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Exercise 15, Enterotube II System (Example: Urinary Tract Infection)
1. When possible, infections should be _____________________ and tested for
______________________________________.
2. In real-life, ________________________________________ are used to identify
bacteria.
3. The Enterotube is a manual procedure that uses ____________________________ and
___________________________ to detect changes due to metabolic activity.
The tube simultaneously tests for 15 biochemical reactions.
4. We will be using bacteria from the family _________________________________.
These bacteria are _______________________ and often found in the intestines. These
are commonly sources of urinary tract infections due to proximity of the urethra
_______________________________________.
5. UTIs must be addressed because bacteria in the kidneys can lead to life-threatening
blood infections.
6. Each UTI bacteria must not only be identified, but then tested for
__________________________________________________, because the bacteria
causing one infection may have different resistance genes than the same kind of
bacteria causing the next infection BECAUSE of ________________________ picked
up from other bacteria.
8. Bacteria commonly causing Urinary Tract Infections:
1. Members of the Family, __________________________, used in this exercise.
2. Pseudomonas aeruginosa
3. Staphylococcus aureus
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9. Steps of urine culture:

Collect sample _____________________, ____________________, or
____________.

____________________________ or _________________________, because
after 2 hours at room temperature the lab rejects the urine. In 2 hours, bacteria can
reproduce several times, leading to a false positive. For example, the generation
time for _________________________ is about 40 minutes.

______________________________________. This tells you which antibiotics
may be good choices to start. Also tells you the shape of the bacteria.

Perform _________________________________, and determine if an infection is
present.

An infection is greater than or equal to _____________________ CFUs/mL.

__________________________________________, because bacteria exist in
mixed populations.

__________________________________________, to know which is the
problem organism.

Determine ____________________________________________, to be sure the
medication will cure the infection.
10. Case study: Baby born with urinary tract reflux to kidneys:

Page 1. Pediatrician has capability of determining whether a UTI is likely. Blood
found, high protein, positive nitrate, moderate leukocytes indicate UTI. Sent out
for C & S (culture & sensitivity).

Page 2. Partial (immediate analysis) results show “GRAM NEGATIVE BACILLI
ISOLATED”, greater than 100,000 CFUs/ mL.

Page 3. FINAL results tell identification of pathogen as Pseudomonas aeruginosa,
which was sensitive to all antibiotics tested.
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
Page 4. FINAL of UTI #2 was Enterococcus sp. which was resistant to
ciprofloxacin, but sensitive to other antibiotics.

Note that a different set of antibiotics were tested when the UTI was caused by a
gram + than a gram – organism.
The big picture for Enterotube identification:
1.
2.
3.
4.
Reading the Enterotube
See Enterotube Review and Practice Power Point in TEST 3 Folder.
Review Ex 15: Enterotube with Urinary Tract Pathogens
1. STEPS OF URINE CULTURE
1.
2.
3.
4.
5.
6.
7.
2. The Enterotube

All “Fake Urines” are members of the Family Enterobactriaceae.
1 – Escherichia coli
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2 – Enterobacter aerogenes
3 – Proteus mirabilis
4 – Citrobacter freundii
5 – Klebsiella pneumonia
6 – Enterobacter cloacae

If the pathogen was NOT an Enterobacteriacae, a different tube would be used.
3. ____________________________________: if there are several organisms listed for a
specific 5-digit number, the book will give extra tests to perform to determine which is
the organism is your pathogen.
Exercise 16: Kirby Bauer Test for Antibiotic Sensitivity
1. ______________________________ are antibiotics that work against several types of
bacteria (i.e. gram + and gram -). Can kill normal flora.
______________________________ kill specific bacteria.
2. Why does the doctor need a list of several antibiotics to choose from?
1.
2.
3.
3. The agar we will use is the _________________________________. The oatmeal base
does not interfere with antibiotics (chemically).
4. The Kirby-Bauer method is a ________________________________, the same as
antiseptic lab
5. You will know the __________________________________, and the disks will tell
you the _______________________________________ you are looking at.
You need both pieces of information to use the Table in Lab Manual.
6. Definitions:
__________________________ (S) = The bacteria is sensitive to the antibiotic.
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__________________________ (I) = Intermediate. The bacteria has some resistance, and
this antibiotic is not the best choice.
__________________________ (R) = The bacteria is resistant, and this antibiotic will
not cure the infection.
6. Main concepts:
AMC
This Week
Next Week (Tomorrow)

