisk of life--
threateningthreatening
disease,disease,
which maywhich may
Ebola virus,Ebola virus,
Lassa virus, and
Lassa virus, andothers that causeothers that cause
hemorrhagichemorrhagic
feversfevers
Requires use of classRequires use of class
III biologic safetyIII biologic safetycabinet or fullcabinet or full--body,body,
airair--supplied positivesupplied positive
pressure suit andpressure suit and
independent unit withindependent unit with
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Biosafety Levels Continued
Level Risk Types of
Agents
Example of
Agents
Precautions
required
be transmitted
by aerosolsand for which
there is no
vaccine or
therapy
specialized
ventilation andwaste management
systems
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Biologic Safety Cabinets
ClassClass Description Description Use Use
II Unsterilized room air passes into theUnsterilized room air passes into the
cabinet. Air passes through a highcabinet. Air passes through a high--
efficiency particulate air (HEPA) filterefficiency particulate air (HEPA) filter
before being exhaustedbefore being exhausted
Provides minimalProvides minimal
personnel protection.personnel protection.
Doesnt protect workDoesnt protect work
surface.surface.
IIII Laminar flow cabinets with variableLaminar flow cabinets with variable
sash opening. Air passes through onesash opening. Air passes through one
HEPA filter before reaching the workHEPA filter before reaching the work
surface and another before beingsurface and another before beingexhausted.exhausted.
Type most commonlyType most commonly
used in hospitalused in hospital
microbiologymicrobiology
laboratories. Provideslaboratories. Providesprotection for both theprotection for both the
laboratorian and thelaboratorian and the
work. Must maintain anwork. Must maintain an
open area for 3 feetopen area for 3 feet
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Biologic Safety Cabinets Continued
ClassClass Description Description Use Use
From cabinet duringFrom cabinet during
operation. Must beoperation. Must be
certified initially,certified initially,whenever moved morewhenever moved more
than 18 inches, andthan 18 inches, and
annually thereafter.annually thereafter.
IIIIII Self Self--contained system, completelycontained system, completely
enclosed, with negative pressure. Air isenclosed, with negative pressure. Air isfilter sterilized coming in and goingfilter sterilized coming in and going
out. Gloves are attached to the frontout. Gloves are attached to the front
for manipulations inside the hood.for manipulations inside the hood.
Provides maximumProvides maximum
protection. Usedprotection. Usedextensively in researchextensively in research
facilities.facilities.
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Physical Methods of Microbial
Control
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Dry Heat Sterilization
kills spores, vegetative forms
ideal for sterilizing glasswares, metalwares,
and anh
ydrous oils Oxidizing to ashes
Burns cell constituents
Denaturing proteins
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Direct flaming
Incineration
Oven
160-170 deg. Celsius- 2-3 hours
200 deg. Celsius 1 hours
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Moist Heat Sterilization
more effective than dry heat method
coagulation of the cell protein
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Autoclaving
most effective method of moist heat sterilization
for vegetative cells, spores
121 deg. Celsius- 30 mins. 15 psi
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Boiling
for vegetative cells and viruses
100 deg. Celsius-10 mins
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Pasteurization
Destroy spoilage organism in wine, vinegar,
milk, juices
62 deg. Celsius- 30 mins 72 deg. Celsius- 15 secs (HTST)
82 deg. Celsius- 20 secs-ice cream
104-150 deg. Celsius- 2-5 secs (UHT)
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Fractional Sterilization Method
for vegetative forms of microorganisms
Tyndallization
Inspissation
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DayDayTemperTemper
atureatureTime Time PurposePurpose
11 8585CC 60 minutes60 minutesDrying of the medium and killing theDrying of the medium and killing the
organisms in their vegetative formorganisms in their vegetative form
Time inTime in
betweenbetween
overnightovernight
incubationincubationGrowth of vegetative forms from sporesGrowth of vegetative forms from spores
2275 to75 to
8080CC20 minutes20 minutes killing the organisms in their vegetative formkilling the organisms in their vegetative form
Time inTime in
betweenbetween
overnightovernight
incubationincubation
Growth of vegetative forms from any sporesGrowth of vegetative forms from any spores
remainingremaining
3375 to75 to
8585CC20 minutes20 minutes killing the organisms in their vegetative formkilling the organisms in their vegetative form
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Filtration
nitrocellulose or cellulose acetate
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Cold
The effectiveness of low temperatures
depends on the particular microorganisms and
the intensity of the application
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Dessication
Removal of H2O
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Osmotic pressure
high concentrations of salts and sugars
undergo plasmolysis
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Radiation
Ultraviolet radiation
reduction of air borne contamination
has poor penetration capability
Ionization Radiation
high energy emitted from radioactive isotopes
such as cobalt 60 (gamma rays) or by cathode or
beta rays
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Chemical Methods of Microbial
Control
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Principles of Effective Disinfection
Careful attention should be paid to the
properties and concentration of the
disinfectant to be used
The presence of organic matter, degree of
contact with microorganisms, and
temperature should also be considered
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Phenol and Phenolics
injuring plasma membranes
inactivating enzymes
denaturing proteins
Triclosan- phenolic compound toothpaste,
lotions, deodorant soaps
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Biguanides (Chlorhexidine)
damages plasma membranes of vegetative
cells
Soaps, lotions, cath
eters, surgical mesh
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Halogens
Iodine combines with the amino acid tyrosine
Tincture
Iodophore
Chlorine is added to water forming
hypochlorous acid
Water treatment
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Alcohols
denaturing proteins
dissolving lipids
Antiseptic, disinfectant
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Heavy Metals and Their Compounds
Silver, mercury, copper, and zinc
Opthalmia neonatorum
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Quaternary Ammonium Compounds
(Benzalkonium chloride, Cetylpyridinium chloride)
disrupting plasma membranes most effective against gram-positive bacteria
Disinfectant, non-food substances
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Organic Acids and Derivatives
Sorbic acid, benzoic acid, and propionic acid
inhibit fungal metabolism
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Aldehydes
inactivating proteins
Gluteraldehyde medical instruments
Formalydehyde vaccine preparation
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Gaseous Chemosterilizers
protein denaturation
Ethylene oxide gas
Easily penetratesh
ard-to-reach
places and fabricsand does not damage moisture-sensitive material
Toxic, explosive and potentially carcinogenic
For medical devices
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Oxidizing Agents
Ozone and peroxide are used as antimicrobial
agents.
oxidizing molecules inside cells
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Antimicrobial Action
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Principles
1. The agent must be in an active form.
This is ensured through the *pharmacodynamic
design of the drug, which takes into account the
route through which the patient will receive the
agent (e.g., orally, intramuscularly, intravenously).
* The study of the action or effects of drugs on living
organisms.
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2. The antibiotic must also be able to achievesufficient levels or concentrations at the siteof infection so that it has a chance to exert an
antibacterial effect (i.e., be in anatomicapproximation with the infecting bacteria).
The ability to achieve adequate levels depends onthe *pharmacokinetic properties of the agent.
* The process by which a drug is absorbed,distributed, metabolized, and eliminated by thebody.
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3. The remaining steps in antimicrobial action
relate to direct interactions between the
antibacterial agent and the bacterial cell
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Mode of Action of Antibacterial
Agents
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Inhibitors of Cell Wall Synthesis
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Beta-Lactam Antimicrobial Agents
Beta-lactam antibiotics are those that contain
the four-membered, nitro-gen-containing,
beta-lactam ring at the core of their structure.
The beta-lactam ring is key to the mode of
action of these drugs that target and inhibit
cell wall synthesis by binding the enzymes
involved in synthesis.
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AntibioticsAntibioticAntibiotic Mode of Mode of
ActionAction
ActivityActivity Comments Comments
BetaBeta--LactamsLactams
PenicillinsPenicillins
Penicillin,Penicillin, ampicillinampicillin,,mezlocillinmezlocillin,,
piperacillinpiperacillin,, AugmentinAugmentin
CephalosporinsCephalosporins
CefazolinCefazolin,, cofotetancofotetan,,
ceftriaxoneceftriaxone,, cefotaximecefotaxime,,
ceftazidimeceftazidime
AztreonamAztreonam
ImipenemImipenem
InhibitionInhibition
of cell wallof cell wall
synthesissynthesis
EffectiveEffective
against gramagainst gram--
positives andpositives and
gramgram--
negativesnegatives
UsuallyUsually
bactericidal.bactericidal.
