Antimicrobial sensitivity testing and

resistant pattern
Dr. Israe A Ali
Specialist Medical microbiology
2022
 Antimicrobial agents

An antimicrobial agent is
defined as a natural or
synthetic substance that kills or
inhibits the growth of
microorganisms such as
bacteria, fungi and algae
 Antimicrobial activity
Not all antimicrobials, at the
concentration required to be
effective are completely non –
toxic to human cells. Most,
however, show sufficient selective
toxicity to be of value in the
treatment of microbial diseases.
 Mechanisms Of Action Of
Antimicrobial Drugs
Antimicrobial drugs act in
one of several ways:
1.Selective toxicity
2.Inhibition
 Selective Toxicity
An ideal antimicrobial agent exhibits
selective toxicity, which means
that the drug is harmful to a pathogen
without being harmful to the host.
Often, selective toxicity is relative
rather than absolute, this implies that a
drug in a concentration tolerated by the
host may damage an infecting
microorganism.
Selective toxicity may be a
function of:
A. A specific receptor required
for drug attachment,
B. It may depend on the
inhibition of biochemical
events essential to the
pathogen but not to the host.
 Mechanisms Of Action Of Antimicrobial Drugs
inhibition

1. Inhibition of cell wall synthesis

2. Inhibition of protein synthesis (i.e.
inhibition of translation and
transcription of genetic material)
3. Inhibition of cell membrane function
4. Inhibition of nucleic acid synthesis
 mode of action of antimicrobial agents
Bacteria have a rigid outer layer.
The cell wall maintains the shape
and size of the microorganism,
which has a high internal osmotic
pressure. Injury to the cell wall
(eg. By Lysozyme) or inhibition of
its formation may lead to lysis of
the cell.
• Gram-positive bacteria have thicker membranes
that are easier to penetrate.
• Gram-negative bacteria have thinner
membranes that are harder to penetrate,
making them more resistant to some antibiotics.
 1.Inhibition Of Cell Wall Synthesis
# Penicillins
(Beta- lactam agents): bactericidal
used to treat infection caused by
Streptococcus, pneumococci
,clostredia and when sensitive, also
staphylococcal infection , meningitis
,gonorrhoea, syphilis and anthrax.
➢Ampicillin and Amoxycillin: EX-spectrum, are broad
spectrum penicillins active against G+ve (including
Enterococcus), H. influenzae (G-ve coccibacili), and many
coliform.
➢Flu cloxacillin and Cloxacillin (anti- Staph.) are used to
treat beta- lactamase (penicillinase) producing
Staphylococcus.
➢Carbenicillin and ticarcillin: Antipseudomonal used for
treating infection caused by Pseudomonas aerugeno
➢Azlocillin and pipracilling: are active against Klebsiell
and are also anti-Pseudomonal.
 What are penicillin antibiotics?

. Extended-spectrum penicillin:
➢ Ampicillin and amoxicillin for are broad spectrum
penicillins active against G+ve (including
Enterococcus), H. influenzae, and many coliform.
• Antistaphylococcal penicillins :
✓ Dicloxacillin ,Nafcillin ,Oxacillin
• Natural penicillin
✓ Pencillin G ,Pencillin V
• Antipseudomonal penicillins:
✓ Pipracillin, Pipracillin+ tazobactam, Ticracillin
✓ lactamase inhibitor + antibiotic combinations
Clavulanic acid + amoxicillin
 anti – bacterial Resistance to
penicillin
• anti – bacterial Resistance to penicillin may occur due
to Beta- Lactamase production , cell membrane
alteration reducing antibiotic uptake (G-VE bacteria).
• Or change in PBP (Penicillin-binding proteins are generally enzymes
involved in peptidoglycan biosynthesis, so contribute essential roles in bacterial
cell wall biosynthesis)
as occur in MRSA ( MRSA are usually
resist to many antibiotic as penicillin, erythromycin
,tetracycline and some times gentamycin , sever
infection require treatment with vancomycin ,which
also reported as resist .
 Inhibition Of Cell Wall Synthesis
# Cephalosporin
Bactericidal and have beta-lactam ring.
They are used to treat sever systemic
infection caused by
aerobic Gram –ve .

