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Presented by
M.Vimali,
Pharm.D(PB)- I year
Microbiology
 Microbiology is the study of microbes that infect
humans, the disease they cause, their diagnosis,
prevention and treatment.
 It also deals with the response of the human
host to microbial and other antigens.
Culture
 Bacteria have to be grown(or cultured) on
culture media for them to be identified, as only
rarely can they be recognized by their
morphology alone.
 They are the invitro procedures used to
detect anti-microbial resistance in individual
bacterial isolates.
 The same can be used for monitoring the
emergence and spread of resistant micro
organisms in a population.
 In modern laboratories, bacteria are usually
identified by characterization of the genome:
identifying the characteristics of the DNA and
RNA of the sample species
 This typing of testing is generally considered
more reliable-and soon, less expensive- than
actually growing bacterial cultures and exposing
them to various types of antibiotics to see which
drugs kill or inhibit the bacterial growth. But if
more than identification is required, and if an
anti
 also called susceptibility testing
 Helps to find the right anti-biotic to kill an
infecting organism. This test determines
the sensitivity of a colony of bacteria to an
antibiotic.
 The efficacy of an antibiotic can be
demonstrated under suitable conditions by
its inhibitory effect on micro organisms.
 Unfortunately, many bacteria are resistant to
common antibiotics.
 Resistant means that the drug cant kill the
bacteria.
 Sensitivity analysis is a useful tool to help
quickly determine if bacteria are resistant to
certain drugs.
 Also be used for fungal infection
 Examples of antibiotic resistant infections
include:
 A persistent sore throat
 A recurring urinary tract infection(UTI)
 An unresponsive case of pneumonia.
 Sensitivity analysis starts with a bacterial
sample.
 The sample is obtained by swabbing the
infected area or secretions of the infected area
and from any area that has an infection.
 Cultures are taken from blood
 Urine
 Sputum
 Inside a cervix
 A wound
 The doctor will send the specimen to a licensed
laboratory in a special culture tube for testing.
 Spread a sample of the infective material onto a
plate of nutrient substance-culture media and
the Bacteria in the culture will grow and multiply
 The bacteria will form colonies-large groups of
bacteria that will be exposed to different
antibiotics
 With a sufficient population of bacteria grown
on the plate in the form of a “lawn", the
technicians will perform two main operations:
1.IdentifyThe Species Of Bacteria:
 This is done with various techniques,
including examination of lawn
characteristics-color, texture, growth pattern,
etc.-
 Gram staining, microscopic examination,
metabolic requirements and even DNA
sequencing.
 Bacterial species commonly isolated depends
on the location and the cause of the infection
2.DetermineThe Bacterial Populations
SensitivityTo A Range Of Antibiotics
 This can be done by p[lacing small disks of
filter paper or the agar impregnated with
various types of antibiotics onto the bacterial
lawn.The bacteria are allowed to incubate for
a specified days and then the late is examined
to see whether the bacterial growth is
inhibited or not by the antibiotics on each
disk.
 Clinical relevance of the isolate
 Purity of the isoate
 Logical panel of anti-microbial agent to be
tested-do ot include antibiotics to which the
isolate is known to have intrinsic resistance
 Availability of test methodology, resourcres, and
trained persons
 Standardisation of testing
 Valid interpretation of results
 Cost efficiency
 Effective means to communicate results and
interpretation to end-users
 Dilution method-broth and agar dilution
method
 Disc-diffusion method
 E-test
 Automated methods
 Mechanism-specific tests such as beta-
lactamase detection test and chromogenic
cephalosporin test
 Genotypic methods such as PCR and DNA
hybridization methods
 The broth dilution method involves subjecting a series
of concentrations of anti-microbial agents in a broth
environment.
 Micro dilution tests uses about 0.05 to0.1 ml total
broth volume and can be conveniently performed in a
micro-titre format.
 Macro dilution testing uses broth volumes at about
1.0ml in standard test tubes.
 For both these dilution methods, the lowest
concentration at which the isolate is completely
inhibited- as evidenced by the absence of bacterial
growth- is recorded as the minimal inhibitory
concentration.
 The MIC is thus the minimum concentration of
the anti-biotic that will inhibit the particular
isolate.
 The test is only valid only if the psitive contro
shows growth and negative control shows no
growth.
 A procedure similar to broth dilution is agar
dilution.
