Exercise 12 – Controlling Microorganisms with Antimicrobials
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Exercise 12 – Controlling Microorganisms with Antimicrobials
5.
6.
7.
8.
Objectives
1. Discuss factors effecting chemotherapeutic agent effectiveness.
2. Compare and contrast narrow and broad spectrum drugs.
3. Use the Sanford guide to identify antibiotic uses and side effects.
4. Describe the major mechanism of actions for antibiotics.
Perform a Kirby Bauer test for antimicrobial sensitivity and interpret zone sizes correctly.
Evaluate sensitivity results with regards to clinical applications.
Explain the characteristic antibiograms of bacterial species, particularly those highly
resistant forms (MRSA, VRE, ERBL, and MDRTb).
Analyze the presence of resistant and contaminating organisms during the testing
procedure.
Our lives have been greatly affected by the use of antimicrobial agents used in the
treatment of infections. Agents produced by living organisms, such as bacteria or fungi, that
inhibit or kill microorganisms are called antibiotics. Some antimicrobials are synthetically
produced and altered, resulting in chemical variations referred to as generations of drugs. For
instance, cephalosporin’s are now in their fourth generation (refer to the Sanford guide) each
alteration enhances the activity of the drug by some means.
Several factors impact the effectiveness of these chemotherapeutic agents:
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organism type
method of use - absorbed (topical), ingested (P.O.), or injected (I.V. or I.M.)
antimicrobial tissue concentration
toxicity level
interaction with other agents (synergy or inhibition)
mode of action - lethal (-cidal) or inhibiting (-static)
microbial resistance
The activity of antimicrobials is usually specific, limited to bacteria, viruses, or fungi,
usually not combinations of microbes. Susceptibility patterns (antibiograms) are so specific that
they can be used to confirm the identity of many species of bacteria. The action of these
substances targets metabolic functions in the organism. The best antimicrobials attack
prokaryotic functions and have little or no effect on eukaryotic cells and are ineffective with
viruses. However in some cases, antibiotics may be toxic to a particular tissue in humans or may
cause other complications such as allergic reactions or disruption of normal flora.
While antibiograms are fairly consistent, resistance factors carried on plasmids often result
in geographically unique species with increased resistance to specific antibiotics. While
physicians often treat empirically, the only way to know a specific antimicrobial’s effectiveness is
to test it against the specific pathogen isolated form the infection. Labs test the true effectiveness
of these substances by dilution tests, called Minimum inhibitory concentrations (MIC) in order to
provide the physician with the most complete information available. Today most of these tests
are automated and interpreted by computers. Our experiment will involve a simpler test, called
the Kirby Bauer test, using sterilized filter discs that have been commercially impregnated with a
given concentration of antimicrobial, placed upon a lawn culture of 3 different types of bacteria.
Three species of bacteria will be studied for antimicrobial inhibition.
Exercise 12 – Controlling Microorganisms with Antimicrobials
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Susceptibility to these agents is often under genetic control and in recent years the most
pathogenic organisms are the ones that have gained resistance to our antimicrobial agents.
Various genes for resistance are carried on plasmids in bacteria, and therefore, easily
transferred. MRSA (methicillin resistant Staph. aureus) plague hospitals and convalescent homes
where the pressure to select for the survival of these organisms is strongest. In some cases there
are organisms resistant to even the newest generations of drugs produced. It is the race against
these organisms that drives antibiotic prices higher, and mandates the testing of every
pathogen's susceptibility to a variety of antimicrobials rather than relying on a standard treatment.
Materials and Methods:
3 Kirby Bauer plates (per group)
4 antimicrobial discs
lab disinfectant discard beaker
6 sterile cotton swabs
3 sterile water
McFarland dilution standard
STOCK CULTURES:
Staphylococcus aureus
Pseudomonas aeruginosa
E. coli
forceps
Bunsen burner
First Lab Session
1. Put on your PPG and use eye protection for this lab.
2. Label 3 sterile water tubes and 3 Kirby Bauer plates for each stock culture Staphylococcus
aureus, Pseudomonas aeruginosa, & E. coli.
3. Using a sterile swab and aseptic technique, prepare a standard concentration of each of
the stock cultures in the sterile water tube by comparing them to the McFarland standard.
(Refer to lab 5).
4. Dispose of the swab in a small beaker of disinfectant.
5. Using a fresh sterile swab dipped one time in the standardized concentration of stock
culture produce a LAWN culture.
6. Use the antimicrobial dispenser to apply the discs to the three plates. Each stock will be
tested for the same antimicrobials. Note that the discs are labeled. Use the forceps to
gentle tap the disc down, sterilize forceps in the incinerator.
7. On the table list the names and concentration of each antimicrobial. Look them up in the
Sanford Guide and indicate key information about each.