Some cephalosporin cause kidney

damage .(cephaloridine > cefotiam > cephalothin >
cefoxitin > cefazolin > cefoperazone > cefotaxime).
 Inhibition Of Cell Wall Synthesis
# Glycopeptides :
Vancomycin: used to treat serious infection suc
as endocarditis and septicemia by G+ve bacteri
particularly MRSA-stain (treatment require
monitoring due to ototoxicity (damage to
hearing).
Teicoplanin: used as in vancomycin in G+ Mlti-
Drug resistant Staphylococcus arueus and
Enterococcus faecalis, and effective treatment
in pseudomembranous colitis and clostridium
difficile associated diarrhea.
 2.Inhibition of protein synthesis
It is established that
erythromycins, lincomycins,
tetracyclines, aminoglycosides,
and chloramphenicol can
inhibit protein synthesis in
bacteria.
 Inhibition of protein synthesis :
1. Aminoglycosides : the exhibit bactericidal effects. Its
showing synergy effect with beta-lactam agent .
Manly reserved for the treatment of sever sepsis due
to coliforms and other Gram –negative aerobic. With
beta-lactams they are sometimes used to treat
endocarditis caused by Staphylococcus and
Streptococcus. Side effect include hypersensitivity ,
ototoxicity and nephrotoxicity. 30S sub unit.
(Gentamicin,tobramycin,amikacin,streptomycin,paromo
mycin,neomycin and plazomicin).
 Inhibition of protein synthesis :
2. Macrolides
Useful Bacteriostatic agent also its bactericidal when used
in high concentration . Many treat Staphylococcus
infection , respiratory infection, non specific urethritis,
and when indicated Campylobacter enteritis . Macrolides
: includes, erythromycin, azithromycin, clarithromycin
and roxithromycin. 50 S. subunit.
They are useful second line drugs for treating patient
with penicillin hypersensitivity .
Resistant may be occur S. aureus , S. pyogenes, and S.
pneumoniae.
Side effect include gastroenteritis upsets and rashes.
 Inhibition of protein
synthesis:3.Lincosamides
Lincomycine, clindomycin
• Useful in treating • Pseudomembranous colitis,
Staphylococcus , bone and joint also called antibiotic-associated
colitis or C. difficile colitis, is
infection and anaerobic inflammation of the colon
infection associated with an overgrowth
of the bacterium Clostridioides
but Lincosamides have difficile.
been associated with • This overgrowth of C. difficile is
pseudomembranous often related to a recent
hospital stay or antibiotic
colitis . treatment.
50S subunit • C. difficile infections are more
common in people over 65
years old.
Inhibition of protein synthesis :
4. Chloramphenicol
• Bacteriostatic broad spectrum drug , act on 50. S
subunit, used in treating typhoid
fever, meningitis, rikettsial and
chlamydial infection and also eye
infection (in eye drops). It can cause
aplastic anaemia and is toxic in
neonates.
 Inhibition of protein synthesis
:5.Tetracycline ,Doxcycline 30 S subunit
• Bacteriostatic widely used broad spectrum
antibiotic with active against G+ve and some G-
ve bacteria and also borreliae, rickettsiae,
chlamydia C. burnetii and mycoplasma. Side effects
include gastrointestinal disturbance , kidney damage and
staining of teeth in children.
# it should not be used in pregnancy.
Resistance to tetracycline is common (e.g. with
H. influenza ,S. pneumoniae and S. pyogenes.
Proteus is always resisant.)
 3. Inhibition of nucleic acid
1. Quinolones: bacteriostatic or bactericidal agent,
Nalidixic acid is used to treat lower G-ve
Urinary infection , Ciprofloxacin
(fluroquinolone) is active against Pseudomonas
, andalso used to treat serious
systemic infection.
 Inhibition of nucleic acid
2. Sulphonamides and trimethoprim: (e.g.)
Co-trimoxazole, sulphadimidine, trimethoprim and sulphadoxine:
bacteristatic agent (may be bactericidal in
combination) with activity against G+ ve, G- ve (P.
aeruginosa is resistant ).
co-trimoxazole Is used to treat urinary and respiratory
tract infection. Pneumocystis pneumonia and invasive
salmonellosis . Many enterobacteria are resistant .
Side infect include nausea and vomiting ,rash ,mouth
ulceration and occasionally thrombocytopenia and
leucopenia . Side effect are less with trimethoprim.
 Inhibition of nucleic acid
3. Metronidazole: bactericidal agent used to treat
anaerobic infection (e.g. caused by Bacteroides
,anaerobic cocci and clostridia. Actinomyces
are resistant .
Also used to treat Vincents angina and
protozoal infection e,g caused by T. vaginalis ,
E. histolytica , G. lamblia
Few side effect.
 Ant mycobacterial agents
Rifampicin, Ethambutol , isoniazid Diamino-
diphenylsulphane (dapsone). Antimycobacterial
agents represent a diverse group of compounds that
have activity against mycobacterial infections,
including tuberculosis , leprosy and Mycobacterium
avium complex (MAC) disease. The 1st-line agents
for tuberculosis are rifampin, isoniazid,
pyrazinamide, and ethambutol.
• The drugs vary in their mechanisms of
action: Rifampin inhibits RNA synthesis,
isoniazid inhibits mycolic acid synthesis,
pyrazinamide acts on membrane
transport and protein synthesis, and
ethambutol prevents cell wall synthesis.
Monotherapy is not recommended
because of the increased risk of
drug resistance.
• Multidrug treatment takes several months and
requires sputum monitoring. As for leprosy, an
infection due to Mycobacterium leprae, rifampin is
also used, with dapsone. The lepromatous
form requires a 3rd agent (clofazimine).
• Pulmonary infections with Mycobacterium
avium complex (MAC) disease are managed
with macrolides (azithromycin), rifampin, and
ethambutol
 Bactericidal and
bacteriostatic agent:
❑ Antibacterial agents are generally described as
bacteriostatic agent, when at usual dosages, they
prevent the active multiplication of bacteria(e.g.
chloramphenicol, tetracycline and erythromycin ).
❑ Antibacterial agents are generally described as
Bactericidal agent, when usual dosage, they kill
bacteria (e.g. penicillins, cephalosporin, glycopeptidea
and aminoglycosides)
❑ Some bacteristatic agent become bactericidal when
used at high concentrations, (e.g. erythromycin and
tertracycline.)
• Broad spectrum antibiotic: The term is
applied to antibacterial with activity against a
wide rang of Gram positive and Gram negative
organism. They include the tetracyclines,
aminoglycosides, sulphonamides and
chloramphenicol.
• Narrow spectrum antibiotic: are those with
activity against one or few types of bacteria
(e.g. vancomycin against Staphylococci and
enterococci.
 Antibiotic used in combination

