ANTIBIHLS
·
TESE
CHEMICAL SUBSTANCES
prebio By
MILPORSENSMS
NAT
Micks
IIHIBIS
GKOMA
of
1158
RGANISM]
CLINICAL BACTERIOLOGY -- LECTURE
PRELIMS TOPIC 8. ANTIMICROBIAL SUSCEPTIBILITY TESTING
Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT, Mr. Gianleri Nor, RMT
FULL TRANSES MASTERLIST: https://bit.ly/masterli_st
ANTIBIOTICS
These chemical substances are produced by
microorganisms with the capacity to inhibit
(bacteriostatic) or kill (bactericidal) other
microorganisms.
•
They can also be synthesized via chemical
procedures that are independent from microbial
activity.
•
ANTIBIOGRAM -- An antibiogram is an overall
profile of antimicrobial susceptibility testing results
Ne
of a specific microorganism to a battery of
antimicrobial drugs.
SOURCES OF ANTIBIOTICS
MICROORGANISM
ANTIBIOTIC PRODUCED
11 Bacitracin
Bacillus subtilis
2 . Bacillus polymyxa
fu Polymyxin
Cephalosphorium
Cephalosporins
Micronospora purpurea
Gentamicin
, Penicillin
A
Penicillum notatum
Erythromycin
⑲ Streptomyces erythraeus
- Neomycin
Streptomyces fradiae
J Amphotericin B
Streptomyces nodusus A
Streptomyces noursei
Nystatin
Streptomyces venuzelae %
Chloramphenicol
**These are natural drugs.
CLASSIFICATION OF ANTIBACTERIAL DRUGS
BACTERICIDAL AGENTS
•
~
•
=
-
•
•
↳
-
-
2
These are antimicrobial agents that usually kill or
destroy organisms
Used as a treatment for life threatening conditions
Ex: Aminoglycosides (Gentamicin, Amikacin, and
&
Streptomycin)
GAM
>
-
-
-
6 Am
>
Bautraun
poly my un
-
Cephalosporum
Centrmy in
e
pencillin
Eretlermyn
emper
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GROUPINGS OF ANTIMICROBIAL AGENTS
GROUP A PRIMARY
Ampicillin, Cefazolin,
TEST
Gentamicin, and
c c
Tobramycin
GROUP B PRIMARY
Amikacin, Cefuroxime,
TEST SELECTIVELY
Ciprofloxacin, Impenem,
Capac
and Piperacillin
GROUP C
Aztreonam,
SUPPLEMENTAL
Chloramphenicol, and REPORT COLLECTIVELY Tetracycline
COMMON TERMINOLOGIES
⑭
I
impofere
A
&
8
-
C
Kanamycin ,
I
.
0
•
Lorbapenicillin
Methicillin
3
.
1984dard
MINIMAL INHIBITORY CONCENTRATION (MIC)
ex
·
SEMI-SYNTHETIC DRUGS
D
These are modified natural drugs with added
chemical
groups
o Ex: Ampicillin, Carbapenicillin, and
Methicillin A E M
0
-
-
-
SYNTHETIC DRUGS
•
These are chemically produced drugs
o Ex: Sulfonamides, Trimethroprim, 42S
Ciprofloxacin, and Isoniazid
CLASSIFICATION OF ANTIMICROBIAL AGENTS
BACTERIOSTATIC AGENTS
B b CERIOSAT2
134ST En , CIDAL
These drugs are produced by bacteria or fungi
o Ex: Amphotericin B, Erythromycin,
,
Kanamycin
KED
-
msicillin
irmethrone
NATURAL DRUGS
Amphoterion B
& Mythre my 2 In
-
O
L
D
:
•
8
•
These are antimicrobial agents that inhibit bacterial
growth; but generally, they do not kill the
microorganisms.
o Ex: Chloramphenicol, Dapsone,
Erythromycin, Sulfonamides, and
-Tetracycline
-
•
Lowest concentration of drug that inhibit bacterial
=
⑪
growth
-
=
85
*
THERAPEUTIC INDEX
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%
%
%
%
%
%
%
1
Gefazdin
Ampicillin
Tobraein
Gentamicin
•
•
•
NUCLEIC ACID SYNTHESIS INHIBITORS
Rifampicin - inhibits RNA polymerase and
synthesis
Quinolones - effective for enteric bacteria (E. coli)
Metronidazole - disrupts DNA and effective against
anaerobic bacteria
•
Ratio of the toxic dose to the therapeutic dose and
as such, the higher the therapeutic index, the more
effective the chemotherapeutic agent
TYPES OF ANTIMICROBIAL AGENTS ACCORDING TO
MECHANISM OF ACTION
1 CELL WALL INHIBITORS
•
These are most selective antibiotics with a higher
therapeutic index
•
They inhibit the activity of transpeptidase enzymes
in which cell growth stops and the death of cells
often follows.
•
These drug are effective against gram positive
bacteria.
o Bacitracin - inhibits synthesis of
peptidoglycan precursors
o B-lactams - inhibits transpeptidation
o Isoniazid - can be bactericidal or
bacteriostatic
o Vancomycin - inhibits translocation and
elongation of peptidoglycan
:
CELL MEMBRANE INHIBITORS
Polymyxin B and E are effective against gram (-) bacteria,
like PAE
ESSENTIAL METABOLITE INIHIBITORS
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•
•
•
•
•
•
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I PROTEIN SYNTHESIS INHIBITORS
•
•
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These antibiotics bind to 30S subunit that results in
the misreading of mRNA
These drugs target aerobic and anaerobic gram (+
and gram (-) bacteria;
Effective against M. tuberculosis, B. fragilis, N.
meningitidis, H. influenza, and S.pneumoniae.
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%
%
%
Folic Acid Inhibitors (Sulfamethoxazole)
Cord factor inhibitor (Isoniazid)
Take note that Trimethroprim and Sulfamethoxazole
are synthetic drugs, they do not occur in nature.
ACTIONS OF ANTIMICROBIAL DRUGS
Inhibits cell wall synthesis
Inhibits cell membrane function
Inhibits protein synthesis
Inhibits nucleic acid synthesis
Inhibitors of Other Metabolic Processes
%
%
%
%
2
•
ANTIMICROBIAL SUSCEPTIBILITY TESTING
PRINCIPLE
•
It measures the ability of an antibiotic or other
microbial agent to inhibit bacterial growth
PRIMARY GOAL
•
To determine whether the bacterial isolate is
capable of expressing resistance to the
antimicrobial agents selected for treatment
Standard: 0.5 McFarland/ Barium Sulfate
Suspension (99.5ml of 1% H,SO4 & 0.5ml of
sure
xx
1.175% BaCiz)
BARIUM SVLEATE
Standard inoculum: 1.5 x 108 organisms/ml
pH: 7.2-7.4
-
•
•
-
-D
PURPOSE
•
To guide the clinician in selecting appropriate
antimicrobial agent
•
To gather epidemiological data on microbial
resistance
STANDARDIZATION
•
To optimize bacterial growth conditions
•
To optimize conditions for maintaining antimicrobial
integrity and activity
•
To maintain reproducibility and consistency in the
resistance profile
DISK DIFFUSION METHOD (KIRBY-BAUER TEST)
•
Medium: Mueller-Hinton Agar
MUELLER HINTON AGAR
•
8~
The standard susceptibility medium for non fastidious bacteria.
-
-
-
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%
%
%
non-fast
%
dors
%
banterna
%
3
beet
•
infusion,
micleincided
,
Mitrinens
,
caren
PROCEDURE AST
Composed of beef infusion, nucleic acid, vitamins,
casein, and agar.
-&
An
MH broth with&
2% NaCI is used to improve the
&
detectionof oxacillin
- resistant staphylococci.
°
An MH broth with 5% lysed horse blood or sheep
blood is utilizedfor testing susceptibility for
streptococci, N. meningitidis, and other fastidious
organisms.
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•
•
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•
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•
f
A MOLN of ANSULLM
.
2
THIOKNESS
3
GROWTH rpis
.
.
%
PH
-5
.
