Identification of an Unknown Pathogen

Francisco Diaz
12pm-2pm Tuesdays, Thursdays
September 15, 2016

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Abstract:
A Student fell ill with diarrhea and vomiting after spring break when he
visited Cancun, Mexico. To determine the causation of these symptoms a
series of biochemical test were conducted to determine the pathogen inside
the host. A gram stain was performed and it seemed that the sample was
contaminated with gram positive microbes. The sample was mixed with
another microbe when it arrived to the lab. The pathogen needed to be
isolated and to do so it was grown in Eosin methylene blue medium (EMB).
Once isolated three different test were conducted to identify if the pathogen
reduced sulfur, if the pathogen reduced nitrate, if the pathogen produced
indole, and to identify if the microbe used citrate as its sole carbon source.
Both the Nitrate reductase and Indole production were positive and the other
two was negative. It was hypothesized that the pathogen that was
associated with these symptoms was (E. coli). The biochemical test
confirmed that the pathogen that was infecting the patient was (E. coli).
Introduction:
Bacteria are single cell organisms that can be found almost
everywhere. Some microbes can found in the human gut without causing
harm, but those that do harm the host are pathogenic. Once inside the host
the pathogen has to find a way to attach itself to the gut tissue. Most
pathogens ligate themselves onto the eukaryotic cell until the pathogen has
a pedestal leading to diarrhea (Slonczewski, Foster, etc. 2014).
To determine what pathogen is found in the patient, laboratory tests of
stool samples from the patient confirmed a gram negative bacterial
pathogen as the cause of infection. Further studies indicated that the
pathogen was a facultative anaerobic, non-endospore forming, non-

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encapsulated, oxidase negative bacteria. After reviewing Burgees manual it
determined that the possible pathogens were: (Enterobacter aergenes),
(Salmonella typhimurium), (Proteus vulgaris), (Escherichia coli), (Shigella
flexneri). The questions that arose were; how can we isolate the gram
negative pathogen which pathogen could be causing theses symptom and
how would they be differentiated? It was hypothesized that (E. coli) was
associated with the symptoms the patient experienced and can be
differentiated using three biochemical test. To solve the problem with the
mixture of gram positive and gram negative the sample was grown in a EMD
medium.
According to the Center for Disease control (CDC) (E. coli) can cause
severe vomiting and diarrhea they also mentioned that people that travel
internationally are at greater risk of getting infected with (E. coli) (United
States, September 2006). If left untreated the this could lead to dehydration;
constant vomiting and watery bowel movements are making the patient lose
fluids. In order for the hypothesis to be correct the pathogen needs to be a
gram negative bacillus, be able to produce indole, and be able to reduce
nitrate.
Materials and Methods
A gram stain and quadrant streak were performed on the sample of the
pathogen. The medium that was used was selective; from that quadrant
streak an isolated colony was then streaked onto a TSL slant for a pure
culture. One the pure culture grew; a dichotomous key was used to identify
the pathogen. The dichotomous key listed the biochemical test that were
necessary to identify which pathogen that was precipitating the symptoms;
Figure 6. The biochemical differential tests that were performed are as

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followed: Citrate, Nitrate Reductase, and the SIM medium were used to
determine sulfur reduction, and indole production, (motility was not relevant
for this investigation). The procedures for these tests were from Microbiology
Laboratory Theory and Application and Bergeys Manual of Determinative
Bacteria was used to consolidate. All of the test were incubated at 37 C.
Results:
The gram stain identified both gram negative and gram positive
microbes, the gram positive were purple cocci, and the gram negative
microbes were pink bacillus as seen in Figure 1. To isolate the pathogen from
the rest of the gram positive microbes it was streaked and incubated in an
EMB medium at 37 C. in the EMB quadrant streak plate
Figure 1. Gram
Negative and Gram
Positive Microbes.