 Agar dilutuion method followa the principle of
establishing the lowest concentration of the
serially diluted antibiotic concentration at which
bacterial growth is stil inhibited.
 Because of convenience, efficiency and cos, the disc diffusion
method is probably the most widely method
 A growth medium, usually Mueller-hinton agar, is first even
seeded throughout the plate with the isolate of interest that has
been diluted at a standard concentration(app 1 to 2 x 10 coony
forming units per ml).
 Commercially prepared discs, each of which are pre impregnated
with a standard concentration of a particular, are then evenly
dispensed and lightly pressed onto yhe agar surface.
 The test antibiotic immediately begins todiffuse ouyward from the
discs, creating a gradient of antibiotic conc, isn the agar such that
the highest conc., is found close to the disc.
 After an overnight incubation, the bacterial growth around each
disc is observed.
 If the test isolate is susceptible to a particular antibiotic, a zone pf
inhibition is observed.
E-Test(AB Biodisk, Solna, Sweden) is a
commercially available test that utilises a
plastic test strip impregnated with a
gradually decreasing concentration of a
particular antibiotic.
The strip also displays a numerical scale that
corresponds to the antibiotic concentration
contained therein.
 This method provides for a convenient
quantitative test of antibiotic resistance of a
clinical isolate.
 However, a separate strip is needed for each
antibiotic, and therefore the cost of this can
be high.
 Most automated antimicrobial susceptibility
testing systems provide automated inoculation,
reading and interpretation.These systems have
the advantage of being rapid and convenient,
but one major limitation for most laboratories is
the cost entailed in initial purchase, operation
and maintenance of the machinery.
 Examples include:
 Avantage test system- abbott laboratories,
Irving,Texas.
 Phoenix-BD Biosciences, Maryland,etc.,
 Resistance may also be established through tests that
directly detectthepresence of a particular resistance
mechanism.
 For example, beta lactamase detection can be
accomplished using an assay such as the chrmogenic
cephalosporinase test(Cefinase disc by BD
Microbiology Systems, Cockeysville, MD and BBL Dry
slide Nitrocefin, Becton Dickinson, Sparks, MD) AND
DETECTION FOR CHLORAMPHENICOL MODIFYING
ENZYME CHLORAMPHENICOL acetyl
transferase(CAT) may utilise commercial colorim,etric
assays such as a CAT reagent kit(Remel, Lenexa,
Kansas)
 Although nucleic acid-based detections systems
are generally rapid and sensitive, it is important
to remember that the presence of a resistance
gene does not necessarily equate to treatment
failure, because resistance is also dependent on
the mode and level of expressions of these
genes.
 Some of the most common molecular
techniques utilized for anti-microbial resistance
detection are as follows:
 Polymerisation chain reaction:
 It is one of the most commonly used molecular
techniques for detecting certain DNA sequences of
interest.This involves several cycles of denaturation
of sample DNA, nnealing of apecific primers to the
target sequence and the extension of this sequence as
facilated by a thermostable polymerase leading to
replicatrion of a duplicate DNA sequence, in an
exponential manner, to a point ehich will be visibly
detectable by gel electrophoresis with the aid of a
DNA- intercalatingh chemical flouresces under uv
light.
 This is based on the fact the DNA
pyrimidines(cytosine and thymidine) specifically
pair up with purines(guanine and adenine; or
uracil for RNA).
 Therefore, a labelled probe with a known
specific sequence can pair up with opened or
denatured DNA from the ytest sample, as long
as their sequences complement each other.
 If this hybridisation occurs, the probe labels this
with a detectable radioactive isotope, antigenic
substrate, enzyme or chemiluminescent
compound.
 Whereas if no target sequence is present or
the isolate does not have the specifi gene of
interest, no attachment of probes will occur,
and therefore no signals will be detected.
 Modifications of the PCR and DNA
hybridization.With these basic principles,
several modifications have been introduced
which further improve the sensitivity and
Specificity of these procedures.
 Examples of such development the use of
flourecence-labeled oligonucleotides, the
development of molecular becons,
development of DNA arrays and DNA chips,
Among many others.