⑳
of THS
OF
of East
PLATE
unobrism
•
-
Da
Only pure cultures can be tested
-
THICKNESS OF SUSCEPTIBILITY PLATE
mSbirM
162 PLRE
Conceiver
plupirt
-
AMOUNT OF INOCULUM/TEST ORGANISM
SUSCOPTIBILIY
NUMBS 09 NIST)
·
O-
Depth: 4mm
Condition: Aerobic, No CO2
Temperature: 35-37°C
Incubation Time: 16-18 hours A
Antibiotic disc: 6mm
Petroff-Hausser: bacterial count
A
FACTORS AFFECTING DISK DIFFUSION
•
as
If the agar is too thick, the zone sizes will be
smaller; if the agar is too thin, the zone sizes will be
larger
Bowis
GROWTH RATE OF TEST ORGANISM
•
•
A temperature of 35 degrees is best for most
bacteria within 16 - 18 hours of incubation
Temperatures
higher than 35 degrees may lead to
false detection of MRSA
Lower temperature may lead to
larger ZOl
Incubation with increased 5% to 10% carbon
dioxide is not recommended except for capnophilic
bacteria.
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•
•
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THE pH OF THE MEDIUM (7.2-7.4)
•
•
•
Incubation of plates in CO2 could result in
decreased pH.
Increased pH results to decreased activity of
tetracycline drugs.
Low pH decreases the activity of aminoglycosides
and erythromycin.
NUMBER OF DISK PER PLATE
•
•
A 150mm plate can have a maximum of 12 disks.
Placement of more than 12 may result in
overlapping of zones
CONCENTRATION OF DIVALENT BONDS (CALCIUM
AND MAGNESIUM)
•
It can affect the testing of aminoglycosides and
tetracycline against PAE.
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MEASUREMENTS
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15mm = Disk distance
•
150mm = 12 discs
•
100mm = 5-6 discs
FALSE RESISTANT
FALSE SENSITIVE
•
15 mins (delay of
•
15 mins (delay of
disc application)
incubation)
•
Increase moisture
•
Increase drying
•
Thick medium
•
Thin medium
•
Increase
•
Decrease
organisms =
organisms =
Decrease Zone of
Increase Zone of
Inhibition
Inhibition
READING OF PLATES AND INTERPRETATION OF
RESULTS
•
•
•
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%
%
%
the lawn must be confluent or almost confluent - if
the growth is poor & nonconfluent, the test should
be repeated
provided growth is satisfactory, the diameter of
each inhibition zone is measured using a caliper or
ruler
appearance of individual colonies is unacceptable
Susceptible: microorganism should respond to
therapy with that antimicrobial agent
Intermediate: microorganism falls into a range of
susceptibility in which the MIC approaches or
exceed the level of antimicrobial agent that can be
achieved and for which clinical response is likely to
be less than with a suscpetible strain
Resistant: no zone or small zone of inhibition antibiotic is not the appropriate choice for treatment
AUTOMATED SYSTEMS
Vitek 2
o It is fully automated equipment designed
for the identification of bacteria and AST
o Optical readings are made every 15
minutes to measure transmitted light
through each well
o Final reading 6-8 hours
Microscan Walkaway System by Beckman
Coulter
%
%
%
%
5
Inocula are manually introduced to the
broth microdilution tray
o Growth patterns are automatically read
and interpreted after incubation.
•
Phoenix System by BD Microbiology Systems
o It uses manual gravity based inoculation
technique.
o Growth patterns are automatically read.
o It has a mechanism to confirm ESBL of
gram negative bacteria.
o Results after 8 to 12 hours.
SPECIFIC TESTING PROCEDURES FOR ORGANISMS OF
MEDICAL INTEREST
•
MRSA infection is caused by a strain of
Staphylococcus bacteria that's become resistant to
oxacillin and methicillin
2 TYPES OF MRSA
•
Health care-associated MRSA (HA-MRSA)
o Are associated with invasive procedures or
devices, such as surgeries, intravenous
tubing or artificial joints
•
Community - associated MRSA (CA-MRSA)
o Often begins as a painful skin boil. Spread
by skin- to-skin contact.
o At risk populations include groups such as
high school wrestlers, child care workers
and people who live in crowded conditions.
TEST FOR MRSA
•
Clinical and Laboratory Standards Institute (CLSI),
recommends:
o Broth microdilution testing
o cefoxitin disk screen test,
o latex agglutination test for PBP2a
o plate containing 6 g/ml of oxacillin in
Mueller-Hinton agar supplemented with
4% NaCI as alternative methods of testing
for MRSA.
o chromogenic agars that can be used for
MRSA detection.
o Nucleic acid amplification tests, ex.
polymerase chain reaction (PCR)
§
to detect the mecA gene, is the
most common gene that mediates
oxacillin resistance in
staphylococci.
o
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