Unknown Organism # 3A Report

November 19, 2014

BIOL 4415-1
Courtney S. Zahn

Introduction

11/20/14

Zahn Unknown Organism # 3A

The case study regarding Unknown Organism #3A involved a 26-year old
man with untreated diabetes who was admitted due to leg pain. Following
admission, painful, erythematous swelling of the right foot and calf were observed,
along with vomiting and abdominal pain. The patient was bitten by a dog 2 weeks
prior, and left the wound untreated. Physical examination revealed a temperature
of 39.3 degrees Celsius and blood pressure of 108/63 mHg. The skin of the lesion
was purple and had blisters. His peripheral blood leukocyte count was 34,900 per
microliter. Cultures taken from the wound revealed an aerobic bacterium that was
moderately resistant to Penicillin G and moderately sensitive to Clindamycin.
Eventually, an above-the-knee amputation was carried out.
Interpretation of the case study began with a search regarding bacteria that
are commonly associated with infected dog bite wounds in humans. Streptococcus
and Staphylococcus organisms are commonly seen in infected dog bite wounds.
Moraxella catarrhalis is seen in infected dog bite wounds as well, but less commonly
than Streptococcus or Staphylococcus (Abrahamian & Goldstein, 2011). A search
was then performed to determine if any of these bacteria cause gangrene in the
infected human. It became evident that Streptococcus and Staphylococcus bacteria
are commonly involved in wet gangrene (Human Diseases and Conditions, 2014).
From this knowledge, suspects from the list of potential unknown organisms were
narrowed down to Staphylococcus aureus, Staphylococcus epidermidis, Moraxella
catarrhalis, Streptococcus agalactiae, and Streptococcus pyogenes.
Methods
Using Bergeys Manual of Determinative Bacteriology as a guide, the process
of identifying Unknown Organism # 3A began. The first step in the identification
process was a Gram stain. The stain showed a Gram positive organism with
coccoidal morphology. The Gram stain was repeated for accuracy. Each subsequent
Gram stain gave the same results. Moraxella catarrhalis is Gram negative, so was
quickly eliminated from the suspects list due to the Gram positive result of the
Gram stain.
Following Gram staining, the organism was streaked via inoculating loop on
blood agar for hemolysis. Beta hemolysis was observed following streaking and
incubation. The organism was also streaked onto plates of nutrient agar, Mannitol
Salt Agar (MSA), Phenylethyl Alcohol (PEA) Agar, and MacConkey agar. After
incubation, results were gathered from each plate. Growth was seen on the nutrient
agar and PEA agar plates. Growth on the PEA plate confirmed a Gram positive
coccoidal organism. The MSA plate showed no growth, which eliminated
Staphylococcus from the suspects list. No growth was observed on the MacConkey
agar, which further confirmed the Gram positive result of the Gram stain.
Once the plates were observed and data was collected, motility tests were
performed. The organism was observed in a microscopic wet mount, and it
appeared to be nonmotile. For certainty, a second motility test was performed with
a Motility Agar stab. This test confirmed the nonmotile result. A Triple Sugar Iron
(TSI) agar test was also performed, and turned yellow in color following incubation.
This provided evidence that the unknown organism was positive for acid production.
A glucose fermentation test also turned yellow in color, indicating the organism to
be positive for glucose fermentation. Catalase and oxidase tests were performed,
both showing negative results.
Streptococcus pyogenes and Streptococcus agalactiae remained on the list of
possible suspects. A CAMP test was performed for final diagnosis of the unknown.
An increase in beta hemolysis (in the shape of an arrowhead) of a Staphylococcus

11/20/14

Zahn Unknown Organism # 3A

aureus indicator strain would have shown Unknown Organism # 3A to be

Streptococcus agalactiae, because only Group B streptococci produce the factor
that cause this hemolysis increase (Fox, 2010). The CAMP test was inconclusive,
and results could not be recorded, so Unknown Organism # 3A will remain unknown.