For a given bacterium, the zone of inhibition (diameter in mm) varies with the
specific antibiotic:

Mueller-Hinton II is the standard agar for this test. The oatmeal base does not
chemically interfere with the antibiotic.

Antibiotic sensitivity is measured by machine, but Kirby-Bauer method is used in
difficult cases, when alternatives are being sought out.
7. Take measurements of the diameter, and complete the charts on pages 104 & 105
in the lab manual.
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Exercise 17: Differentiation of Staphylococcus species
1. General characteristics of Staphylococci
• Gram positive, cocci
• Pyogenic (puss forming)
• Form clusters of cells, like grapes

Staphylococcus aureus with resistance genes is _____________________.
Methicillin-resistant Staphylococcus aureus. Resistant to all the -illins.

Staphylococcus epidermis is a normal flora on skin. It is pathogenic when it is
_________________________ the body.

Staphylococcus saphrophyticus can cause ______________________.
2. The Staphyloslide Test is a test for the presence of ____________________________.
It is a _______________________________________.

Antibody- A protein molecule secreted by B plasma cells which bind to specific
antigens.

Antigen- A molecule (i.e. protein or sugar) that triggers and immune response (i.e
Protein A in Staph aureus, Antigen A in Strep pyogenes).

Agglutination Test - The clumping of bacteria cells in the presence of an
antibody.
Agglutination = ______________________________
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3. Coagulase Test for Staph aureus

Coagulase – an enzyme which causes plasma* portion of blood to clot.
*Blood plasma is the liquid portion of blood with no red or white blood cells.
Coagulase = ______________________________________

The jelly like substance ______________________, coats the bacteria cell so the
body’s immune system doesn’t “see”it.

Only Staph aureus has the coagulase enzyme.

A positive coagulase test is a ________________________ test for Staph aureus.
It is reported as Staph aureus.

A negative coagulase test is reported as non-Staph aureus.
It could be Staph epidermidis or Staph saphrophyticus or other
non-Staph aureus bacteria.
Exercise 18 : Differentiation of Streptococcus Species
1. Characteristics of Streptococci
 Gram positive, cocci
 Pyogenic (puss forming)
 Forms chains of cells
 Hemolysis pattern, Agglutination concepts apply.
2. Species of Streptococcus are separated from one another by two different methods:
1. __________________________ and 2. ______________________________.
3. Hemolysis Pattern (Hemo- blood, lysis- break) in blood agar
 Beta hemolysis- Complete breaking of RBC (red blood cells).
Agar looks clear around bacterial growth.

Alpha hemolysis- Partial breaking of RBC.
Agar looks cloudy around bacterial growth. Often it is green or
brownish. Incomplete lysis of blood cells (left trash behind).

Gamma hemolysis- no breaking of RBC.
There is no hemolytic pattern around growth.
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o Group A : Strep pyogenes –which is responsible for 25% of upper
respiratory infections. Other S. pyogenes strains cause “Flesh-eating
Strep”. This has “Antigen A”.
o Group B: Strep agalactiae – Commonly colonizes the vagina, and can be
passed on to newborns. #Semmelweiss. OBGYNS test for this in 3rd
trimester pregnant women and treat to prevent meningitis in newborn.
o Group D Enterococcus: Enterococcus faecalis, Enterococcus faecium –
They carry antibiotic resistance genes and pass plasmids around.
o Non-Grouped (no reactive antigens on membrane): Strep pneumoniae –
frequent cause of bacterial pneumonia with high death rate in elderly.
Confirmed by P disc (Optichin) sensitivity.
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Vaccine with 23 different capsular antigens.
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Exercise 19: Antibiotic Resistant Bacteria
1. Infections have become harder to treat due to:
1. ___________________________________________________________
2. Antibiotics use and misuse in __________________________________,
_________________________, and _____________________.
3. The public needs to be educated in antibiotic resistance. Completing medicine
is extremely important.
4. People have pressured healthcare providers, and antibiotics are prescribed
even if the illness is viral. What can be done? How can we know if something
is viral or bacterial?
Poor communication among healthcare facilities has spread these infections around.
The resistance story:
The drug of last resort…..
2. Antibiotic Resistant Tuberculosis
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3. HA-MRSA – Hospital Associated Methicillin Resistant Staphylococcus aureus, found
in ____________________, after surgery. Internal infection in body.
4. CA-MRSA- Community Acquired Methicillin Resistant Staphylococcus aureus, found
in
______________________, _____________________, ______________________, and
_________________. Surface skin infection.
5. PRSP- Penicillin Resistant Streptococcus pneumoniae. One type of bacterial
pneumonia.
6. VRE – Vancomycin resistant Enterococcus sp. Healthy people do not get VRE.
Immunocompromised, elderly, and recovering patients are susceptible. Common in
___________________________________ settings. The infection can be anywhere in
the body.
7. ESBLs- Extended-spectrum beta-lactamases are most often found in hospitals in
________________________________.
Most Common are 1) __________________ and 2) Klebsiella pneumonieae.
Definitely resistance is passed through ________________ genes.
8. Bacteria that are resistant to carbapenam antibiotics:
CRE (Carbapenam-resistant Enterobacteriaceae)
KPC (Klebsiella carbapenemase)
9. Enzyme that breaks down carbapenem antibiotics.
NDM (New Delhi Metallo-beta-lactamase)
10. What procedures should a medical facility have in place to protect both patients and
employees?
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11. What sort of information do you think is important for patients and families to
understand about superbugs?
12. What other alternatives might there be to infections other than antibiotics?
Video: Frontline: The Hunt for Nightmare Bacteria
Story 1: Addie had CA-MRSA, which developed into boils on the skin and then into
pneumonia. She needed the ECMO Machine to oxygenate her blood and pump it through
her body to allow the lungs and heart to rest. Due to the tubes, an infection from
Stenotrophomonas (gram negative & not many drugs can be used) developed.
Pan-resistant – resistant to all antibiotics.
Story 2: David, a missionary working in India. Was caught and the train ran over his leg.
He had a bacterium with the NDM-1 enzyme, which makes many bacteria resistant and is
easily transferred by plasmid. Areas where water and sanitation are poor, bacteria can
swap resistance genes in a puddle of water. Colistin from the 1940’s, is an antibiotic that
is also toxic to human cells.
Story 3: KPC in New York hospital. Patient transferred to NIH (National Institute for
Health) Maryland hospital. KPC spread in the intensive care unit. Checked for equipment
and caregivers, but there was no crossing. They used DNA sequencing to map how the
spread may have occurred. Some people were silent carriers (living in the digestive tract
without causing symptoms). KPC had spread into the general patient population.
“Hospitals are not required to report antibiotic resistant infections.”
“Antibiotics are the only class of drugs that the more we use, the more we lose because of
resistance”.
“Public Health Officials estimate that half of the antibiotics used in the US is not
necessary or inappropriate.”
-In the 1980’s and 1990’s, bacteria became hard to treat. No new antibiotics were being
developed.
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-Antibiotics are only used briefly, so the company does not get the economic return to
make bringing a new drug to market costs. Companies answer to SHAREHOLDERS &
make portfolio management decisions.
-Who’s in charge of combatting antimicrobial resistance? There is not one single group
dedicated to antimicrobial resistance.
-No reporting mechanisms for reporting antibiotic resistance in the US. Not reported on
death certificates.
-Antibiotic resistance is never going to end.
Exercise 20: Transmission of Infection
Epidemiology –
Index Case –
CDC Atlanta and WHO –
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