ANTIMICROBIAL AGENTS of the activity is less than the activity of the most active

individual drug).
Historical Highlight 3. Autonomous: results obtained when two drugs is equal
• 1907 - Paul Ehrlich speculated about some “magic bullet” to the result with most effective drugs by itself.
• 1928 - significant discovery of the “miracle” drug,
Penicillin by Alexander Fleming
• 1932 – discovery of compound termed Prontosil (first sulfa
drug)
• World War II - Allied armies made wide use of protonsil
• 1940 - first clinical trials of penicillin took place

Terminologies
• CHEMOTHERAPEUTIC AGENTS - chemical substances
used in treating diseases PROPERTIES OF A GOOD ANTIBIOTIC
• ANTIBIOTIC - substance produced by microorganisms that 1. Selective toxicity - harmful to microorganism but without
in small amounts inhibits another microorganism being harmful to the host; relative rather than absolute
• ANTIMICROBIAL AGENTS - natural and synthesized 2. Broad spectrum - inhibits many different species of
substances that destroys microorganisms or suppresses their pathogenic microorganisms
multiplication or growth 3. Prevents development of genetically and phenotypically
• ANTIBACTERIAL SPECTRUM: Range of activity of an resistant strains
antimicrobial against bacteria. 4. Bactericidal rather than Bacteriostatic
• BROAD-SPECTRUM DRUG - inhibit a variety of 5. Non-allergenic and no adverse reaction
gram-positive and gram-negative bacteria 6. Should remain active in the presence of plasma and other
• NARROW-SPECTRUM DRUG - active against a limited body fluids.
variety of bacteria 7. Stable and water soluble
• BACTERIOSTATIC ANTIBIOTIC: Antibiotic that inhibits
the growth of bacteria but does not kill. Dangers of indiscriminate use of Antibiotics
• BACTERICIDAL ANTIBIOTIC: Antibiotic that kills 1. Widespread sensitization (Hypersensitivity reaction)
bacteria. 2. Changes in normal flora of the body (super-infection due to
• MINIMUM INHIBITORY CONCENTRATION (MIC): The drug- resistant strain)
lowest antibiotic concentration that inhibits the growth of the 3. Masking serious infection
bacteria. 4. Direct drug toxicity (renal damage, auditory nerve damage ,
• MINIMUM BACTERICIDAL CONCENTRATION (MBC): etc.)
The lowest antibiotic concentration that kills 99.9% of the 5. Development of drug resistance in microbial population
population
• β-LACTAMASE: An enzyme that hydrolyzes the β-lactam TARGET SITES
ring in the β- lactam class of antibiotics, inactivating the • CELL WALL SYNTHESIS – inhibit peptidoglycan
antibiotic. The enzymes specific for penicillins, • Beta-lactam: penicillin, cephalosporins, carbapenem,
cephalosporins, and carbapenems are the penicillinases, monobactams
cephalosporinases, and carbapenemases, respectively. • Glycopeptides: Vancomycin, Teicoplanin
• Cycloserine
Antibiotic Combinations • Bacitracin
1. Antibiotic synergism: • PROTEIN SYNTHESIS
• Combinations of two antibiotics that have enhanced • Binds to 30S RNA
bactericidal activity when tested together compared with the - Aminoglycosides: Gentamicin, Tobramycin,
activity of each antibiotic. Kanamycin, Amikacin, Streptomycin
2. Antibiotic antagonism: - Tetracyclines (broad spectrum): Doxycycline,
• Combination of antibiotics in which the activity of one Minocycline (Mycoplasma, Chlamydia)
antibiotic interferes with the activity of the other (e.g., the sum
 • Binds to 50S RNA
- Macrolide-Lincosamide-Streptogranin:
Erythromycin, FusidicAcid
- Chlorampenicol (for serious gram Negative
infections like meningitis)
• NUCLEIC ACID SYNTHESIS
- Fluoroquinolones – binds to DNA gyrase:
ciprofloxacin, ofloxacin
- Rifampicin – Inhibits RNA synthesis
• FOLIC ACID SYNTHESIS
- Sulfonamides (synthetic)
- Trimethoprim – given in combination with
sulfonamides (SXT)
• CELL MEMBRANE FUNCTION
- Polymyxin: Colistin (for gram negative bacteria)