8. Incubate these plates at 35C for 24 hours.
9. Replace equipment, discard stock cultures & contaminated materials as biohazards.
Hospitals are required to collect the data concerning their specific isolates and their sensitivity
each year and we refer to this pattern of resistance and sensitivity as the antibiogram. This data
is made available to the physician's so that they can begin early treatment empirically. The
antibiograms are specific to geographic regions and even individual hospitals within an area. The
following antibiogram is used with permission of Kern Medical Center Microbiology Lab; please
refer to following link KMC 2006 Antibiogram. N= the number of total isolates of that species of
bacteria in the previous calendar year. The numbers for each species below the antibiotic
represents the percent of sensitive isolates for that species to that specific antibiotic. For
instance, only 44% of the E.coli isolated in during 2005 were sensitive to Ampicillin. The KMC
antibiogram was determined by computerized techniques on a Vitek machine. We are using the
standard Kirby Bauer disk diffusion technique but the results are comparable.
Exercise 12 – Controlling Microorganisms with Antimicrobials
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The diameter of the zone of inhibition is measured in millimeters and then interpreted using the
chart below. Interpretation of the zone size depends upon the concentration of the antibiotic and
the type of organism. When the lab reports out sensitivity results they will describe an organism
as resistant, intermediate, or sensitive when doing a Kirby Bauer Sensitivity. In other cases a
Minimum Inhibitory Concentration (MIC) will be reported indicating the smallest concentration that
can be used to inhibit the growth of the organism. Use the chart below to interpret the zone sizes
for the organisms tested in this lab.
Antimicrobial Interpretation Chart
Disc
Antimicrobial Agent
AM 10
Ampicillin 10mcg
Gram Negative
Staphylococci
Enterococci
Streptococci
(not S. pneumoniae)
Haemophilus sp.
Listeria monocytogenes
Methicillin 5mcg
Staphylococci
Penicillin 10 units
Gram Negative(E.coli)
Staphylococci
Enterococci
Streptococci(not S.
ME5
P10
Resistant
Intermediate
Lactams - Penicillins
Sensitive
13
28
16
21
14-16
18
19
19-21
22
20
9
10-13
14
13
28
14
19
14-16
17
29
15
28
Neisseria gonorrhea
Listeria monocytogenes
26
19
27-46
47
20
Pipericillin 100mcg
P. aeruginosa
17
17
18-20
21
18
22-29
20-27
17
29
17
30
pneumoniae)
PIP100
AMC30
SAM20
CEC30
CFP75
CTX30
CRO30
CXM 30
FOX 30
IMP10
Lactam/Lactamase Inhibitors
Amoxicillin 20mcg/Clavulanic Acid 10 mcg
Staphylococci
19
Other organisms
13
14-17
Ampicillin 10mcg/Sublactam 10mcg
Gram Negative enterics &
11
12-14
staph
Cephalosporins
Cefaclor 30 mcg
14
15-17
Haemophilus sp.
16
17-19
Cefoperazone 75 mcg
15
16-20
Cefotaxime 30 mcg
14
15-22
Ceftriaxone 30 mcg
13
14-20
Haemophilus sp
N. gonorrhea
18
20
21
23
21
26
35
Cefuroxime 30 mcg
Cefoxitin 30 mcg
15-17
15-17
18
18
14-15
16
Impenem 10mcg
14
14
Carbapenems
13
20
18
15
Exercise 12 – Controlling Microorganisms with Antimicrobials
Disc
Antimicrobial Agent
Haemophilus sp
Resistant
VA30
Vancomycin
Enterococci
Other Gram Positives
S. pneumoniae
4
Intermediate
Sensitive
16
15-16
10-11
17
12
17
13-14
15
14-17
11-12
17-20
18
13
21
13
15
13
14-22
16-20
14-17
13
14-17
23
21
18
12
18
13-15
15-18
26-28
31-37
18-21
16
19
29
38
22
16-20
19-21
13-16
21
21
36
22
17
9-12
13-17
13
18
20
14
10
10
15-16
11-15
11-15
21
17
16
16
10
15
11-15
16-18
16
19
Glycopeptides
GM10
Gentamicin 10mcg
CLR15
Clarithromycin 15 mcg
Haemophilus sp
S. pneumoniae
E15
Erythromycin 15 mcg
S. pneumoniae
Azithromycin
Haemophilus sp
S. pneumoniae
AZM15
DO30
TE30
Doxycycline 30 mcg
Tetracycline 30 mcg
Haemophilus sp
N. gonorrhea
S. pneumoniae
CIP5
Ciprofloxacin 5mcg
Haemophilus sp
N. gonorrhea
Lomefloxacin 10 mcg
Norfloxacin 10 mcg
LOM10
NOR10
B10
C30
FD 300
TMP 5
SxT
14
9
Aminoglycosides
12
Macrolides
13
10
16
Bacitracin 10 units
Chloramphenicol
30mcg
S. pneumoniae
Nitrofurantoin 300mcg
