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1. What steps would be taken in the clinical laboratory before setting a susceptibility test?

A sample for culture and susceptibility testing should be collected before the start of any treatment with
an antimicrobial unless the test is used to monitor the effectiveness of treatment.


The pH of each batch of Meller-Hinton agar should be checked when the medium is prepared. The exact method used
will depend largely on the type of equipment available in the laboratory. The agar medium should have a pH between
7.2 and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g.,
aminoglycosides, quinolones, and macrolides), while other agents may appear to have excessive activity (e.g.,
tetracyclines). If the pH is too high, the opposite effects can be expected. The pH can be checked by one of the following

* Macerate a sufficient amount of agar to submerge the tip of a pH electrode.

* Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup.

* Use a properly calibrated surface electrode.

If, just before use, excess surface moisture is present, the plates should be placed in an incubator (35C) or a laminar
flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually 10 to 30
minutes). The surface should be moist, but no droplets of moisture should be apparent on the surface of the medium
or on the petri dish covers when the plates are inoculated.

Effects of Thymidine or Thymine

Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and
trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in false-resistance
reports. Meller-Hinton agar that is as low in thymidine content as possible should be used. To evaluate a new lot of
Meller-Hinton agar, Enterococcus faecalis ATCC 29212, or alternatively, E. faecalis ATCC 33186, should be tested with
trimethoprim/sulfamethoxazole disks. Satisfactory media will provide essentially clear, distinct zones of inhibition 20
mm or greater in diameter. Unsatisfactory media will produce no zone of inhibition, growth within the zone, or a zone
of less than 20 mm.

Effects of Variation in Divalent Cations

Variation in divalent cations, principally magnesium and calcium, will affect results of aminoglycoside and tetracycline
tests with P. aeruginosa strains. Excessive cation content will reduce zone sizes, whereas low cation content may result
in unacceptably large zones of inhibition. Excess zinc ions may reduce zone sizes of carbapenems. Performance tests
with each lot of Meller-Hinton agar must conform to the control limits.

Testing strains that fail to grow satisfactorily

Only aerobic or facultative bacteria that grow well on unsupplemented Meller-Hinton agar should be tested on that
medium. Certain fastidious bacteria such as Haemophilus spp.,

N. gonorrhoeae, S. pneumoniae, and viridans and -haemolytic streptococci do not grow sufficiently on unsupplemented
Meller-Hinton agar. These organisms require supplements or different media to grow, and they should be tested on
the media described in separate sections.

Susceptibility Testing. Retrieved from

Manual on Antimicrobial Susceptibility Testing.Retrieved from https://

2. Of what significance is the size of the zone of inhibition surrounding a disc?

Zone of inhibition: This is an area of media where bacteria are unable to grow, due to presence of a drug that
impedes their growth.
If the bacteria are susceptible to the particular antibiotic from a wafer, an area of clear media where
bacteria are not able to grow surrounds the wafer, which is known as the zone of inhibition. A larger zone
of inhibition around an antibiotic-containing disk indicates that the bacteria are more sensitive to the
antibiotic in the disk. If the observed zone of inhibition is greater than or equal to the size of the standard
zone, the microorganism is considered to be sensitive to the antibiotic. Conversely, if the observed zone
of inhibition is smaller than the standard size, the microorganism is considered to be resistant.

Measuring Drug Susceptibility. Retrieved from


3. What are the specific modes of action of the antimicrobial agents used in the exercise? do not answer

4. What is meant by the terms: bacteriostatic; bactericidal; narrow spectrum; board spectrum

Products that act fighting bacteria are usually divided in bactericidal and bacteriostatic. Definition: bacteriostatics
inhibit the growth of bacteria and bactericidal are used to eliminate a big part of the bacteria population (usually
more than 99,9%).

Broad spectrum antibacterials are active against both Gram-positive and Gram-negative organisms. Examples
include: tetracyclines, phenicols, fluoroquinolones, third-generation and fourth-generation cephalosporins.
Narrow spectrum antibacterials have limited activity and are primarily only useful against particular species of
microorganisms. For example, glycopeptides and bacitracin are only effective against Gram-positive bacteria,
whereas polymixins are usually only effective against Gram negative bacteria. Aminoglycosides and sulfonamides
are only effective against aerobic organisms, while nitroimidazoles are generally only effective for anaerobes.



Spectrum of Activity. Retrieved from

5. Why is the term susceptibility preferable to sensitivity in particular test?

Because susceptible is used to describe the affects an antibiotic has on the growth of an organism while
Sensitive is used to describe any reaction a patient may have to the chemotherapeutic agent.

Ex 13: Antimicrobial Agent Susceptibility Testing and ResistanceRetrieved from.


6. Give the definition/s of the term: antibiotics and antimicrobial

The word antimicrobial was derived from the Greek words anti (against), mikros (little) and bios (life) and
refers to all agents that act against microbial organisms. This is not synonymous with antibiotics, a similar
term derived from the Greek word anti (against) and biotikos (concerning life). By strict definition, the
word antibiotic refers to substances produced by microorganisms that act against another
microorganism. Thus, antibiotics do not include antimicrobial substances that are synthetic (sulfonamides
and quinolones), or semisynthetic (methicillin and amoxicillin), or those which come from plants
(quercetin and alkaloids) or animals (lysozyme).