INHIBITING THE CELL WALL SYNTHESIS

• Target: PEPTIDOGLYCAN
• Beta-lactam antibiotics – a four-member ring, nitrogen
containing, Penicillin
beta-lactam ring at the core of their structure • With beta-lactam ring called the nucleus
• Beta-lactam: four-member ring that functions as a • Prevent the cross-linking of the peptidoglycans, primarily of
structural analogue of the normal substrate gram-positive bacteria
acyl-D-alanyl-D-alanine and inhibits the • Types are differentiated
transpeptidation reaction. by the chemical side
• Similar to acyl-D-alanyl-D-alanine (normal substrate) chains attached to their
• Death results from osmotic instability caused by nucleus
faulty synthesis
• Broad spectrum of gram (+) and gram (-) bacteria Natural Penicillins
• In gram-negative cells, the β-lactams must pass through cell • Penicillins extracted from cultures of Penicillium fungi
wall porin channels to reach the target PBPs. • Penicillin G – prototype compound of all the penicillins
• Narrow spectrum of activity and their susceptibility to
BETA-LACTAM ANTIBIOTICS penicillinases
• active moiety of β-lactam: four-member β-lactam ring Semisynthetic Penicillins
• act by forming covalent complexes with enzymes that • Developed to overcome the disadvantages of natural
generate the mature peptidoglycan molecule (PBPs) penicillins
• transpeptidation and cell wall synthesis is halted • Part of the penicillin is produced by the mold and part is
• examples: added synthetically
• Penicillins
• Cephalosporins
• Monobactams
• carbapenems
Penicillinase-resistant Penicillins POLYPEPTIDE ANTIBIOTICS
• Applied to strains that have developed resistance to a wide • Glycopeptides – act by binding to the terminal D-ala-D-ala
range of penicillins and cephalosporins. of the pentapeptidyl-glycosyl peptidoglycan intermediates.
Extended-Spectrum Penicillins • prevents their incorporation into the peptidoglycan chain by
• To overcome the problem of the narrow spectrum activity of blocking the transpeptidation step in cell wall biosynthesis
natural penicillins. • Cannot penetrate the outer membrane of most gram-negative
• Effective against many-negative and gram-positive bacteria bacteria
• Not resistant to penicillinases • Vancomycin – has a very narrow
• Aminopenicillins and carboxypenicillin spectrum of activity; last line of
antibiotic defense for Tx of S. aureus
infections that are resistant to other
antibiotics; red man syndrome
(RMS)/vancomycin-infusion reaction
(VIR)
• Lipoglycopeptides – semisynthetic molecules that contain
Penicillins plus-b-lactamase inhibitors hydrophobic chemical groups.
• Combination of penicillins • Bacitracin –inhibit the synthesis of cell walls at an earlier
with potassium clavulanate stage than penicillins and cephalosporin with the synthesis of
• Clavulanic acid – product the linear strands of the peptidoglycan
of streptomycete; non
competitive inhibitor of ANTIMYCOBACTERIAL ANTIBIOTICS
penicillinase with no • Isoniazid (INH) - inhibits the synthesis of mycolic acids,
antimicrobial activity on its which are components of cell walls only of the mycobacteria
own. • has little effect on nonmycobacteria
a. Cephalosporins - in structure, the nuclei resemble those of • Ethambutol is effective only against mycobacteria.
penicillin; more widely used than any other β -lactam • inhibits incorporation of mycolic acid into the cell wall
antibiotics making the cell wall weaker.
b. Carbapenems –substitutes a carbon atom for a sulfur atom • Principal use is as the secondary drug to avoid
and add a double bond to the penicillin nucleus. resistance problems.
• have an extremely broad spectrum of activity.
c. Monobactams – a synthetic antibiotic that has only a
single ring rather than the conventional β-lactam double ring
• Frist member: aztreonam (avoids the effects of
penicillinase)
• has unusually low toxicity, affects only certain
gram-negative bacteria, including pseudomonads and E. coli.