 Selection of the appropriate method will
depend on the intended degree of accuracy,
convenience, urgency, bioavailability of
sources, availability of expertise and cost
 The colonies show up as:
 Susceptible, resistant, or intermediate
 Susceptible- in this case, a clear, circular
“halo”-technically known as a “plaque” or
zone of inhibition- will appear around the
antibiotic disc, indicating an absence of
bacteria.The antibiotic has inhibited their
growth and/or killed them, means that they
cant grow if the drug is present.This indicates
an effective antibiotic
 Resistant : in this case, the filter Paper will have
no discernable plaque around it, meaning that
the bacteria can grow even in the presence of a
antibiotic.This indicates an ineffective anti-
biotic
 Intermediate: a somewhat cloudy plaque
indicates that not all the bacteria in the area
around the disc have been killed. this means that
a higher dose of the antibiotic is needed to
prevent the growth.
 The doctor can use the results to determine the
best antibiotic to treat your infection
 Few risks are associated with this test. Blood collection
comes with small risks.
 Rare risks of taking a blood sample include:
 Lightedness or fainting
 Hematoma- a bruise where blood accumulates
under the skin
 Infection-usually prevented by the skin being
cleaned before the needle is inserted
 Excessive bleeding- bleeding for a long period
afterwards may indicate a more serious
bleeding condition and should be reported to
the doctor
 The doctor will advise the patient about the potential
risks associated with the sample
 An antibiotic that bacteria, fungus, or
another micro organism shows resistance to
shouldn’t be used to treat the infection.Your
doctor will decide which drug is best if several
antibiotics are shown to be effective in killing
the micro organism causing the infection
 To provide a reliable prediction of whether an
infection caused by a bacterial isolate will
respond therapeutically to a particular
antibiotic treatment.
 This may be utilized as guidelines for
chemotherapy, or at the population level as
indicator of emergence and spread of
resistance based on passive or active
surveillance.
 Becos of the required culture time, sensitivity
testing may take several days, which is not
ideal in critical cases demanding urgency.
 Practitioners switch ove rto anti-biograms
 The culture media are prepared according to
the specifications of the USP, European
Pharmacopoeia, British Pharmacopoeia and
Indian Pharmacopoeia.
 The antibiotic media are identified
numerically with names assigned by Grove
and Randall Seed agar
 Base agar
 Assay broth
 Yeast beef agar
 Aspects of quality control include:
 Standardized bacterial inoculum size
 Culture conditions-growth medium, pH,
cation concentration
 Blood and serum supplements andThymidine
contents
 Incubation conditions- atmosphere,
temperature, duration
 Concentration of anti microbials for testing
 Guidelines and recommendations for these
continuously updated by certain
organizations worldwide, such as CLSI,
EUCAST, OIE, BSAC, SFM, SRGA and CDS.
Microbiological Culture sensitivity tests

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Microbiological Culture sensitivity tests

  • 2. Microbiology  Microbiology is the study of microbes that infect humans, the disease they cause, their diagnosis, prevention and treatment.  It also deals with the response of the human host to microbial and other antigens. Culture  Bacteria have to be grown(or cultured) on culture media for them to be identified, as only rarely can they be recognized by their morphology alone.
  • 3.  They are the invitro procedures used to detect anti-microbial resistance in individual bacterial isolates.  The same can be used for monitoring the emergence and spread of resistant micro organisms in a population.
  • 4.  In modern laboratories, bacteria are usually identified by characterization of the genome: identifying the characteristics of the DNA and RNA of the sample species  This typing of testing is generally considered more reliable-and soon, less expensive- than actually growing bacterial cultures and exposing them to various types of antibiotics to see which drugs kill or inhibit the bacterial growth. But if more than identification is required, and if an anti
  • 5.  also called susceptibility testing  Helps to find the right anti-biotic to kill an infecting organism. This test determines the sensitivity of a colony of bacteria to an antibiotic.  The efficacy of an antibiotic can be demonstrated under suitable conditions by its inhibitory effect on micro organisms.
  • 6.  Unfortunately, many bacteria are resistant to common antibiotics.  Resistant means that the drug cant kill the bacteria.  Sensitivity analysis is a useful tool to help quickly determine if bacteria are resistant to certain drugs.  Also be used for fungal infection
  • 7.  Examples of antibiotic resistant infections include:  A persistent sore throat  A recurring urinary tract infection(UTI)  An unresponsive case of pneumonia.