Antimicrobials: An Introduction. Retrieved from

7. Fill in the table on the classification of the antibiotics.

Classification Examples Mechanism of action Generic + Brand names Susceptib
le organs
Penicillins Aminopenicillin kills susceptible bacteria Amoxicillin-Amoxicot
s by specifically inhibiting
the transpeptidase that
catalyzes the final step in
cell wall biosynthesis, the
cross-linking of
Cephalosporin First They bind to and block Duricef
s generation the activity of enzymes generic name: cefadroxil
cephalosporins responsible for making
peptidoglycan, an
important component of
the bacterial cell wall.
Aminoglycosid tobramycin work by inhibiting Tobi
es protein synthesis inside generic
bacteria name: tobramycin
Macrolides azithromycin inhibit protein synthesis Zmax
in bacteria by reversibly generic
binding to the P site of name: azithromycin
the 50S unit of the
Tetracyclines Doxycycline inhibit protein synthesis Adoxa TT
in the microbial RNA by generic
reversibly binding to the name: doxycycline
30S ribosome and
preventing the
attachment of aminoacyl-
tRNA to the ribosomal
acceptor site.
Quinolones lomefloxacin inhibit the enzyme Maxaquin
topoisomerase II, a DNA generic name:
gyrase that is necessary lomefloxacin
for the replication of
Other Beta- CEPHALOSPORI Inhibition of cell wall Avycaz
lactams NS synthesis. generic
name: avibactam/ceftazid
Lincomycins Lincomycin inhibits protein Lincocin
derivatives synthesis in susceptible generic name: lincomycin
bacteria by binding to the
50 S subunits of bacterial
ribosomes and
preventing peptide bond
formation upon
Chlorampheni chloramphenic inhibiting protein Chloramphenicol-
col ol otic synthesis. It prevents Chloromycetin Otic
protein chain elongation
by inhibiting the peptidyl
transferase activity of the
bacterial ribosome.

Vancomycin Drugs and prevention of cell-wall Vancomycin-Vancoled

Supplements biosynthesis of bacteria.
Vancomycin It is specially effective
(Intravenous against gram-positive
Route) bacteria and has been
known to be effective
against resistant strains
Linezolid Linezolid tablet disrupts bacterial growth Linezolid-Zyvox
by inhibiting the initiation
process of protein
Metronidazole Metronidazole It inhibits nucleic acid Metronidazole-Flagyl
oral tablet synthesis by disrupting
the DNA of microbial cells
Miscellaneous aztreonam Bactericidal antibiotic ins Cayston
erts into the bacterial cell generic name: aztreonam
membrane, causing
depolarization and
potassium efflux.

Penicillins.Retrieved from

Cephalosporins . Retrieved from

Yocum RR, Rasmussen JR, Strominger JL. The mechanism of action of penicillin. Penicillin acylates the active site
of Bacillus stearothermophilus D-alanine carboxypeptidase.Retrieved from

Aminoglycosides. Retrieved from

Macrolides.Retrieved from

Tetracyclines.Retrieved from

Quinolones. Retrieved from

Metronidazole oral tablet.Retrieved from

Chloramphenicol.Retrieved from
Beta Lactam Antibiotics Retrieved from

Mechanism of Action of Vancomycin.Retrieved from

8. What is antibiotic resistance ? Describe the mechanism of how antibiotic resistance occurs.

The inactivation or modification of the antibiotic;

An alteration in the target site of the antibiotic that reduces its binding capacity;
The modification of metabolic pathways to circumvent the antibiotic effect;
The reduced intracellular antibiotic accumulation by decreasing permeability and/or increasing active
efflux of the antibiotic.
Bacteria can develop resistance to antibiotics by mutating existing genes (vertical evolution),[6,7] or by acquiring
new genes from other strains or species (horizontal gene transfer).[8,9] The sharing of genes between bacteria by
horizontal gene transfer occurs by many different mechanisms. Mobile genetic elements, including phages,
plasmids and transposons mediate this transfer, and in some circumstances the presence of low levels of the
antibiotic in the environment is the key signal that promotes gene transfer, [10] perhaps ensuring that the whole
microbial community is protected from the antibiotic.[11]
Mechanism of antibiotic resistance. Retrieved from

8. What can be done to avoid or prevent drug resistance?

Patients can:

Ask if tests will be done to make sure the right antibiotic is prescribed.
Take antibiotics exactly as the doctor prescribes. Do not skip doses. Complete the prescribed course of
treatment, even when you start feeling better.
Only take antibiotics prescribed for you; do not share or use leftover antibiotics. Antibiotics treat specific
types of infections. Taking the wrong medicine may delay correct treatment and allow bacteria to multiply.
Do not save antibiotics for the next illness. Discard any leftover medication once the prescribed course of
treatment is completed.
Do not ask for antibiotics when your doctor thinks you do not need them. Remember antibiotics have side
Prevent infections by practicing good hand hygiene and getting recommended vaccines.

Healthcare providers can:

Prescribe antibiotics correctly get cultures, start the right drug promptly at the right dose for the right
duration. Reassess the prescription within 48 hours based on tests and patient exam.
Document the dose, duration and indication for every antibiotic prescription.
Stay aware of antibiotic resistance patterns in your facility.
Participate in and lead efforts within your hospital to improve prescribing practices.
Follow hand hygiene and other infection control measures with every patient.
Mission Critical: Preventing Antibiotic Resistance. Retrieved from