Differential Grouping of Cephalosporins

INHIBITOR OF PROTEIN SYNTHESIS

• Because protein synthesis is central to cellular function, it is
an excellent target for antibiotic drug product development.
• 50S ribosomal subunit
• (e.g., macrolides, oxazolidinones, streptogramins)
• 30S ribosomal subunit
• (i.e., aminoglycosides, tetracyclines, glycylcyclines)
 and polypeptide translation ceases.
• erythromycin – drug of choice for the treatment of
legionellosis, mycoplasmal pneumonia, and several other
infections

30s RIBOSOMAL SUBUNIT

a. AMINOGLYCOSIDE
• interfere with the initial steps of protein synthesis by
changing the shape of the 30S portion of the 70S
prokaryotic ribosome which causes the genetic code of
the mRNA to be read incorrectly.
• incorporation of aberrant proteins into the cell wall
results in cell
leakage and enhanced cellular penetration of additional
Nitrofurantoin antibiotic
• Consists of a nitro group on a heterocyclic ring available as Streptomycin – best-known aminoglycoside
an oral suspension or capsule • can affect hearing by causing permanent damage to
• it concentrates in urine as the the auditory nerve, and damage to the kidneys
kidney removes it in the blood • Gentamicin
• Converted by bacterial • TETRACYCLINE
nitroreductase to intermediates • binds to 16S ribosomal RNA near the amino-acyl tRNA
that attach bacterial ribosomal acceptor (A) site, thus inhibiting the rotation of bound
proteins tRNA into the A site during translation
• treatment of urinary bladder • physical blocking results in premature release of
infections tRNA and termination of peptide bond formation
• often suppress the normal intestinal microbiota, causing
50s RIBOSOMAL SUBUNIT GI upsets and often leading to superinfections,
a. CHLORAMPHENICOL particularly by the fungus Candida albicans
• inhibits the addition of amino acids to the growing peptide • most common antibiotics added to animal feeds, where
chain by reversibly binding to the 50s ribsosomal subunit, their use results in significantly faster weight gain
inhibiting transpeptidation
• has serious adverse effects; most important is the DISRUPTION OF BACTERIAL MEMBRANE
suppression of bone marrow activity (aplastic anemia) Lipopeptides
b MACROLIDE-LINCOSAMIDE-STREPTOGRAMIN • binding to and disrupting the cell membrane of
(MLS) Group gram-positive bacteria. The drug binds to the cytoplasmic
• Streptogramin membrane and inserts its hydrophobic tail into the membrane,
• enter bacterial cells by passive diffusion, bind disrupting the cell membrane and increasing its permeability,
irreversibly to the 50s subunit, and induces a which results in cell death
conformational change in the ribosome • Daptomycin - effective only against gram-positive bacteria
• Clindamycin (Lincosamide) – assoc with clostridium • resistance is uncommon
difficile-associate diarrhea • has potent activity against gram-positive cocci, including
• inhibits protein synthesis at 50s ribosomal subunit those resistant to other agents such as beta-lactams and
• effective against anaerobes (use in the treatment of acne) glycopeptides (MRSA, VRE, VRSA)
• Macrolides (erythromycin, clarithromycin, • cannot penetrate the outer membrane of gram-negative
andazithromycin) bacilli and thus is ineffective
• Binds to the peptidyltransferase cavity in the proximity POLYMYXIN
of the a and p loops, near adenine of 23s rRna • cyclic lipopeptide agents that disrupt bacterial cell
• by blocking the exit tunnel of the elongating peptides, membranes by act as detergents, interacting with
premature release of peptidyl-trna intermediates occurs phospholipids in the cell membranes to increase permeability
• Polymyxin B – bactericidal antibiotic effective against TRIMETHOPRIM
gram-negative bacteria; it acts by binding to the outer • inhibits a different enzyme, dihydrofolate reductase
membrane of the cell wall • SMZTMP – a single formulation to produce an
• Polymyxin E (colistin) – treat antibiotic-resistant antibacterial agent that can simultaneously attack two targets in
ventilator-associated pneumonia caused by gram-negative the same folic acid metabolic pathway
bacteria • the combination also has a broader spectrum of action
• regarded as a “ last resort ” antibiotic to treat extremely and greatly reduces emergence of resistant strains
drug-resistant infections
• emergence of plasmid-mediated polymyxin resistance in
gram-negative bacteria is currently a global concern
• polymyxins pose a risk of toxicity (neurotoxicity and
nephrotoxicity)

INTERFERENCE WITH NUCLEIC ACID SYNTHESIS

RIFAMYCINS
• Rifampin - best-known derivative of the rifamycin family
• structurally related to the macrolides and inhibit the
synthesis of mRNA
• against mycobacteria (treatment of tuberculosis and leprosy)
• penetrate tissues and reach therapeutic levels in
cerebrospinal fluid and abscesses
• unusual side effect: orange-red urine, feces, saliva, sweat,
and even tears
QUINOLONES AND FLUOROQUINOLONES
• unique bactericidal effect by selectively inhibiting an
enzyme (DNA gyrase) needed for the replication of DNA
• bind to and interfere with DNA gyrase enzymes involved in
the regulation of bacterial DNA supercoiling, a process
essential for DNA replication, recombination, and repair
• newer fluoroquinolones also inhibit topoisomerase IV
• tendinitis and rupture of the Achilles tendon (in general
population)

Competitive inhibition of essential metabolites

SULFONAMIDES
• Sulfa drugs are structurally similar to a folic acid precursor
called para- aminobenzoic acid (PABA)
• competitively bind with the enzyme meant for PABA and
thereby block folic acid production
• target and bind to one of the enzymes, dihydropteroate
synthase, and disrupt the folic acid pathway
• bacteriostatic and do not harm human cells
• moderately toxic, causing vomiting, nausea, and
hypersensitivity reactions. Sulfonamides are also antagonistic
for several other medications, including warfarin, phenytoin,
and oral hypoglycemic agents
 ANTIBIOTIC RESISTANCE
• Phenomenon in which formerly effective medications have
less and less impact on bacteria
Why is drug resistance a concern?
• Proliferation of resistant strains in Health care institution
• Treatment failure which causes serious consequences
especially critically ill person
• Broader infection control problems for both in Healthcare ASPECTS OF ANTIMICROBIAL RESISTANCE
Institutions and communities as well • biologic versus clinical antimicrobial resistance
• Added burden in Health care cost • environmentally mediated antimicrobial resistance
• microorganism-mediated antimicrobial resistance
REASONS WHY ANTIBIOTIC RESISTANCE OCCURS
1. Over-prescription of antibiotics TYPE OF RESISTANCE
2. Non-completion of prescribed antibiotics Biologic resistance
3. Use of antibiotics in animals as growth enhancers • changes that result in observably reduced susceptibility of an
4. Poor hospital hygiene organism to a particular antimicrobial agent
5. Indiscriminate use of antibiotics Clinical resistance
• antimicrobial susceptibility has been lost to such an extent
Emergence and Dissemination of Antimicrobial Resistance that the drug is no longer effective for clinical use
• Antibiotic resistance mechanisms: part of the evolution of ENVIRONMENTALLY MEDIATED RESISTANCE
bacteria as a means of survival among antibiotic-producing • resistance directly resulting from physical or chemical
competitors characteristics of the environment that either directly alter
• All bacterial resistance strategies are encoded on one or the antimicrobial agent or alter the microorganism ’ s normal
more genes. physiologic response to the agent
• Resistance may spread to a wide variety of bacteria, and any • environmental factors that mediate resistance include pH,
single organism may acquire multiple genes and become atmosphere, cation concentrations, and thymidine content
resistant to the full spectrum of available antimicrobial agents. • used to establish standardized testing methods that
minimize the effect of environmental factors and for the • Vertical gene transfer
accurate determination of microorganism-mediated resistance • Lateral or Horizontal gene transfer
mechanisms c. A combination of mutational and gene transfer events