  • 8.  Sensitivity analysis starts with a bacterial sample.  The sample is obtained by swabbing the infected area or secretions of the infected area and from any area that has an infection.  Cultures are taken from blood  Urine  Sputum  Inside a cervix  A wound
  • 9.  The doctor will send the specimen to a licensed laboratory in a special culture tube for testing.  Spread a sample of the infective material onto a plate of nutrient substance-culture media and the Bacteria in the culture will grow and multiply  The bacteria will form colonies-large groups of bacteria that will be exposed to different antibiotics  With a sufficient population of bacteria grown on the plate in the form of a “lawn", the technicians will perform two main operations:
  • 10. 1.IdentifyThe Species Of Bacteria:  This is done with various techniques, including examination of lawn characteristics-color, texture, growth pattern, etc.-  Gram staining, microscopic examination, metabolic requirements and even DNA sequencing.  Bacterial species commonly isolated depends on the location and the cause of the infection
  • 11. 2.DetermineThe Bacterial Populations SensitivityTo A Range Of Antibiotics  This can be done by p[lacing small disks of filter paper or the agar impregnated with various types of antibiotics onto the bacterial lawn.The bacteria are allowed to incubate for a specified days and then the late is examined to see whether the bacterial growth is inhibited or not by the antibiotics on each disk.
  • 12.  Clinical relevance of the isolate  Purity of the isoate  Logical panel of anti-microbial agent to be tested-do ot include antibiotics to which the isolate is known to have intrinsic resistance  Availability of test methodology, resourcres, and trained persons  Standardisation of testing  Valid interpretation of results  Cost efficiency  Effective means to communicate results and interpretation to end-users
  • 13.  Dilution method-broth and agar dilution method  Disc-diffusion method  E-test  Automated methods  Mechanism-specific tests such as beta- lactamase detection test and chromogenic cephalosporin test  Genotypic methods such as PCR and DNA hybridization methods
  • 14.  The broth dilution method involves subjecting a series of concentrations of anti-microbial agents in a broth environment.  Micro dilution tests uses about 0.05 to0.1 ml total broth volume and can be conveniently performed in a micro-titre format.  Macro dilution testing uses broth volumes at about 1.0ml in standard test tubes.  For both these dilution methods, the lowest concentration at which the isolate is completely inhibited- as evidenced by the absence of bacterial growth- is recorded as the minimal inhibitory concentration.
  • 15.  The MIC is thus the minimum concentration of the anti-biotic that will inhibit the particular isolate.  The test is only valid only if the psitive contro shows growth and negative control shows no growth.  A procedure similar to broth dilution is agar dilution.  Agar dilutuion method followa the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is stil inhibited.
  • 16.  Because of convenience, efficiency and cos, the disc diffusion method is probably the most widely method  A growth medium, usually Mueller-hinton agar, is first even seeded throughout the plate with the isolate of interest that has been diluted at a standard concentration(app 1 to 2 x 10 coony forming units per ml).  Commercially prepared discs, each of which are pre impregnated with a standard concentration of a particular, are then evenly dispensed and lightly pressed onto yhe agar surface.  The test antibiotic immediately begins todiffuse ouyward from the discs, creating a gradient of antibiotic conc, isn the agar such that the highest conc., is found close to the disc.  After an overnight incubation, the bacterial growth around each disc is observed.  If the test isolate is susceptible to a particular antibiotic, a zone pf inhibition is observed.
  • 17. E-Test(AB Biodisk, Solna, Sweden) is a commercially available test that utilises a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein.
  • 18.  This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate.  However, a separate strip is needed for each antibiotic, and therefore the cost of this can be high.
  • 19.  Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading and interpretation.These systems have the advantage of being rapid and convenient, but one major limitation for most laboratories is the cost entailed in initial purchase, operation and maintenance of the machinery.  Examples include:  Avantage test system- abbott laboratories, Irving,Texas.  Phoenix-BD Biosciences, Maryland,etc.,
  • 20.  Resistance may also be established through tests that directly detectthepresence of a particular resistance mechanism.  For example, beta lactamase detection can be accomplished using an assay such as the chrmogenic cephalosporinase test(Cefinase disc by BD Microbiology Systems, Cockeysville, MD and BBL Dry slide Nitrocefin, Becton Dickinson, Sparks, MD) AND DETECTION FOR CHLORAMPHENICOL MODIFYING ENZYME CHLORAMPHENICOL acetyl transferase(CAT) may utilise commercial colorim,etric assays such as a CAT reagent kit(Remel, Lenexa, Kansas)
  • 21.  Although nucleic acid-based detections systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure, because resistance is also dependent on the mode and level of expressions of these genes.  Some of the most common molecular techniques utilized for anti-microbial resistance detection are as follows:
  • 22.  Polymerisation chain reaction:  It is one of the most commonly used molecular techniques for detecting certain DNA sequences of interest.This involves several cycles of denaturation of sample DNA, nnealing of apecific primers to the target sequence and the extension of this sequence as facilated by a thermostable polymerase leading to replicatrion of a duplicate DNA sequence, in an exponential manner, to a point ehich will be visibly detectable by gel electrophoresis with the aid of a DNA- intercalatingh chemical flouresces under uv light.
  • 23.  This is based on the fact the DNA pyrimidines(cytosine and thymidine) specifically pair up with purines(guanine and adenine; or uracil for RNA).  Therefore, a labelled probe with a known specific sequence can pair up with opened or denatured DNA from the ytest sample, as long as their sequences complement each other.  If this hybridisation occurs, the probe labels this with a detectable radioactive isotope, antigenic substrate, enzyme or chemiluminescent compound.
  • 24.  Whereas if no target sequence is present or the isolate does not have the specifi gene of interest, no attachment of probes will occur, and therefore no signals will be detected.
  • 25.  Modifications of the PCR and DNA hybridization.With these basic principles, several modifications have been introduced which further improve the sensitivity and Specificity of these procedures.  Examples of such development the use of flourecence-labeled oligonucleotides, the development of molecular becons, development of DNA arrays and DNA chips, Among many others.
  • 26.  Selection of the appropriate method will depend on the intended degree of accuracy, convenience, urgency, bioavailability of sources, availability of expertise and cost
  • 27.  The colonies show up as:  Susceptible, resistant, or intermediate  Susceptible- in this case, a clear, circular “halo”-technically known as a “plaque” or zone of inhibition- will appear around the antibiotic disc, indicating an absence of bacteria.The antibiotic has inhibited their growth and/or killed them, means that they cant grow if the drug is present.This indicates an effective antibiotic
  • 28.  Resistant : in this case, the filter Paper will have no discernable plaque around it, meaning that the bacteria can grow even in the presence of a antibiotic.This indicates an ineffective anti- biotic  Intermediate: a somewhat cloudy plaque indicates that not all the bacteria in the area around the disc have been killed. this means that a higher dose of the antibiotic is needed to prevent the growth.  The doctor can use the results to determine the best antibiotic to treat your infection
  • 29.  Few risks are associated with this test. Blood collection comes with small risks.  Rare risks of taking a blood sample include:  Lightedness or fainting  Hematoma- a bruise where blood accumulates under the skin  Infection-usually prevented by the skin being cleaned before the needle is inserted  Excessive bleeding- bleeding for a long period afterwards may indicate a more serious bleeding condition and should be reported to the doctor  The doctor will advise the patient about the potential risks associated with the sample
  • 30.  An antibiotic that bacteria, fungus, or another micro organism shows resistance to shouldn’t be used to treat the infection.Your doctor will decide which drug is best if several antibiotics are shown to be effective in killing the micro organism causing the infection
  • 31.  To provide a reliable prediction of whether an infection caused by a bacterial isolate will respond therapeutically to a particular antibiotic treatment.  This may be utilized as guidelines for chemotherapy, or at the population level as indicator of emergence and spread of resistance based on passive or active surveillance.
  • 32.  Becos of the required culture time, sensitivity testing may take several days, which is not ideal in critical cases demanding urgency.  Practitioners switch ove rto anti-biograms
  • 33.  The culture media are prepared according to the specifications of the USP, European Pharmacopoeia, British Pharmacopoeia and Indian Pharmacopoeia.  The antibiotic media are identified numerically with names assigned by Grove and Randall Seed agar  Base agar  Assay broth  Yeast beef agar
  • 34.  Aspects of quality control include:  Standardized bacterial inoculum size  Culture conditions-growth medium, pH, cation concentration  Blood and serum supplements andThymidine contents  Incubation conditions- atmosphere, temperature, duration  Concentration of anti microbials for testing
  • 35.  Guidelines and recommendations for these continuously updated by certain organizations worldwide, such as CLSI, EUCAST, OIE, BSAC, SFM, SRGA and CDS.