Trimethoprim 5mcg
Trimethoprim1.25mcg/
Sulfamethoxazole
23.75mcg
Haemophilus
S. pneumoniae
Tetracyclines
12
14
25
30
17
Quinolones
15
18
12
Others
8
10
List of Antibiotics typically available in lab
Amikacin
Ampicillin
Ampicillin/Sublactam (Synercid)
Amoxicillin/Clavulanic Acid
(Augmentin)
Ceftriaxone (Rocephin)
Cefoperazone (Cefobid)
Cefuroxime (Zinacef)
Chloramphenicol
Clindamycin
Nitrofurantoin (Macrodantin)
Oxacillin or Nafcillin
Pipercillin
Penicillin
Quinupristin (Synercid)
Exercise 12 – Controlling Microorganisms with Antimicrobials
Azithromycin
Bacitracin
Cefaclor (Ceclor)
Cefazolin (Ancef, Kefzol)
Cefixime (Suprax)
Ciprofloxacin
Doxycycline
Erythromycin
Gentamicin
Lincomycin
Nafcillin (Unipen) or Oxacillin
5
Sulfamethoxazole trimethoprim
(SXT-Bactrim)
Tetracycline
Unasyn
Vancomycin
Lab Report #12
Name________________________
1. Data Chart
Record the zone of inhibition by measuring the diameter in millimeters and referring to the interpretation chart, write S
(for sensitive), I (for intermediate), or R (for resistant) next to the measured results.
Antimicrobial
Concentration
Staphylococcus
aureus
mm
Interp
Pseudomonas
aeruginosa
mm
Interp.
Escherichia
coli
mm Interp.
Exercise 12 – Controlling Microorganisms with Antimicrobials
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NOTE: The size of the diameter can not be compared between organisms and antibiotics because the concentration
of the antibiotics is different.
Exercise 12 – Controlling Microorganisms with Antimicrobials
2. Refer to the text, Sanford guide or Prescription Drug Guide for the antimicrobials below and
their mode of action.
Antimicrobial
Mode of Action
Drugs in
the same
family
Precautions &
Warnings
Penicillin
Any
Cephalosporin
Ciprofloxacin
Erythromycin
Tetracycline
Trimethosulfa
(Bactrim, SXT)
3. Using the interpretation chart decide which antibiotic would be most effective against Staph
aureus who’s zone sizes are: Penicillin 25mm, Ampicillin 26mm, Methicillin 18mm
4. Is the above organism an MRSA? Why or why not?
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Exercise 12 – Controlling Microorganisms with Antimicrobials
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5. At KMC the number of MRSA isolates in 1970 was nearly zero. But over the years, the
number of MRSA isolates has increased to 65% out of 1719 isolates.
Look at the increase in MRSA isolates at these North Carolina Hospitals. What are the
implications of the data?
Table 1. Streptococcus pneumoniae susceptibility to nine antimicrobial agents, North Carolina, 1996–2000
% of all susceptible isolates
Year
Penicillin Erythromycin Cefotaxime Levofloxacin
Tmpsmxb
Tetracycline Clindamycin Vancomycin Chloramphenicol
1996
65
78
85
100
64
96
90
100
93
1997
63
69
80
100
57
79
76
100
95
1998
56
64
83
92
51
83
88
100
89
1999
54
61
80
94
51
84
85
100
92
2000
52
61
77
98
50
81
88
100
94
b
Tmp-smx, trimethoprim-sulfamethoxazole
6. What do you think explains the increase in MRSA across the United States?
Exercise 12 – Controlling Microorganisms with Antimicrobials
7. How would you explain a zone where there are a few colonies of organisms growing within an
apparent area of inhibition? As seen in the top two zones on this sensitivity
8. Antibiotics that affect the growth of both gram negative and gram positive organisms are
referred to as broad spectrum antibiotics because they affect a wide variety of organisms.
Antibiotics affecting only gram negative organisms or only a few families of organisms, are
referred to as narrow spectrum antibiotics. Why wouldn't a physician use a broad spectrum
antibiotic to treat an infection and save the patient the time and money involved in culturing and
testing for sensitivities?
For another sample antibiogram see
http://www.ohsu.edu/pharmacy/DraftAntibiogram2004.pdf#search='antibiogram'
For antibiotic resistance
http://www.microbeworld.org/htm/cissues/resist/resist_0.htm
http://www.cdc.gov/Ncidod/eid/vol9no2/02-0123.htm
Date last updated 2/18/2016
©Janet Fulks
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