Low toxicity.Low toxicity.
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Glycopeptides
Glycopeptides bind to precursors of cell wall
synthesis, the binding interferes with the
ability of the PBP enzymes, such as
transpeptidases and transglycosylases, to
incorporate the precursors into the growing
cell wall
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Antibiotics Continued
AntibioticAntibiotic Mode of Action Mode of Action Activity Activity Comments Comments
GlycopeptidesGlycopeptides
VancomycinVancomycin
Inhibition of cellInhibition of cell
wall synthesiswall synthesis
EffectiveEffective
against gramagainst gram--
positivespositives
resistant toresistant to
other betaother beta--
lactams.lactams.
UsuallyUsually
ineffectiveineffective
against gramagainst gram--negatives.negatives.
Used to treatUsed to treat
methicillinmethicillin
resistantresistant
staphstaph
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Inhibitors of Cell Membrane
Function
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Inhibitors ofProtein Synthesis
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Aminoglycosides
Aminoglycosides binds to protein receptors on
the organisms 30S ribosomal subunit
gentamicin.
tobramycin, amikacin, netilmicin,
streptomycin, and kanamycin
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Macrolide-Lincosamide-Streptogramin
(MLS) Group
macrolides, such as erythromycin,
azithromycin, and clar-ithromycin, and
clindamycin (a lincosamide).
binds to receptors on the bacterial 5OS
ribosomal subunit
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Oxazolidones
currently represented by linezolid
Linezolid is a synthetic agent that inhibits
protein synthesis through a unique
mecha-nism.
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Chloramphenicol
binds to the 5OS ribosomal subunit
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Tetracyclines
binds to the 30S ribosomal subunit
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Inhibitors ofDNA and RNA
Synthesis
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Fluoroquinolones
simply referred to as quinolones, are
derivatives of nalidixic acid, an older
antibacterial agent.
bind to and interfere withDNA gyrase
enzymes
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Metronidazole
direct interactions between the activated drug
and DNA that results in breakage ofDNA
strands
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Rifampin
binds to the enzyme DNA-dependent RNA
polymerase and inhibits synthesis of the RNA
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Inhibitors of Other Metabolic
Processes
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Sulfonamides
bind to dihydropteroate synthase and disrupt
the folic acid pathway
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Trimethoprim
dihydrofolate reductase is inhibited
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Nitrofurantoin
have several targets involved in bacterial
protein and enzyme & synthesis and the drug
also may directly damage DNA
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Testing Methods
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Disk Diffusion/Kirby Bauer Technique
Using the disk diffusion susceptibility test,
antimicrobial resistance is detected by
challenging bac-terial isolates with antibiotic
disks that are placed on the surface of an agar
plate that has been seeded with a lawn of
bacteria.
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BrothDilution Tests
Using the broth dilution test, the microorganismis grown in liquid media containing differentconcentration of antibiotics.
The minimum inhibitory concentration (MIC) isthe lowest concentration of chemotherapeuticagent capable of preventing microbial growth.
The minimum bactericidal concentration (MBC) is
the lowest concentration of chemotherapeuticagent that kills bacteria
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Broth Microdilution Methods
Commercial panels are designed to receive
the stan-dard inoculum and are incubated
using conditions and durations recommended
for conventional broth microdilution.
They are growth-based systems that require
overnight incubation, and NCCLS interpretive
criteria apply for interpretation of mostresults.
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Agar Dilution Derivations
Uses an instrument to apply antimicrobial
agent to the surface of an already prepared
agar plate in a concentric spiral fashion.
Start-ing in the center of the plate the
instrument deposits the highest concentration
of antibiotic and from that point drug
application proceeds to the periphery of theplate
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Serum bactericidal test
The serum bactericidal test (SBT) is analogous
to the MIC-MBC test except that the medium
used is patients serum that contains the
thera-peutic antimicrobial agent(s) the patient
has been receiving
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