• Occasionally, combination of antimicrobials

are used to treat mixed infection to prevent
treatment failure and drug resistance from
developing, to treat sever infections when the
organism is not known or it is necessary to
obtain a grater antimicrobial effect from two
bactericidal drugs acting together (synergistic
effect). Like aminoglycoside with beta- lactam .
Antimicrobial resistance :

There are many different

mechanisms by which
microorganisms might
exhibit resistance to drugs:
1.Microorganisms produce
enzymes that destroyed
the active drug.
Examples: Staphylococci
resistant to penicillin G
produce a β-lactamase
that destroys the drug
2. Microorganisms change
their permeability to the drug.
Examples: Tetracyclines
accumulate in susceptible
bacteria but not in resistance
bacteria. Resistance to
polymyxins is also associated
with change in permeability to
the drug. polymxins interact with
lipopolysaccharid (LPS)of outer
membrane of G-ve
3. Microorganism develop an altered
structural target for the drug.
Examples: Erythromycin-resistant
organisms have an altered receptor
on the 50S subunit of the ribosome,
resulting from methylation of a 23S
ribosomal RNA. Resistance to some
penicillin's and cephalosporin may be
a function of the loss or alteration of
PBPs.
4. Microorganism develop an
altered metabolic pathway
that bypasses the reaction
inhibited by the drug.
Examples: Some
sulfonamide-resistant
bacteria do not require
extracellular PABA but, like
mammalian cells, can utilize
preformed folic acid.
5. Microorganism develop an
altered enzyme that can still
perform its metabolic function
but is much less affected by the
drug. Examples: In
trimethoprim-resistant
bacteria, the dihydrofolic acid
reductase is inhibited far less
efficiently than in
trimethoprim-susceptible
bacteria.
 Origin of drug resistance