COMMON PATHWAYS FOR ANTIMICROBIAL

RESISTANCE
Resistance to Beta-Lactam Antimicrobials
• mediated by enzymatic destruction (e.g., beta-lactamases) -
most common
• altered antibiotic targets, resulting in low affinity or
decreased binding of antibiotic to the target PBPs -
• decreased intracellular uptake or increased cellular efflux of
the drug - beta-lactam resistance in gram-negative bacteria

Resistance to Aminoglycosides
• accomplished by enzymatic, altered target, or decreased
uptake pathways
• three general types of enzymes catalyze one of the following
modifications of an aminoglycoside molecule:
1. Phosphorylation of hydrozyl groups
ORGANISM-BASED RESISTANCE 2. Andenylation of hydroxyl group
Microorganism-Mediated Antimicrobial Resistance 3. Acetylation of amine groups
• antimicrobial resistance from genetically encoded traits of • mechanism of resistance is rare in bacteria exposed to
the microorganism common aminoglycosides

INTRINSIC (OR INHERENT) RESISTANCE Resistance to Quinolones

• Antimicrobial resistance resulting from the normal genetic, • mediated either by a decrease in uptake or in
structural, or physiologic state of a microorganism is accumulation or by production of an altered target
intrinsic resistance • exhibit a mechanism by which the antimicrobial is actively
• resistance factors are often chromosomally encoded in the pumped out of the cell, keeping the intracellular concentration
organism’s genome and are not readily transferrable of the antimicrobial below an effective level.
• resistance pattern may be predictable • reduces the intracellular accumulation of the antibiotic rather
• useful for determining which antimicrobial agents should be than decreased uptake.
included in the battery of drugs tested against specific types of • primary pathway: mutational changes in the targeted
organisms subunits of the DNA gyrase

Acquired resistance ANTIMICROBIAL SUSCEPTIBILITY TESTING

• results from altered cellular physiology and structure caused • determine whether the bacterial isolate expresses resistance
by changes in a microorganism’s genetic makeup to the antimicrobial agents commonly selected for treatment at
• may be a trait associated with specific strains of a particular the site of infection
organism group or species • determine acquired resistance in clinically important
• resistance pattern is unpredictable organisms for which the antimicrobial susceptibility profile is
• acquired resistance mechanisms are all genetically encoded, unpredictable
the methods for acquisition involve genetic change or • used to select effective antimicrobial drugs for treatment and
exchange control of infectious diseases especially when caused by
• Resistance may be acquired by: resistant pathogens
a. Successful genetic mutation • predicts the in-vivo success or failure of antibiotic therapy
b. Acquisition of genes from other organisms via gene transfer • MIC and MBC determination
mechanisms:
• major focus of in vitro AST: to measure an organism ’ s 2. cAST
expression of resistance 3. Special screen and indicator tests
• To control the impact of environmental factors, testing
conditions for AST are extensively standardized. DISK DIFFUSION METHOD
Standardization serves three important purposes: aka KIRBY-BAUER TEST
1. Optimizes bacterial growth conditions • Principle: Performed by inoculating a standardized
2. Optimizes conditions for maintaining antimicrobial integrity amount of organism into agar, followed by adding
and activity antibiotic disks. Diameter of ZOI is measured around the
3. Maintains reproducibility and consistency in the resistance disks and evaluates as to whether the organism is R, I, or S.
profile of an organism • FILTER PAPER DISKS impregnated with antimicrobial
agents of specific concentrations are carefully placed on an
Standardized components of antimicrobial susceptibility agar plate that is previously inoculated with the bacteria being
testing testing.
• Bacterial inoculum size • The larger the zone of inhibition, the lower the MIC.
• Growth medium (typically a Mueller-Hinton base) • pH • Limited to aerobic and facultative anaerobic bacteria
• Cation concentration • Not recommended for testing slow-growing organisms like
• Blood and serum supplements mycobacteria and anaerobes.
• Thymidine content
• Incubation atmosphere STANDARDIZED AMOUNT OF BACTERIA
• Incubation temperature • Important component: pure culture and standardized
• Incubation duration inoculum.
• Antimicrobial concentrations • McFarland turbidity standard: 1% sulfuric acid and
1.175% barium chloride
PRINCIPLES • 0.5 McFarland standard, which is commercially available,
• Directly measure the activity of one or more antimicrobial provides an optical density comparable to the density of a
agents against a bacterial isolate bacterial suspension of 1.5 × 10^8 CFU/mL
• Directly detect the presence of a specific resistance • Standardized inoculum: used within 15 minutes after
mechanism in a bacterial isolate standardization; spread using swab all throughout the MHA
• Measure complex antimicrobial organism interactions plate.