there was evidence of what looked like dark

blue colored colonies in all of the four

quadrants; this was evident that the pathogen which grown in the medium
was only gram negative. From the quadrant streak as seen in Figure 2., the
pathogen was then grown in a TSA slant to maintain a pure culture.
The colonies for biochemical test were gathered from the TSA slant;
the results can be seen in Table 1 below, and the outcome was as followed:
Citrate test had no growth or color change in Simmons Citrate agar, which
can be seen in Figure 3. as a negative result. This meant that the pathogen
was not able to use citrate as its sole source of carbon. The SIM
medium indicated that the pathogen was not able to reduce sulfur; as
seen from Figure 4. There was no change in the color of the medium
indicating a negative result. The pathogen was able to produce indole.
Once the Kovac reagent was added the liquid the liquid layer that
accumulated from the reagent turned red. (Motility was not relevant for this

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investigation). The Nitrate Reductase test is displayed in Figure 5. This test
indicated that the pathogen was positive for reducing nitrate.
Biochemical
Test:
Citrates Test
SIM Medium
Sulfur
Reductase
Figure 3. Citrate
SIM Medium
Test Result
NegativeIndole
Production
Nitrate
Figure
4. The (SIM)
Reductase
Medium
(+) indole
Production (-) sulfur
reduction.

Resu
lt:
(-)
(-)
(+)

(+)

Figure 2. EMB
agar

Observations:

Test for:

No growth or color change.

Positive result would have
growth or turn blue.
There was no color change in
the Medium. Positive result
would be black.
The Kovacs reagent turned red
once it was added to the
medium. Negative result no
color change.
Once both reagents were
added to the broth it instantly
changed color (red).

Use citrate as a carbon source

If the pathogen was able to
reduce Sulfur.

This test was used see if the

pathogen could produce indole
Hydrolyze tryptophan to indole
with tryptophanase
This test was performed to see
the pathogen was able to redu
nitrate. Convert NO 3 NO 2

Discussion:
The biochemical deferential test concluded that the pathogen causing
the patients symptoms was E. coli. The hypothesis was supported by the
biochemical test using the dichotomous key shown in Figure6. The results
from the Citrate test indicated that the pathogen did not contain the enzyme
citrate-permease to transport citrate into the cell. This eliminated the
possibility of the pathogen being either (E. aergenes), or (S. typhimurium)
since these organisms are positive for this test. The nitrate reductase test
was positive. As soon as both of the reagents were added the broth turned
pink, so zinc was not required to reduce nitrate into nitrite, but not into
nitrogen gas. This pathogen possess the enzyme nitrate reductase.
NO3 + Nitate reductates NO 2 + Nitrite Reductae N 2 gas .

This test confirmed that

the pathogen could not have been (P. vulgari) since this pathogen does not

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have either enzyme. The test that supported the hypothesis was the SIM
medium which indicated that the pathogen was able to produce indole, this
means that this pathogen has the enzyme trytophanase. This enzyme
allowed the organism to hydrolysis tryptophan to pyruvate, ammonia, and

Figure 5.
Nitrate
Reduction (+)

indole. This medium also indicated that the pathogen did

not contain either cysteine desulfurase, or
thiosulfate reductase. So this
organism was not able to reduce
sulfur indicated by the equation:
+Thiosulfate Reductase 2 SO 3 +2 H 2 S
.
3 S2 O 3+ 4 H
(Leboffe &Pierce, 2014)
Once the pathogen was confirmed,

Figure 6. The
dichotomous key used
to identify the

I consulted two other colleagues: Miguel Zamorano, and Evelyn Turcote.

According to Miguel Zamorano the sample that he attained was concluded to
be (P. vulgari), he mentioned that when he conducted the SIM medium test
he got a positive result for Sulfur reductase. Evelyn on the other hand came
to a similar conclusion to the one I came across. According to her test her
pathogen was also identified to be (E. coli) after she conducted the Citrate
test.
From this lab investigation it was concluded that the pathogen
infecting the patient is (E. coli). The patient should take Carbapenem which
targets gram negative pathogens and prevents cell wall growth.
(Slonczewski, Foster, etc. 2014).

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References:
Slonczewski, J., Foster, J. W., & Gillen, K. M. (n.d.). Microbiology: An evolving science.
Leboffe, Michael J., and Burton E. Pierce. Microbiology Laboratory Theory and
Application.

Ongoing Multistate Outbreak of Escherichia coli serotype O157:H7 Infections

Associated with Consumption of Fresh Spinach United States, September
2006. (2006, September 26). Retrieved September 11, 2016, from
http://biotech.law.lsu.edu/cdc/mm55d926.pdf