1.1.Non-Gentic origin
2.2.Gentic origin
Non-gentic origin
1. Active replication of bacteria is
required for most antibacterial drug
actions. Consequently,
microorganism that are metabolically
inactive (non-multiplying) may be
phenotypically resistance to drug.
However their offspring are fully
susceptible.
Examples: Mycobacterium
often survive in tissues for
many years after infection
yet are restrained by the
hosts defenses and do not
multiply. Such “persisting”
organism are resistant to
treatment and cannot be
eradicated by drugs.
2.Microorganisms may .2
lose the specific target
structure for a drug for
several generations and
thus be resistant.
Example: Penicillin-susceptible
organisms may change to cell
wall-deficient. L-forms during
penicillin administration.
Lacking cell walls, they are
resistant to cell wall-inhibitor
drugs (penicillins,
cephalosprins) and may remain
so for several generations.
 3.Microorganisms .3
may infect the host at
sites, where
antimicrobials are
excluded or are not
active.
Examples: Aminoglycosides
such as gentamicin are not
effective in treating
salmonella enteric fevers
because the salmonellae
are intracellular and the
aminoglycosides do not
enter the cells.
Genetic origin
Most drug-resistant microbes
emerge as a result of genetic
change and subsequent
selection processes by
antimicrobial drugs.
A.Chromosomal Resistance:
This develops as result of
spontaneous mutation in a locus
that controls susceptibility to a
given antimicrobial drug. The
presence of the antimicrobial drug
serves as a selecting mechanism to
suppress susceptible organisms and
favor the growth of drug-resistant
mutants.
Spontaneous mutation occurs
with a frequency of 10ˉ¹² and
thus is an infrequent cause of
the emergence of clinical
drug resistance in a given
patient. However,
chromosomal mutants
resistant to rifampin occur
with high frequency.
Consequently, treatment of
bacterial infections with
rifampin as the sole drug often
fails. Chromosomal mutants
are most commonly resistant
by virtue of a change in a
structural receptor for a
drug.
Thus, the P12 protein on the 30S
subunit of bacterial ribosome
serves as a receptor for
streptomycin attachment.
Mutation in the gene controlling
that structural protein results in
streptomycin resistance. Mutation
can also result in the loss of PBPs
making such mutants resistant to
β-lactam drugs.
B- Extrachromosomal Resistance
Bacteria often contain extrachromosomal
genetic elements called plasmids. Some
plasmids carry genes for resistance to
one – and often several – antimicrobial
drugs. Plasmid genes for antimicrobial
resistance often control the formation
of enzymes capable of destroying the
antimicrobial drugs.
Thus, plasmids determine resistance to
penicillins and cephalosporins by carrying
genes for the formation of β-lactamases.
Plasmids code for enzymes that acetylate,
adenylate, or phosphorylate various
aminoglycosides; for enzymes that determine
the active transport of tetracyclines across the
cell membrane; and for others.
Genetic material and plasmids can be
transferred by transduction, transformation,
and conjugation.
 Antimicrobial
Sensitivity Test
Sensitivity (susceptibility)
testing is used to select
effective antimicrobial
drugs. Sensitivity test is not
usually indicated when the
sensitivity reactions of a
pathogen can be predicted
for example:
• Proteus species are generally
resistant to nitrofurantoin and
tetracyclines.
• S.pyogenes is usually sensitive to
penicillin
• K.pneumoniae is generally
ampicillin resistant.
• Anaerobes are sensitive to
metronidazole.
• Organism that are susceptible to tetracycline are
also consider susceptible to doxycycline and
minocycline.
• However , some organism that are intermediate
or resistant to tetracycline may be susceptible to
doxycycline ,minocycline or both.
• RX: Refampin should not used alone for
antimicrobial therapy.
•
❑When fecal sample isolates of
salmonella and shigella spp. ,are tested
only ampicillin, a fluoroquinolone ,and
trimethoprim /sulfamethoxazole should
be reported routinely.
❑In additional for extra intestinal isolates of
salmonella spp. , a third –generation of
cephalosporin should be tested and reported
,and chloramphenicol may be tested and
reported , if requested.
• Resistance to cephalosporins
can attributed to:
1. poor permeation of
bacteria by the drug
2. lack of PBP for a specific
drug
3. degradation of drug by β-
lactamases
Antimicrobial Subclass
1.First-Generation Cephalosporins:
Very active against gram-positive
cocci – except enterococci and
methicillin-resistant
staphylococci(MRSA), and
moderately active against some
gram-negative rods – primarily E.
coli, Proteus, and Klebsiella.
None of the 1st generation drugs
penetrate the CNS and they are
not drugs of choice for any
infection
a)Cephalothin
b)Cephapirin
c)Cefazolin
d)Cephradine
2. Second Generation Cephalosporins:
All are active against organisms
covered by 1st generation drugs
but have extended coverage
against gram-negative rods
a)Cefamandole
b)Cefuroxime (Parenteral)
c) Cefonicid
3. Third-Generations Cephalosporins:
Have decreased activity against gram-positve
cocci, except for Strep. Pneumoniae,
enterococci but enhanced activity against
gram-negative rods .
3rd generation drugs – except Cefoperazone is
ability to reach the CNS
a)Cefotaxime
b)Ceftizoxime
c) Ceftriaxone
d)Ceftazidime
e) Cefoperazone
4. Fourth – Generation Cephalosporins:
Cefepime is the only 4 th –
generation cephalosporin. It
has enhanced activity against
Enterobacter and Citrobacter
species that resistant to 3rd
generation cephalosporins.
5. Cephalosporins with Anti-MRSA activity:

Ceftaroline
Ceftobiprole
 Cross Resistance
• Microorganisms resistant to a certain drug may
also be resistance to other drugs that share a
mechanism of action. Such relationships exist
mainly between agents that are closely related
chemically (eg, different aminoglycosides) or
that have a similar mode of binding or action
( eg, macrolides-lincomycins)
 Cross Resistance
In certain classes of drugs, the
active nucleus of the chemical is
so similar among many
congeners (eg, tetracyclines) that
extensive cross-resistance is to be
expected.
 Warning: the following antimicrobial /organism may
appear active in vitro, but are not effective clinically
and should not reported as susceptible.

Salmonella spp, shigella spp. 1st and 2ed –generation

cephalosporins, cephamycins and
aminoglycosides.

Oxacillin –resistant Penicillins, ß-lactam/β-lactamase

staphylococcus spp. inhibitor combination,
antistaphylococcal cephems , and
carbapenems.
Enterococcus spp. Aminoglycosidase (except high
concentration), cephalosporins,
clindamycin , and trimethoprim
sulfamethoxazole.
Disc diffusion sensitivity tests
Dsic diffusion techniques are used by most
laboratories to test routinely for
antimicrobial sensitivity. A disc of blotting
paper is impregnated with a known volume
and appropriate concentration of an
antimicrobial, and this is placed on a plate
of sensitivity testing agar uniformly
inoculated with the test organism.
The antimicrobial diffuses from the disc
into the medium and the growth of the
test organism is inhibited at a distance
from the disc that related (among
other factors) to the sensitivity of the
organism. Strains sensitive to the
antimicrobial are inhibited at a
distance from the disc whereas
resistant strains have smaller zones of
inhibition or grow up to edge of the
disc.
Kirby-Bauer NCCLS modified
disc diffusion technique
The validity of this carefully
standardizes technique depends on
using discs of correct antimicrobial
content, an inoculums which gives
confluent growth, and a reliable
Mueller Hinton agar. The test method
must be followed exactly in every
detail.
After incubation at 35 Cº for 16 – 18
hours, zone sizes are measured and
interpreted using NCCLS (National
Committee for Clinical Laboratory
Standards) standards. These are
derived from the correlation which
exists between zone sizes and MICs.
The NCCLS kirby-Bauer technique
should only be used for well-evaluated
bacterial species. It is not suitable for
bacteria that are slow-growing, need
special nutrients, or require CO2 or
anaerobic incubation.
 Stokes disc diffusion
technique
In this disc technique both the test and control organisms are
inoculated on the same plate. The zone sizes of the test
organism are compared directly with that of the control.
This method is not as highly standardizes as the kirby-
Bauer technique and is used in laboratories particularly
when the exact amount of antimicrobial in a disc cannot be
guaranteed due to difficulties in obtaining discs and
storing them correctly or when the other conditions
required for the Kirby-Bauer technique cannot be met.
One way laboratories in developing countries
performing the Stokes technique could
change to a technique comparable to the
WHO recommended Kirby-Bauer technique
is to use highly stable Rosco Diagnostica
antibiotic tablets (Neo-Sensitabs) instead of
less stable paper discs.
Rosco Neo-Sensitabs susceptibility testing. The
tablets are 9 mm in diameter and colour-
coded. The formulae used to produce the
tablets gives them a shelf-life of about 4 years
and many Neo-Sensitabs can be stored at
room temperature.
Modified Kirby-Bauer sensitivity testing
technique

Mueller Hinton sensitivity testing agar •

Prepare and sterilize the medium as instructed
by the manufacturer. The pH medium
should be 7.2 – 7.4 pour into 90 mm
diameter sterile petri dishes to a depth of 4
mm (about 25 ml per plate). Care must be
taken to pour the plats on a level surface so
that the depth of the medium is uniform.
Note: if the medium is too thin the
inhibition zones will be falsely large
and if too thick the zones will be
falsely small.
Control each new batch of agar by
testing it with a control strain of
E.faecalis (ATCC 29212 or 33186)
and co-trimoxazole disc. The zone
of inhibition should be 20 mm or
more in diameter.
Store the plates at 2 – 8 Cº in sealed plastic
bags. They can be kept for up to 2 weeks.
For use, dry the plates with their lids slightly
raised in 35 – 37 Cº incubator for about 30
minutes.
Fastidious organisms: Unmodified Mueller
Hinton agar is not suitable for sensitivity
testing H.influenzae, S.pneumoniae,
N.gonorrhoeae. Isolates of these organisms
should be sent to a specialist microbiology
laboratory for testing.
 ▪ Antimicrobial discs
The choice of antimicrobials to be
included in sensitivity tests will depend
on the pathogen, the specimen, range of
locally available antimicrobials, and
local prescribing policies. Consultation
between laboratory, medical, and
pharmacy staff is required. The range of
1st choice drugs should be limited and
reviewed at regular intervals
Additional drugs should be
included only by special request.
Where there is cross-resistance,
only one member from each
group of related antimicrobials
need be selected. An oxacillin
disc is representative of the
whole group of beta-bactamase
resistant penicillins when testing
staphylococci.
Note: paper antimicrobial discs
stable sensitivity and can be
used for 1 year or longer from
date of manufacture providing
they are stored correctly (-20 Cº
or working stock at 2 – 8 Cº in
an airtight container with an
indicating desiccant. Discs that
have expired should not be used.
About 1 hour before use, the working
stock of discs should be allowed to
warm to room temperature,
protected from direct sunlight.
Important: Decreasing control zone
size with a particular antimicrobial
disc is often an indication of
deterioration of the antimicrobial
due to moisture or heat.
▪ Turbidity standard equivalent to
McFarland 0.5
This is a barium sulphate standard
against which the turbidity of the test
and control against which the turbidity
of the test and control inocula can be
compared . When matched with the
standard, the inocula should give
confluent or almost confluent growth,.
Shake the standard immediately before
use.
Preparation of turbidity standard:
1. Prepare a 1 % v/v solution of sulphuric
acid by adding 1 ml of concentrated
sulphuric acid to 99 ml of water. Mix
well.
Caution: concentrated sulphuric acid is
hygroscopic and highly corrosive,
therefore do not mouth pipette, and
never add the water to the acid.
2.Prepare a 1 % w/v
solution of barium
chloride by dissolving 0.5
g of dihydrate barium
chloride (BaCl2.2H2O) in
50 ml of distilled water.
3. Add 0.6 ml of the
barium chloride
solution to 99.4 ml
of the sulphuric
acid solution, and
mix.
4. Transfer a small volume of the turbid
solution to a capped tube or screw-cap
bottle of the same type as used for
preparing the test and control inocula.
When stored in a well-sealed container in
the dark at room temperature (20 – 28
Cº), the standard can be kept for up to
6 months.
Thank you