BREAKPOINTS VS. EPIDEMIOLOGICAL CUTOFF

VALUES (ECV)
CLINICAL BREAKPOINTS
• specific concentrations that separate or define the
different categories
• Interpretative category only, no MIC
• Based on MIC distributions,
pharmacokinetic/pharmacodynamics data, and clinical
outcomes
ECV
• May signal emergence of evolution of non-wild-type
strains with acquired mechanisms or resistance
• Based on the MIC distribution of the organism

METHODS THAT DIRECTLY MEASURE

ANTIMICRBIAL ACTIVITY
1. Traditional susceptibility
• disc • broth
• agar
 STANDARD MEDIUM FOR DISK DIFFUSION
• Standard medium: Mueller Hinton Agar
• ph: 7.2 – 7.4
• Low pH: Low pH: dec. activity of Aminoglycoside,
Erythromycin, Clindamycin, inc. activity of tetracycline
• High pH: decreased activity of tetracycline
• Standard depth: 4mm thick (3-5 mm)
• Standard petri plate: 150mm in diameter (12 antimicrobial
disk)
• Pseudomonas AST (calcium: 25 mg/L & magnesium: 12.5
mg/L)
• Increased conc. : decreased activity of aminoglycoside
against P. aeruginosa and decreased activity of
tetracyclines against all organisms
• Decreased conc. : Opposite effect
• Thymidine content: minimal (or absent)
• Inc. conc.: false resistance to sulfonamides and
trimethoprim
• Check QC first before reading
STREAKING AND PLACEMENT OF DISKS • The diameter of each inhibition zone is measured using a
• Inoculate MHA in multiple directions caliper or ruler.
• Within 15 minutes of inoculation, the antimicrobial agents • Plates are examined two to three inches above a black,
are applied into the MHA. non-reflective surface and the zones are measured from the
• The disks must be positioned no closer than 24 mm from back side of the plate illuminated with reflected light
disk center to disk center and no closer than 10 to 15 mm • Zone measurement is compared with the interpretative tables
from the edge of the plate. of CLSI and the results are interpreted as susceptible,
• surface of the medium should be allowed to dry for 3-5 intermediate or resistant.
minutes but not longer than 15 minutes then plates are • Presence of ZOI: indicates susceptibility
inverted. • Absence of ZOI: indicates resistance
• Incubated req: 35 + 2 degrees Celsius for 16 to 18 hours to a • Diameter of zones not to be interpreted quantitatively.
maximum of 24 hours in humidified ambient air
• Increased CO2 (5-7%) is required for N. meningtidis or S. A wide zone surrounding the disk signifies a greater
pneumoniae • Plate not be stacked more than 5 plates high susceptibility of the organism to the antibiotic.

zones are measured from the

back side of the plate
illuminated with reflected
light
 6. Concentration of divalent cations (calcium and
magnesium) in susceptibility media
• It can affect the testing of aminoglycosides and
tetracycline against P. aeruginosa
• Elevated concentrations of the divalent cations may
result in diminished activity of the aminoglycosides
Colonies appearing within a clear zone should not be against P. aeruginosa and the decreased activity of
ignored; the original colony must be tested. tetracycline against all bacteria.

Possible causes of error

• Use of mixed culture
• Inoculum too light: f(S)
• Inoculum too heavy: f(R)
• Too much moisture on agar: f(R)
• Very dry agar surface: f(S)
• Improper storage of disk
• Reading and clerical error
• Deterioration of turbidity standard or control strain

Errors in reporting
• Minor Error: Susceptible isolate erroneously reported as
intermediate or vice versa
• Major Error: Susceptible isolate erroneously reported as
Resistant
• Very major error: Resistant isolate reported as Susceptibl

Modifications for Fastidious microorganisms

• Incubated in 5-7% CO2 for 18-20 hrs
• S. pneumoniae: MHA with 5% sheep RBCs
• H. influenzae: HTM (MHA base supplemented with
FACTORS AFFECTING THE ZONE OF INHIBITION hematin, NAD, and yeast extract
1. Amount of inoculum or test organism. • MRSA: MHA with 2% NaCl incubated for 24hrs @ 30-35
• Only pure cultures can be tested. degC
2. Thickness of the susceptibility agar plate (4mm) • N. meningitidis: MHA with 2-5%lysed horse RBC
3. Growth rate of the test organism • N. gonorrhea: GC agar with supplements
• A temperature of 35 degrees Celsius is recommended • Mycobacteria: Middlebrook 7H10
for most bacteria with 16-18 hours of incubation. • Anaerobes: Brucella blood agar with hemin in incubated
• Temperatures higher than 35 degrees Celsius may lead anaerobically up to 48hrs
to the false detection of MRSA
• Lower temperatures may lead to larger zones of BROTH DILUTION
inhibition. • challenging the organism of interest with antimicrobial
4. pH of the medium (pH 7.2-7.4) agents in a liquid environment
• High ( ↑ ) medium pH results in the decreased activity of • Principle: Quantitative test in which a serial
tetracyclines twofold-dilutions of antibiotics are prepared and standard
• Low ( ↓ ) medium pH results in diminished activity of concentration of bacteria are added. MIC is then determined.
aminoglycosides and erythromycin. • BMD final concertation: 5.0 x 10^5 CFU/mL
5. Number of disk per plate • Incubated: 30 degC or 16-20 hrs
• A 150 mm plate can have a maximum of 12 disks.
• Valuable in: • Macrodilution - 1 mL or greater (rarely used)
• Organism is from blood culture • Key component: proper preparation and dilution of the
• Patients who replase while on therapy antimicrobial agents into the broth medium
• Organism fails to respond to antibiotic therapy • antimicrobial agent is tested using a range of concentrations,
commonly expressed as micrograms ( µ g) of active drug per
Medium and Antimicrobial Agents. milliliter (mL) of broth (i.e., µg/mL
• For the procedure, a specific amount of antibiotic is prepared • the lowest antimicrobial concentration that completely
in a decreasing concentration in broth by a serial dilution inhibits visible bacterial growth, as detected visually or with an
technique in which a standard amount of the test organism is automated or semi- automated method, is recorded as the
then inoculated. minimal inhibitory concentration (MIC).
• 2.5% to 5% laked horse blood is used for MIC • Advantages: Testing fastidious bacteria and identifying
testingmethods MBC endpoints
• MH Broth with 2% NaCl - Improve the detection of
oxacillin-resistant staphylococci MACROBROTH DILUTION
• microdilution (broth microdilution or BMD) - total broth
volume is 0.05 to 0.1 mL
• Use commercially supplied microdilution panels in
which the broth is already supplemented with appropriate
antimicrobial concentrations.
• Advantages: Testing different antimicrobials at the
same time

MICROBROTH DILUTION

AGAR DILUTION METHOD

• Dilutions of the antimicrobial agents are prepared in agar.
Bacteria are inoculated onto the agar plates.
• a standard inoculum of bacteria (1 x 10^4 CFU/spot) is
spot-inoculated onto a 100-mm plate
• Reference method for testing anaerobes and Neisseria
gonorrhoeae
• Frequently used in research studies
• Disadvantages:
• Not helpful in organisms tend to spread such as
Proteus and Pseudomonas
• Have a shelf life of only 1 week (stored at 2-8 degC)
• Plate preparation is laborious
E-TEST (Strip dilution test) • Flattening (D-shaped)of
• Provides quantitative antimicrobial susceptibility testing the clindamycin zone
results. between 2 disks indicates the
• It is a dilution test that is based on the diffusion of a isolate has inducible
continuous varying concentrations of an antimicrobial agents. clindamycin resistance
• Uses thin plastic strips • Clindamycin must be
reported as resistant
PROCEDURE • No flattening indicates
• The strip is placed on the surface of the culture medium that isolate is erythromycin
is inoculated with the desired organism and the antibiotic is resistant only.
diffused into the surrounding agar.
• The result is an elliptic inhibitory areas. BETA LACTAMASE
• The MIC is read at the point on the scale where the ellipse • An enzyme that confers resistance to penicillin and some of
intersects the E-test strip. the semisynthetic penicillins (e.g., ampicillin).

Uses thin plastic strips impregnated on the undersurface with PROCEDURE

an antibacterial concentration gradient and marked on the • Bacteria applied to moistened disk impregnated with
surface with a concentration index or scale cephalosphorin nitrocefin (cefinase diks).
• Red color if Beta-lactam ring is broken

TEST FOR MRSA

D-ZONE TEST • Oxacillin is used as class representative for
• To detect inducible clindamycin resistance in MRSA isolates penicillinase-resistant penicillins.
that are resistant to erythromycin & susceptible to • Oxacillin Screen Plate: MH with 4% NaCl & Oxacillin (6µ
clindamycin on initial testing. g/ml)
• Differentiate clindamycin reisitance resulting from • Any growth=Resistant
efflux(mcrA or MLSB)
SERUM BACTERICIDAL TEST
PROCEDURE Schlichter Test
• Erythromycin & clindamycin disks are placed 15-26mm • It determines the activity of one or more antimicrobial
apart on MHA inoculated with organism. agents that are present in the serum against an organism that is
• After overnight incubation, flattened zone between disks obtained from the patient.
(D-shaped zone of inhibition around clindamycin disk) means • The culture medium is the patient ’ s serum with the
erythromycin induces clindamycin resistance. antibiotics given to the patient.
• Rarelyperformed Highest concentration of drug is found on the area _______ to
the disk.
PROCEDURE a. Farthest
• Serial dilutions of patient ’ s peak & trough specimens b. Closest
inoculated with standardized amount of patient’s pathogen and
incubated overnight. Vitek 2 (bioMérieux, Inc.)
• Serum static titer= highest dilution that inhibits growth • MIC results are validated with the AES software, category
• Serumcidal titer= highest dilution with 99.9% decrease in interpretation is assigned, and the organism ’ s antimicrobial
CFU/ml resistance patterns are reported.
• optical readings are made every 15 minutes to measure
Supplemental methods for detecting antimicrobial resist transmitted light through each well; 64 wells are used.
• Oxacillin agar – detection of Staphylococcal resistance to
penicillinase-resistant penicillins
• Vancomycin agar – detection of enterococcal resistance to
vancomycin
• Aminoglycoside screens – detection of acquired
enterococcal high- level resistance to aminoglycoside that
would compromise synergy with a cell wall-active agent as
ampicillin or vancomycin.
• Oxacillin disk screen – detection of S. pneumoniae
resistance to penicillin

AUTOMATED A.M.S. TESTING

• It follow the principle of turbidimetric detection of bacterial
growth in a broth medium by using a photometer to examine
test wells.
• For the interpretation of results, growth endpoints of broth
microdilution panels are detected using an automated reader
device.
• Growth detection can also be performed through the
hydrolysis of a fluorogenic growth substrate that is
incorporated in a special test medium.
• Fluorometer is also used for growth detection.
• Microbial growth is detected as an emission of a fluorescent
signal
• Systems vary with respect to:
• Extent of automation of inoculum preparation &
inoculation
• Methods used to detect growth
• Algorithms used to interpret and assign MIC values and
categorical findings
• Detects growth in microvolumes of broth with various
dilutions of antimicrobials
• Detection via photometric, turbidimetric or fluorometric
methods
• SMART incubator: separates bacterial plates allowing for
optimal temp, separate racks