Identification of Proteus vulgaris from an Unknown Sample

Praise Selah G. Dagoc

Mambajao, Camiguin

Abstract
Identification of microorganisms from unknown sample is a routine work for a Registered Medical
Technologist assigned in the Microbiology section of the laboratory. For medical technology students, this activity
serves as an important tool in exercising the students’ skills and knowledge in the processing and analysis of the
various methods used in proper identification of isolates. In this activity, the student was able to identify Proteus
vulgaris from an unknown sample with the use of scientific steps and procedures for proper identification of
microorganisms in bacteriology.

Objectives environmental habitats, including long-term care

Through this activity, the student should be able
to:
 Practice skills on proper identification of
microorganisms from sample.
 Properly execute the steps in identification of
microorganisms.
 Know the background and diseases associated
with the microorganism isolated and identified
from the sample.
 Describe the characteristics of the isolate.
 Discuss the infections caused by the isolate and
its treatment.
facilities and hospitals. In hospital settings, it is not
unusual for gram-negative bacilli to colonize both
the skin and oral mucosa of both patients and
hospital personnel wherein infection primarily
occurs. (Struble, et al., 2009)
Patients with recurrent infections, those with
structural abnormalities of the urinary tract, those
who have had urethral instrumentation, and those
whose infections were acquired in the hospital have
also been found to have an increased frequency of
infection caused by Proteus and other organisms like
Klebsiella, Enterobacter, Pseudomonas, Enterococci,
Staphylococci. (Struble, et al., 2009)

P. vulgaris is an enteric bacterium, which means

Introduction it is found in the intestinal tract (Deacon, J.); it is also
found in the soil, contaminated water, or
Proteus vulgaris is one of the most commonly
decomposing organic substances (University of
isolated members of Proteus sp., along with Proteus
Texas, 1995). While it is a pathogenic bacterium and
mirabilis. The genus Proteus is a member of a large
has been shown to cause urinary tract infections, it is
gram-negative bacilli family, Enterobacteriaceae.
also part of the natural flora of the intestinal tract
Proteus organisms are known to be one of those to
(Struble, et al., 2009); and because the microbe can
cause serious infections in humans, along with
live on the skin of some people, P. vulgaris is a
Escherichia, Klebsiella, Enterobacter, and Serratia
common pathogen in hospital wound infections,
species. (Struble, et al., 2009)
especially in the immunosuppressed. (Struble, et al.,
Proteus species are normal flora of the human 2009)
intestinal tract, along with Escherichia coli and
Conversely, Proteus vulgaris is easily isolated
Klebsiella species, of which E. coli is the predominant
from individuals in long-term care facilities and
resident. Proteus is also found in multiple
hospitals and from patients with underlying diseases

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or compromised immune systems. (Struble, et al.,
2009)
But, while P. vulgaris does contribute to the
Proteus infections, it is not the most likely candidate
for community-bourn disease; Proteus mirabilis
causes a majority (90%) of those diseases (Struble, et
al., 2009). However, P. vulgaris is the lead pathogen
in causing hospital-bourn Proteus diseases.
Unfortunately, it is not susceptible to ampicillins or
cephalosporans. (University of Texas, 1995)
The reason the urinary tract is such a hospitable
environment for the colonization of the microbe
includes the microbes ability to degrade urea to
ammonia with the enzyme urease. (Deacon, J.,
Struble, et al., 2009)
The bacterium is a gram-negative rod with
flagella. As a gram-negative rod, it has an
extracytoplasmic outer membrane. It creates an
endotoxin, which can cause a deadly systemic
inflammatory response in 20 to 50 per cent of its
victims. (Struble, et al., 2009)
It has been shown that its optimal growth
temperature was at 37 C. (Struble, et al., 2009) P.
vulgaris is a chemoheterotroph, which means it uses
carbon sources like glucose for energy and carbon
(American Academy of Family Physicians, 2004); as a
chemoheterotroph, it ferments glucose but not
lactose or mannitol. (Clayton State University, 2004)
However, because it is a facultative anaerobe, the
glucose fermentation only occurs in anaerobic
conditions; if placed in non-ideal, aerobic conditions,
the microbe will use a variety of organic molecules
to survive. (Deacon, J.)
When identifying the microbe, several tests can
be used. It will test positive on the citrate test
(Deacon, J.) and urease test (Struble, et al., 2009).
Because it ferments glucose but not mannitol or
lactose, it will only test positive in the
glucose/carbohydrate utilization tests.
Likewise, when observing the plated colonies, it
will be noticed on non-selective media a "swarming"
behavior, where the microbe grows in waves
(Deacon, J.). The bacterium grows and stops in
waves, creating what appears to be distinctive rings
in the manner of tree rings. This feature is the result
of the microbe’s flagella, which allow it to be
extremely motile. The plate will also smell like burnt
chocolate. (American Academy of Family Physicians,
2004)
These biochemical properties and physical
observations helped in the indentification of the
unknown, which turned out to be P. vulgaris.
Materials
To identify the bacteria from the unknown
sample, the following materials were used:
 Sheep Blood agar plate (SBAP)
 MacConkey Agar plate (MAC)
 Test tube containing a Tryptic Soy broth with
an unknown sample
 Test Battery for identification of gram negative
bacilli:
- IMViC (Sulfide Indole Motility medium,
Methyl Red – Voges-Proskauer
Medium, and Simmons Citrate Agar)
- Phenylalanine Deaminase Agar Slant
- Triple Sugar Iron (TSI) Agar
- Lysine Iron Agar (LIA)
- Urea broth
- OF-Carbohydrates (Mannitol, Sucrose,
Lactose???)
 Inoculating loop
 Inoculating needle
 Alcohol lamp
 Test tube rack
 Forceps
 Glass slides
Methods
As a part of the final activity for the bacteriology
laboratory class, each bacteriology student was
given a Tryptic Soy Broth containing an unknown
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isolate which the student must identify using the
appropriate methods he/she has learned from class.
This activity serves as an application of all the
lessons that were taught and to test if the student
understood and remembered the various tests that
were mentioned in the laboratory and lecture
classes in bacteriology. The author received the TSB
tube number 8.
On the first day of the activity, the student
inoculated sample using an inoculating loop from the
TSB broth into the Sheep Blood Agar Plate (SBAP)
and MacConkey plate (MAC) through the multiple
interrupted streak method. After inoculation, SBAP
was placed in a candle jar and incubated at 37C for
24 hours. Whereas, MAC was directly placed into the
incubator and incubated at 37C for 24 hours.
The next day, SBAP and MAC were removed
from the incubator and colony appearance from
each of the plates was noted. Afterward, gram
staining was performed to identify gram stain
reaction of the isolate and guide in biochemical
testing. A colony from SBAP was transferred onto a
pre-heated (sterile) glass slide with the use of a
sterile inoculating loop. Smearing was done by
spreading the colony in a circular motion. The smear
was then air-dried and heat-fixed before staining.
Next, gram stain was applied following the
appropriate order and time for each reagent: Crystal
Violet (primary stain) – 1 minute, Gram’s Iodine
(mordant) – 1 minute, 95% Ethyl Alcohol
(decolorizer) – 30 seconds, and Safranin (counter
stain) – 45 seconds. Upon drying of the stained
smear, the slide was examined under the
microscope set on oil immersion objective. Colonies
observed were pink rods, indicating gram negative
reaction, and were arranged in singles, pairs and
clusters. Because of this finding, the student worker
performed biochemical testing using the test battery
for gram negative bacilli which consists of: IMViC
(SIM, MR-VP & Citrate), Amino Acid Degradation
tests (Phenylalanine & Lysine Iron Agar), Urea
hydrolysis test, Triple Sugar Iron Agar, and
Carbohydrate Fermentation Tests (Mannitol,
etc..????). Sample was inoculated from MAC plate
into MR-VP and Urea using a sterile inoculating loop.
The loop was used to get a part of the colony and
aseptically transferred into each broth by tapping
the loop inside the tubes. On the other hand, sterile
inoculating needles were used to transfer samples
from the plate (MAC) into the solid and semi-solid
media. Transfer into the SIM medium was done by
stabbing the medium until 3/4th to the bottom.
Inoculation on citrate and phenylalanine was done
by streaking the slant in a zigzag pattern using a
sterile inoculating needle. On TSI, transfer was done
using a sterile inoculating needle by stabbing the
butt area and streaking the slant in a zigzag pattern.
Stabbing was done twice in LIA after which the slant
is streaked in a zigzag pattern only once. Full stabs
were done on the OF tests. These tubes were then
placed in an incubator and incubated at 37C for 24
hours.
On the following day, the test batteries were
removed from the incubator and results observed.
On SIM, 2-5 drops of
paradimethylaminobenzaldehyde (PDAB) were
added for Indole test. For MR-VP, 1-2 drops of
methyl red were put into the MR tube, and 10 drops
of 5% alpha-naphthol followed by 15-20 drops of
40% KOH were added to the VP tube. On
phenylalanine slant, 4-5 drops of 10% aqueous ferric
chloride was added.
Results
Swarming, circular, flat, undulate pink colonies
were observed in the MacConkey Agar plate (figure
1.3) whereas swarming, flat, gray colonies were seen
on the Sheep Blood Agar plate (figure 1.1 & figure
1.2). This colony characteristic illustrates a
presumptive identification of a Proteus sp. giving the
student a guide on what to expect.
Gram negative (pink) rods (figure 2.1 & figure
2.2) were seen on the gram stain of a colony from
SBAP. These colonies were found to be arranged in
singles and clusters.
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Table 1-1. Biochemical Test Results
SIM
MR VP Cit Urea
Sulfide Indole Motility
+ + - + - +
For the biochemical testing, SIM showed a
nonmotile reaction demonstrated by the lack of a
brushlike appearance. With the addition of 2-5 drops
of paradimethylaminobenzaldehyde (PDAB), red
color developed on the surface indicating a positive
reaction for Indole. Sulfide reaction was also
observed as demonstrated by the blackening of the
medium (See figure 3.1 in appendix). For MR-VP
(figure 3.2), 1-2 drops of methyl red were put into
the MR tube and 10 drops of 5% alpha-naphthol
followed by 15-20 drops of 40% KOH were added to
the VP tube. A positive reaction (red color) was seen
in the MR tube whereas VP tube exhibited a negative
reaction (no change in color). Citrate tube (figure
3.3) showed a positive reaction as evident in the
prussian blue color of the agar slant. The isolate was
also found to be positive for urea hydrolysis (figure
3.4) as shown in the development of a red-violet
color in the urea tube. In the LIA butt/slant (figure
3.6), a K/A reaction or negative lysine
decarboxylation was observed (purple slant/yellow
butt). H2S reaction was seen in TSI butt/slant (figure
3.5) as demonstrated in the blackening of the
medium (A/A H2S). Gas production was not seen in
TSI. For phenylalanine deaminase test, 4-5 drops of
10% aqueous ferric chloride was added to the slant
after which a green color developed indicating a
positive result. Fermentation (yellow color) was seen
on OF-Sucrose and OF-Maltose whereas OF-
Mannitol showed an inert reaction evident in the
presence of a blue color near the surface of the butt.
A summary of the biochemical test results is shown
in table 1.
OF-Carbohydrates
LIA Phenyl TSI
SUC MAL MAN
+ (A/A
+ - (K/A) + ferm. ferm. ferm.
with H2S)
Discussion
In addition to these methods, Ultrasonography
of the kidneys or a CT scan could also be considered
as part of a workup for Proteus infection of the
urinary tract that does not resolve quickly with
antimicrobial therapy. Calices and/or perinephric
abscesses should be excluded. (Struble, et al., 2009)
For treatment, the recommended empirical
treatment includes an oral quinolone for 3 days or
trimethoprim/sulfamethoxazole (TMP/SMZ) for 3
days for uncomplicated UTIs in women on an
outpatient basis. Acute uncomplicated
pyelonephritis in women can be treated with oral
quinolones for 7-14 days, single-dose ceftriaxone or
gentamicin followed by TMP/SMZ, or an oral
cephalosporin or quinolone for 14 days as outpatient
therapy. For hospitalized patients, therapy consists
of parenteral (or oral once the oral route is available)
ceftriaxone, quinolone, gentamicin (plus ampicillin),
or aztreonam until defervescence. Then, an oral
quinolone, cephalosporin, or TMP/SMZ for 14 days
may be added to complete treatment. Complicated
UTIs in men and women can be treated with a 10- to
21-day course of oral therapy (in the same manner
as for hospitalized patients) as long as the follow-up
is adequate. (Struble, et al., 2009)
However, serious and occasionally fatal
hypersensitivity (ie, anaphylactoid) reactions have
occurred in patients receiving antibiotics. These
reactions are more likely to occur in persons with a
history of sensitivity to multiple allergens. Cross-
sensitivity between penicillins and cephalosporins
has occurred. If a reaction occurs, discontinue the
implicated drug unless the condition is life
threatening and amenable only to therapy with that
antibiotic. Serious anaphylactoid reactions require
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immediate emergency treatment with epinephrine.
Oxygen, intravenous steroids, and airway
management, including intubation, should also be
used as indicated. (Struble, et al., 2009)
P. vulgaris, along with P. penneri, is resistant to
ampicillin and first-generation cephalosporins.
Activation of an inducible chromosomal beta-
lactamase occurs in up to 30% of these strains.
Imipenem, fourth-generation cephalosporins,
aminoglycosides, TMP/SMZ, and quinolones have
excellent activity (90%-100%). (Struble, et al., 2009)
In addition, the use of chlorhexidine and
triclosan in closed urinary catheterization systems
and drug-impregnated catheters reduce the
incidence of Proteus UTI in patients with long-term
indwelling urinary catheters.3,4 While the use of
these types of catheters for Proteus UTIs is helpful in
containing the migration of Proteus in experimental
models, this practice is not widespread, as other,
more common, uropathogens are resistant to the
drugs used in these systems. (Struble, et al., 2009)
A vaccine derived from purified mannose-
resistant Proteus -like (MR/P) fimbriae proteins has
been proven to prevent infection in mouse models
and is under clinical research, but it is not available
commercially. (Struble, et al., 2009)
In presence of struvite renal calculus associated
with Proteus infection, surgery must be done to
remove it. (Struble, et al., 2009)
Most nonurologic infections result in abscesses.
Radical surgical debridement is the cornerstone of
successful therapy. Amputation may be necessary if
skin or muscle necrosis of an extremity is the
presenting infection, but tissue recovery is often
better than expected. Broad-spectrum antimicrobial
therapy is started empirically and is modified by the
results of smears and cultures. Mortality and
morbidity rates are high, even with adequate
treatment. (Struble, et al., 2009)
The discovery of stones requires an evaluation
by a physician knowledgeable in the short- and long-
term management of stones, typically a urologist or
nephrologist. (Struble, et al., 2009)
Summary and Conclusion
Based on the different tests performed, the
student concludes that the organism isolated was
Proteus vulgaris, an organism commonly associated
with urinary tract infections. Moreover, infections
caused by P. vulgaris are treatable with antimicrobial
drugs and, in the case of renal stones, surgery.
References
Struble, K., et al. (2009). "Proteus Infections." E-
medicine.com. Retrieved on March 14, 2010
from http://www.emedicine.com/med/
byname/proteus-infections.htm.
Deacon, Jim. The Microbial World: Proteus vulgaris
and clinical diagnostics. Institute of Cell and
Molecular Biology, University of Edinburgh.
Retrieved on March 16, 2010 from
http://helios.bto.ed.ac.uk/bto/microbes/
roteus.htm.
AAFP.com. Bacterial Identification. American
Academy of Family Physicians 2004. Retrieved
on March 16, 2010 from
http://www.aafp.org/x2215.xml.
University of Texas, Houston (1995). Proteus.
Retrieved on March 16, 2010 from
http://medic.med.uth.tmc.edu/path/00001517.
htm.
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Appendices

Appendix 1. Culture Plates

Figure 1.1 P.vulgaris colonies on Sheep Blood Agar

plate. Colonies show a swarming, gray-colored
appearance

Figure 1.2 A closer look on the colonies in SBAP will

show the flat, undulate characteristic of the P.
vulgaris colonies.

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 Figure 1.3 P.vulgaris colonies on
MacConkey Agar show the characteristic
pink swarming colonies.

Appendix 2 Gram Staining

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 Figure 2.1 Gram staining of the isolate shows gram negative (pink) rods, arranged in singles, pairs and clusters.
Figure 2.2 Gram negative rods of Proteus vulgaris.
Appendix 3 Biochemical Testing
Figure 3.1
SIM Medium
Isolate of P. vulgaris
shows a positive
reaction for Sulfide
(darkening of the
medium), Indole
(reddish/pinkish
surface upon addition
of Kovac’s reagent),
and nonmotile (lack of
brushlike growth).
Figure 3.2
MR-VP Medium
MR (left) shows a positive reaction – red color upon
addition of methyl red.
VP (right) showed a negative reaction – no change in
color after adding 5% alpha naphthol and 40% KOH
Figure 3.3 Citrate
P. vulgaris exhibits a
positive reaction on
Simmon’s Citrate
medium as shown in
the prussian blue color
of the agar.
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 Triple Sugar Iron tubes with P. vulgaris shows a
positive reaction for H2S, evident in the blackening of
the slant, along with the yellow color on the butt and
Figure 3.4 Urea part of the slant. The reaction on TSI is A/A with H2S.
P. vulgaris shows a
positive reaction on
urea hydrolysis test as
demonstrated by the
red-violet color of the
Figure 3.6 LIA
broth.
P. vulgaris shows a negative (K/A) reaction on Lysine
Iron Agar as demonstrated by the purple slant and
yellow butt.

Figure 3.5 TSI

Figure 3.7 Phenylalanine

P. vulgaris is positive on phenylalanine slant, evident in the presence of a green color
on the slant after the addition of 10% aqueous ferric chloride.

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 Figure 3.8 OF-Carbohydrate Tests
Fermentation is observed in OF-Sucrose (left) and OF-Maltose (center) as seen in the yellow color of these two
tubes. Whereas, reaction in OF-Mannitol (right) is inert in P. vulgaris isolate as shown in the blue color near the
surface of the tube.

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