Results
Table 1 lists all microbiological tests, results, evidence confirming the results, and
images (if available) of the results relating to Unknown Organism # 3A.
Table 1
Test
Gram
Stain
Morpholo
gy
Catalase

Result
Positive

Evidence Confirming Result

Purple

Cocci

Viewed with microscope

following Gram stain
No bubbles; indicative of
anaerobes and facultative
anaerobes mainly
Streptococci

Oxidase

Negative

Motility

Negative

Negative

No color change; no production

of cytochrome-c oxidase;
cannot reduce oxygen;
indicative of anaerobic bacteria
Only Brownian motion; no selfpropelled motility

Well-defined growth along stab

line with no cloudy, diffuse
growth away from stab line;
indicitive of no flagellated cells

MSA

Negative

Pink; no color change/no acid;

intolerance for high salt
environment; cannot ferment
mannitol

Image

11/20/14

Zahn Unknown Organism # 3A

Blood
Agar

Beta
Hemolysis

Clear, colorless zone around

colonies indicative of
complete lysis of Red Blood
Cells

Nutrient
Agar

Positive

Growth

PEA

Positive

Growth indicative of Gram

positive coccoidal bacteria

Lactose
(MacConk
ey)

Negative

No growth; indicative of Gram

positive bacteria

Glucose
Fermenta
tion

Positive

Phenol red turned yellow;

indicative of carbohydrate
fermentation and acid
production

TSI

Positive

Turned yellow; indicative of the

ability to ferment sugars and
produce hydrogen sulfide

CAMP

Inconclusiv
e

11/20/14

Zahn Unknown Organism # 3A

Discussion and Conclusion

After performing several tests and following Bergeys flow charts, it was
assumed that Unknown Organism # 3A was either Streptococcus pyogenes or
Streptococcus algalactiae. Some problems were encountered as we performed our
tests on the organisms, which halted our investigation and resulted in an
inconclusive difinitive diagnosis. The organism appeared to be gamma hemolytic at
first glance, but with the assistance of our professor, we were able to see it was
actually beta hemolytic. All tests were important in narrowing down the list of
possible suspects, but the CAMP test was our final and most important test. The
results of the CAMP test would have provided clarity on the exact species of
Streptococcus. Due to an excessive incubation period, the results of the CAMP test
were inconclusive, and we were unable to identify Unknown Organism # 3A.
Additional tests that could have been performed on Unknown Organism # 3A
include Bacitracin sensitivity testing, and using streak-stab technique when plating
the organism on blood agar. Streptococcus agalactiae is bacitracin resistant and
Streptococcus pyogenes is bacitracin sensitive. A zone of inhibition surrounding the
disc would have shown the organism to be S. pyogenes. The streak-stab technique
is used for detection of streptolysin O, which is produced by Streptococcus
pyogenes (Fox, 2010). A PYR test would have been helpful as well. The PYR tests
the presence of L-pyrrolidonyl arylamidase and is only positive in S. pyogenes
(Murray, 2013).
Stretococcus pyogenes is a Group A Streptococcus (GAS) with Gram positive
cocci arranged in chains. It colonizes in the upper respiratory tract and on the skin
surfaces, and is associated with many diseases including pharyngitis, scarlet fever,
pyoderma, erysipelas, cellulitis, Necrotizing fasciitis, and Toxic Shock Syndrome. S.
pyogenes is spread person-to-person by respiratory droplets, or through breaks in
the skin (Murray, 2013).
Streptococcus agalactiae is a Gram positive Group B Streptococcus (GBS)
with Gram positive cocci arranged in chains. It colonizes the upper respiratory tract
and genitourinary tract, and is associated with neonatal disease and disease in
pregnant women, as well as bacteremia, pneumonia, bone and joint infections, and
skin and soft-tissue infections (Murray, 2013).
Appendix A
Tests
Gram Stain
Morphology
Catalase
Oxidase
Motility
MSA
MacConkey
Agar
PEA
Blood Agar
Nutrient Agar
Glucose
Fermentation
TSI

Results
Positive
Cocci
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive

11/20/14
CAMP

Zahn Unknown Organism # 3A

Inconclusi
ve

Work Cited
Abrahamian, F. M. & Goldstein, E. J. C., Microbiology of animal bite wound
infection. Clinical Microbiology Review. 24:2. April 2011. Web. 19 November
2014. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122494/.
Bergeys Manual of Determinative Bacteriology. Bergeys Manual Trust. Springer,
26. May 2014. Web. 19 November 2014.
http://www.antibmcrobe.org/b93.org/.
Fox, A. Bacteriology chapter 12: Strepococci groups A, B, D and others.
Microbiology and Immunology Online: University of South Carolina School of
Medicine. 2010. Web. 19 November 2014.
http://pathmicro.med.sc.edu/fox/streptococci.htm.
Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. Streptococcus. Medical
Microbiology. Elsevier Saunders. 7th edition. 2013.
Watson, R. Summary of biochemical tests. General and Medical Microbiology.
http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm.